In extravasation of T cells, little is known about the mechanisms of transendothelial migration
subsequent to the T cells' tight adhesion to endothelium. To investigate these mechanisms, we
developed a monoclonal antibody (mAb), termed anti-4C8, that blocks transmigration but not
adhesion in a culture system in which high CD26-expressing (CD26hi) T cells preferentially
migrate through human umbilical vein endothelial cell (HUVEC) monolayers cultured on collagen gels. Anti-4C8 reacted with all CD3+ T cells and monocytes but not neutrophils or HUVECs. The structure defined by this antibody was an 80-kD molecule. The mAb at 1 µg/ml inhibited 80-90% of migration of CD3+ T cells through unstimulated and interferon
-stimulated HUVEC monolayers without interfering with adhesion and cell motility. When added to
the cultures after the adhesion, anti-4C8 completely blocked subsequent transmigration of adherent T cells. Phase-contrast and electron microscopy revealed that T cells are arrested at the
intercellular junctions of HUVECs in the presence of anti-4C8. Anti-4C8 exhibited agonistic
effects on resting T cells without other stimuli under culture conditions in which anti-4C8 can
stimulate T cells. First, in the checkerboard assay using collagen gels, the antibody promoted
chemokinetic migration of the cells in a dose-dependent manner from 0.1 to 10 µg/ml. The
predominant population of T cells that migrated into collagen gels with impregnated anti-4C8 were CD26hi. Second, solid-phase-immobilized anti-4C8 induced adhesion of T cells to the
substrate, often with polarizations in cell shape and large pseudopods rich in filamentous (F-)
actin. Third, soluble anti-4C8 augmented F-actin content preferentially in CD26hi T cells
when added to T cells at a high dose of 10 µg/ml. Finally, both anti-4C8-induced chemokinetic migration and transendothelial migration were inhibited by pretreatment of T cells with
pertussis toxin. These findings suggest that stimulation via the 4C8 antigen increases cell motility of CD26hi cells with profound cytoskeletal changes through signaling pathways including G
proteins. The 4C8 antigen may be involved in preferential transmigration of CD26hi cells adherent to HUVECs.
Key words:
 |
Introduction |
Adhesion of leukocytes to the luminal surface of vascular endothelium is the first step for their extravasation,
which is essential for immune surveillance and inflammatory reactions (1, 2). The adhesion process is thought to be
a multistep process. Initially, leukocytes roll along endothelium by selectin-mediated interactions. The leukocyte integrins are activated during this contact with the endothelial
surface, which facilitates tight adhesion and spreading of
the leukocytes to the endothelial cell (EC)1 surface. The
second step is transmigration of the adherent leukocytes across the vessel wall. In general, this step consists of leukocyte diapedesis and penetration of the subendothelial basement membrane; the former involves locomotion of the
adherent cells to and then through nearby endothelial cell-to-cell junctions.
Little is known about the mechanisms of the transmigration process. However, it is becoming evident that leukocyte-EC interactions at the junctional level play an important role in the process (3). A candidate for supporting
these interactions is CD31-platelet-endothelial cell adhesion molecule-1 (PECAM-1), which is an immunoglobulin superfamily expressed by platelets and leukocytes and
localized at the EC junctions (7, 8). Several in vitro and in
vivo studies suggest that homophilic PECAM-1-CD31 adhesion between leukocytes and ECs mediates the transmigration process of leukocytes, including neutrophils, monocytes, and NK cells (9). Although the contribution of
CD31 to T cell transmigration is controversial (4, 15), it
has been demonstrated that naive type T cells (CD45RA+)
migrate through CD31-PECAM-1-transfected murine fibroblast monolayers (8).
With an in vitro system using human umbilical vein EC
(HUVEC) monolayers, we and other investigators have
shown that memory type T cells (CD45RO+) expressing high
CD26 (CD26hi) predominantly migrate through HUVEC
monolayers without a chemokine gradient (16). In
contrast, although CD26-negative (CD26
) T cells also adhere to the monolayers, most of them do not transmigrate. This finding indicates that there are mechanisms other than
CD31-PECAM-1-mediated transmigration that promote
transmigration of adherent CD26hi T cells but not CD26
cells. Transmigration is directional movement of leukocytes
from the apical surface of ECs to the subendothelial space.
It is therefore assumed that molecules involved in transmigration mechanisms selectively stimulate cell motility of
CD26hi T cells. Here we report that a mAb, anti-4C8, inhibits transmigration of CD26hi T cells subsequent to their
adhesion to HUVEC monolayers and induces cell movement similar to chemokinesis as well as an increase in filamentous (F-) actin content in CD26hi cells. This is the
first report suggesting that the 4C8 antigen is involved in
the process of postadhesive transendothelial migration of
CD26hi T cells.
 |
Materials and Methods |
Reagents.
Type I collagen solution extracted from porcine
skin (Cellmatrix I-A) was purchased from Nitta Gelatin Co. EC
growth supplement (ECGS) and porcine heparin were purchased
from Collaborative Research and Nakarai Chemical Co., respectively. Recombinant human IFN-
was provided by Shionogi
Pharmaceutical Co. Monocyte chemoattractant protein-1 (MCP-1)
was provided by T. Kasahara (Kyoritsu College of Pharmacy,
Tokyo, Japan; 19). Rhodamine-conjugated anti-CD26 mAb and
phycoerythrin-Cy5-conjugated anti-CD3 mAb were obtained from Coulter Corporation. Anti-CD11a mAb and purified
mouse IgG3 were obtained from Becton Dickinson and Zymed
Laboratories, Inc., respectively. FCS was purchased from Cell
Culture Laboratories. BSA, Hepes buffer, gelatin, diisopropyl fluorophosphate (DFP), papain, L-cysteine, and collagenase (type
1-A) were obtained from Sigma Chemical Co. Pertussis toxin
(PT) and M199 were obtained from Seikagaku Corporation and
GIBCO BRL, respectively.
Preparation of Cells.
PBMCs were prepared from heparinized
healthy human venous blood by Ficoll-Conray density gradient
centrifugation as described previously (16). The T cell-enriched
fraction was obtained by passing the mononuclear cells through a
nylon wool column. CD3+ cells were negatively selected by inclusion of the fraction with magnetic anti-CD16 mAb (Advanced
Magnetics, Inc.) The selected cells contained >96% CD3+ cells,
as determined by flow cytometry. Neutrophils were prepared by
dextran sedimentation, centrifugation with Ficoll-Conray, and hypotonic lysis of contaminating erythrocytes (20). Neutrophil fractions contained >95% neutrophils. Endothelial cells were obtained from human umbilical cord veins treated with 0.1% collagenase as described previously (16). Cells were grown on gelatin-precoated dishes in M199 containing 20% heat-inactivated FCS,
60 µg/ml ECGS, 100 µg/ml heparin, 1% penicillin and streptomycin solution, and 15 mM Hepes buffer. Culture medium was
changed every 3 d. These experiments used cells in passages 2 and
3 only.
Production of Anti-4C8 mAb.
The anti-4C8 mAb was produced by standard techniques after immunization of BALB/c
mice with PBMCs cocultured with HUVEC monolayers. In
brief, after removal of nonadherent PBMCs from the cocultures, the cocultured adherent cells containing at least 107 lymphocytes
were intraperitoneally injected five times at 2-3 wk intervals.
The final immunization was performed by intravenous injection
of 7 × 106 transmigrated T cells isolated by using an in vitro vessel
model as previously described (16). 3 d later, the spleen was removed and cells were fused with NS-1 cell line. Hybridoma cultures producing antibodies that inhibited T cell migration across
but not adhesion to HUVEC monolayers were selected, cloned,
and recloned by limiting dilution methods in the presence of IL-6.
Malignant ascites were then developed and further purified by an
IgG purification kit (Pierce Chemical Co.). The anti-4C8 mAb
was shown to be of the IgG3 subclass by an ELISA method for
determining subclasses of mouse IgG. Fab fragments were produced by incubating purified IgG with 10 µg/ml papain, 5 mM
L-cysteine, and 2 mM EDTA and then purified by passing over
DEAE-cellulose. SDS-PAGE performed under nonreducing conditions proved that the fragments were properly cut and that no
extraneous bands were present on Coomassie blue stain. Fluorescein-conjugated anti-4C8 mAb was prepared by using a fluorescein labeling kit (Sigma Chemical Co.).
Adhesion and Transmigration Assays.
For the adhesion and transmigration assays, we modified the original system that was described elsewhere (16). HUVEC monolayers were grown to confluence on collagen gels (50 µl/well) in 96-well flat bottom plates
(Becton Dickinson), followed by treatment for 48 h with or
without IFN-
(500 U/ml) before assay. Freshly isolated CD3+
T cells suspended in M199-0.1% BSA were or were not pretreated with mAbs for 20 min on ice. The cells were added to the
wells without washing (3 × 105 cells/100 µl/well). The plate was
centrifuged for 1 min at 50 g and incubated for 3-4 h at 37°C in a
5% CO2-humidified incubator. In the adhesion assay, unbound T
cells were gently washed out, and then adherent cells were immediately fixed with 1% paraformaldehyde in PBS. The transmigration assay was performed simultaneously with the adhesion assay. To count migrated cells, adherent T cells and HUVECs were
removed from the surface of collagen gels by 0.4% EDTA treatment. In some experiments, mAbs were added after unbound cells had been washed from the cultures. Adherent cells on the apical surfaces of HUVECs or cells that had transmigrated into the collagen gels were counted by phase-contrast microscopy in a
blinded manner. The cells in a field of 0.25 mm2 were counted at
a magnification of 100. Adhesion or migration index (%) was calculated as follows: the number of cells with antibody/the number
of cells without antibody × 100. All experiments were performed in triplicate.
Release of Adherent and Transmigrated T Cells.
After T cells (2 × 107 cells) were cultured for 5 h with a confluent HUVEC monolayer on 2 ml of collagen gels (60 mm dish), adherent and transmigrated cells were collected as described (16). In brief, after unbound T cells were removed, T cells bound to HUVECs were
incubated for 20 min with 0.4% EDTA. Almost all adherent T
cells could be obtained by this treatment. The HUVEC monolayer was then removed from the surface of collagen gels by the
EDTA treatment for another 30 min. The collagen gels containing transmigrated T cells were incubated with 0.05% collagenase
in PBS for 3 min to release the cells. This collagenase treatment
was repeated twice. No changes in the expression of surface proteins of T cells were found following the treatment.
Chemotaxis and Checkerboard Assays Using Collagen Gels.
We
modified a chemotaxis assay using collagen gels as described by
others (21). Resting and activated T cells were prepared by culturing freshly isolated T cells for 2 d in RPMI 1640 containing 10% FCS and for 6 d on anti-CD3-coated dishes (0.4 µg/ml) in
medium with 100 U/ml of IL-2, respectively. The cells were
washed, resuspended in M199 plus 0.1% BSA, and added directly
onto collagen gels (50 µl/well), with or without impregnated
MCP-1 (100 ng/ml), in 96-multiwell plates (1-4 × 105 cells/
well). After 1.5-2 h, unbound cells and cells attached on the surfaces of the gels were washed out with 0.4% EDTA in PBS. Cells
that migrated into the gels were counted under a phase-contrast microscope at 200× as described above. In the checkerboard assay, mAbs were impregnated into collagen gels and/or added directly to freshly isolated T cells (4 × 105 cells/well) above the
gels at varying concentrations in 96-multiwell plates. T cells were
incubated for 4 h under these conditions. In these experiments,
to reduce spontaneous migration of T cells, the collagen gels
were prepared with collagen solution at a concentration of 3 mg/
ml, which is three times higher than in the transmigration assays.
Migrated cells in the gels were carefully counted and the migration index was calculated as described above. All experiments were performed in triplicate.
Flow Cytometric Analysis.
Cells were treated for 20 min with
saturating amounts of fluorescein-conjugated mAb and washed
three times with PBS containing 0.1% BSA and 0.01% sodium
azide. The stained cells (10,000 cells) were analyzed on a FACScan® flow cytometer (Becton Dickinson) with gating on the lymphocyte, monocyte, or neutrophil population. All staining procedures were performed at 4°C. Lysis II software (Becton Dickinson)
was used to analyze the data obtained.
Western Blotting.
PBMCs or neutrophils (107 cells) were incubated for 30 min with intermittent agitation with or without
DFP (1 mM ) on ice. The pellets of the cells were resuspended
for 60 min in 100 µl of ice cold extraction buffer containing 50 mM Hepes (pH 7.4), 2 mM sodium orthovanadate, 100 mM sodium fluoride, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100,
1 mM PMSF, 100 µg/ml aprotinin, and 10 µg/ml leupeptin.
Cells were treated for 30 min with 1 mM DFP on ice before cell
lysis. After centrifugation, the supernatant was mixed 1:1 with
2× sample buffer (4% SDS, 20% glycerol, 10% mercaptoethanol,
and a trace amount of bromophenol blue dye in 125 mM Tris-HCl, pH 6.8), heated at 100°C for 5 min, and loaded onto an 8%
SDS-polyacrylamide gel. After electrophoresis, proteins were
transferred onto a nitrocellulose membrane (Pierce Chemical Co.). Residual binding sites on the membrane were blocked by
incubating the membrane in Tris-buffered saline (pH 7.6) containing 0.1% Tween-20 and 5% nonfat dry milk for 2 h at room
temperature. The membranes were incubated with anti-4C8
mAb and then with biotin-conjugated anti-mouse IgG antibody.
After incubation, enzymatic development was performed by using peroxidase-conjugated streptoavidin (GIBCO BRL) and the
ECL system (Amersham).
Cell Morphology Assay and F-actin Staining.
The anti-4C8-
induced changes in cell shape and F-actin formation were visualized by staining with TRITC-labeled phalloidin (Sigma Chemical
Co.) as described previously by others (22). Glass slides (uncoated
eight-well CultureSlide; Becton Dickinson) were coated with
anti-4C8 mAb (10 µg/ml, 250 µl/well) or control IgG3 overnight. T cells (5 × 105 cells/well in M199 with 0.1% BSA) were
added to the slides and incubated for 2 h at 37°C. After incubation, attached cells were fixed with 3.7% paraformaldehyde, permeabilized with 0.1% Triton X-100/PBS, and stained for F-actin
with TRITC-labeled phalloidin. Microscopic analysis was performed using an Olympus microscope equipped with fluorescence accessories and photographed with an ×100 oil immersion
objective. In some experiments, T cells (5 × 105 cells/500 µl of
0.1% BSA-M199/Eppendorf tube) were incubated for 3 h with
anti-4C8 mAb or control IgG3 at 37°C. After fixation and permeabilization, the cells were double-stained with FITC-conjugated phalloidin and rhodamine-conjugated anti-CD26 mAb.
The stained cells were then analyzed by a flow cytometer.
Scanning Electron Microscopy.
The transmigration assay was
performed in the presence of anti-4C8 (1 µg/ml) in 24-multiwell
plastic plates (Falcon Labware). After a 5-h incubation, cultures
were fixed overnight in 2% electron microscopy-grade glutaraldehyde and 5% sucrose in 0.1 M sodium cacodylate buffer (pH
7.4), followed by postfixation with 1% OsO4. The fixed cells and
collagen gels were removed from the plate and processed for
scanning electron microscopy by critical-point drying and gold coating.
Statistical Analysis.
All values are represented as means ± SD.
When comparing two groups, P values were calculated by Student's t test. P values <0.05 were considered to indicate a significant difference.
 |
Results |
Expression of the 4C8 Antigen on Human PBLs and HUVECs
and Its Structure.
We first examined immunofluorescence
profiles of 4C8 expression on PBLs and HUVECs by a
flow cytometer. As shown in Fig. 1, the 4C8 antigen was
expressed intensely on CD3+ T cells and to a lesser extent
on CD3
cells (largely CD16+ cells). Staining of the cells
gated on the monocyte population was positive and two
peaks were seen. In contrast, anti-4C8 did not react with
neutrophils or unstimulated HUVECs. The negative expression of neutrophils was unaffected by stimulation with
LPS or TNF-
(data not shown). Immunofluorescence microscopy also showed no significant staining of confluent
monolayers of HUVECs (not shown). To address the molecular weight of the 4C8 antigen, Western blotting analysis was performed using lysates from PBMCs and neutrophils (Fig. 2). Anti-4C8 reacted with a single band of 80 kD in lysates from PBMCs but not neutrophils. It is possible that the 4C8 antigen was cleaved by numerous proteolytic enzymes released from neutrophils during the procedure of cell lysis. However, anti-4C8 did not react with
the lysates in the presence of DFP, a serine protease inhibitor. There were no differences between blots from gels
electrophoresed under reducing and nonreducing conditions (data not shown). In addition, immunoprecipitation failed to detect the 4C8 antigen by a standard method (not
shown). This finding is consistent with fluorescence profiles of the 4C8 antigen on these cells and further suggests
that the antigen is not present in the intracellular contents
of neutrophils.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 1.
Immunofluorescence profiles of the 4C8 antigen on human
PBLs and HUVECs. PBMC and neutrophils from a healthy donor were
stained with FITC-conjugated anti-4C8 and phycoerythrin-Cy5-conjugated anti-CD3 mAb or control FITC-conjugated IgG3. The stained leukocytes as well as HUVECs (10,000 cells) were analyzed by a FACScan®
(Becton Dickinson) flow cytometer with gating on the lymphocyte,
monocyte, and neutrophil populations.
|
|

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 2.
Western blot analysis of
the 4C8 antigen. SDS-gel electrophoresis of lysates from PBMC and
neutrophils (5.5 × 106 cells/lane)
was performed under reducing conditions. DFP, a serine protease inhibitor, was used to prevent proteolytic activity in the neutrophil
lysates. Control Ab was purified
mouse IgG3 of the same isotype as
anti-4C8. See Materials and Methods for details.
|
|
Anti-4C8 mAb Inhibits T Cell Transmigration Subsequent to
LFA-1-mediated Adhesion to a HUVEC Monolayer.
In our
system, 20-30% of total added CD3+ T cells adhered to
unstimulated HUVEC monolayers after 3-5 h of incubation, and 10-20% of the adherent cells transmigrated during this period (16, 17). IFN-
augments the expression of
intercellular adhesion molecule-1 (ICAM-1), the ligand for
CD11a/CD18 (LFA-1
/
), on HUVEC. Stimulation of
HUVEC with IFN-
increased T cell adhesion and transmigration to 1.5- and 3-fold the base line values, respectively (Fig. 3). We then assessed the changes in adhesion
and transmigration in the presence of antibodies. Anti-CD11a mAb (10 µg/ml) inhibited T cell adhesion to and
transmigration through unstimulated and IFN-
-stimulated HUVEC monolayers by 50-60 and 80%, respectively. This indicates that T cell transmigration is largely dependent upon LFA-1-mediated adhesion to HUVECs. On the
other hand, anti-4C8 mAb (1 µg/ml) did not inhibit T cell
adhesion but rather induced a small increase in adhesion to
IFN-
-stimulated HUVEC monolayers (Fig. 3 A). The
small increase was in accord with the number of T cells that
were retained on HUVECs by the transmigration-blocking effect of anti-4C8. However, transmigration was strikingly
inhibited: 79 and 87% with unstimulated and IFN-
-stimulated HUVEC monolayers, respectively (Fig. 3 B). Control
IgG3 (the same isotype as anti-4C8) showed neither inhibition of T cell adhesion nor transmigration. To determine
even more directly whether anti-4C8 mAb acts subsequently to LFA-1-mediated adhesion, the antibody blocking study was performed following removal of unbound T
cells 1 h after coculturing T cells and IFN-
-stimulated
HUVEC monolayers. As shown in Fig. 4, both anti-4C8
IgG (1 µg/ml) and Fab fragments (10 µg/ml), but not anti-CD11a or control IgG3, completely blocked subsequent
migration of adherent T cells during a 5-h incubation. The
blocking effect was not due to detachment of the adherent
cells from the apical surfaces of HUVECs (data not shown).
However, it is possible that the blockage is caused by a direct suppressive effect of anti-4C8 on cell motility. To examine this, we next performed chemotaxis assays using a
three-dimensional collagen matrix (collagen gels), with or
without impregnated MCP-1. Resting and anti-CD3-activated T cells were added to the gels and incubated for
1.5-2 h in the presence or absence of anti-4C8 at 1 µg/ml.
Although spontaneous migration of both resting and activated T cells into the gels was enhanced two to three
times by MCP-1, anti-4C8 had no effect on spontaneous
or MCP-1-induced migration (Fig. 5). Taken together, the
data suggest that anti-4C8 mAb inhibits postadhesive transmigration of T cells without affecting adhesion or suppressing cell motility.

View larger version (39K):
[in this window]
[in a new window]

View larger version (45K):
[in this window]
[in a new window]
|
Fig. 3.
Anti-4C8 mAb inhibits T cell migration through,
but not adhesion to, HUVEC
monolayers. Freshly isolated CD3+
T cells were incubated with unstimulated and IFN- -stimulated
(500 U/ml, 48 h) HUVEC
monolayers cultured on collagen
gels in the continuous presence of
anti-4C8 (1 µg/ml), anti-CD11a
(10 µg/ml), or control IgG3 (10 µg/ml). The numbers of adherent
cells and migrated cells were determined as described in Materials
and Methods. Results of T cell
adhesion (A) and migration (B)
are expressed as the adhesion and
the migration index, respectively, and represent the mean ± SD of five independent experiments. The index (%) is calculated as follows: the number of
adherent or migrated T cells with antibody/the number of adherent or migrated T cells without antibody × 100. The numbers of the background adhesion and migration with unstimulated HUVEC are 376 ± 84 and 131 ± 4, respectively.
|
|

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 4.
Anti-4C8 mAb inhibits postadhesive transmigration of T
cells. T cells were incubated for 1 h with IFN- -stimulated HUVEC
monolayers. After nonadherent T cells were washed out, the cultures
were further incubated with anti-4C8 IgG (1 µg/ml), anti-4C8 Fab fragments (3 and 10 µg/ml), anti-CD11a (10 µg/ml), or control IgG3 (10 µg/ml). Results are expressed as the migration index calculated as described in the Fig. 3 legend and represent the mean ± SD of four independent experiments.
|
|

View larger version (65K):
[in this window]
[in a new window]

View larger version (76K):
[in this window]
[in a new window]
|
Fig. 5.
Anti-4C8 mAb has no effect on spontaneous or chemotactic
migration of resting or activated T cells into collagen gels. Resting and
activated T cells were prepared by incubation for 2 d without stimuli and
for 6 d on anti-CD3-coated dishes with IL-2, respectively. The cells were
placed on collagen gels with or without 100 ng/ml of impregnated MCP-1
in 96-multiwell plates and incubated for 1.5-2 h in the presence or absence of 1 µg/ml of anti-4C8 in medium. After incubation, T cells that
had migrated into the gels were counted. Results are expressed as the migration index (calculated as described in the Fig. 3 legend), and represent
the mean ± SD of three independent experiments. Gray bar, Nil; black
bar, +Anti-4C8.
|
|
Anti-4C8 mAb Inhibits T Cell Transmigration at the Intercellular Junctions of HUVECs.
It has been reported that
monocytes treated with anti-CD31 remained bound to the
apical surface of HUVEC monolayers at the intercellular
junctions (10). We therefore examined where the blockage
of T cell transmigration by anti-4C8 occurs on the apical surface of IFN-
-treated HUVEC monolayers. Although
numerous T cells migrated across the monolayer into collagen gels below in the presence of control IgG3, the migration was strongly inhibited in the presence of anti-4C8
at 1 µg/ml (Fig. 6). Compared to the controls, T cells that
remained on the apical surface of the monolayer increased
in number, and most of them appeared to be arrested at the
intercellular junctions of HUVECs. Scanning electron microscopy revealed that these T cells were firmly attached
and flattened on the EC surface, with pseudopods extending into the junction (Fig. 7).

View larger version (167K):
[in this window]
[in a new window]
|
Fig. 6.
Anti-4C8 mAb inhibits T cell transmigration at the intercellular junctions of HUVECs. T cells were incubated on IFN- -stimulated
(500 U/ml, 48 h) HUVEC monolayers cultured on collagen gels in the
presence of anti-4C8 (1 µg/ml; B and D) or control IgG3 (1 µg/ml; A
and C). After 4 h, monolayers were washed to remove nonadherent cells
and fixed with 1% paraformaldehyde in PBS or further treated with 0.4%
EDTA to remove monolayers from the surface of collagen gels. Cells
were photographed under a phase-contrast microscope (×100). In the
control Ab sample, although a number of T cells still adhered to the apical
surface of the HUVEC monolayer (A), numerous cells that migrated into
the collagen gel below could be seen (C). However, in the anti-4C8-
treated sample, migration was strongly inhibited (D), whereas the number
of adherent cells was increased (B) compared to the control. Most adherent cells appear to be arrested at the EC junctions (B). The black arrows
indicate these T cells.
|
|

View larger version (171K):
[in this window]
[in a new window]
|
Fig. 7.
Anti-4C8 mAb blocks transmigration of T cells at the junctional level. T cells were incubated for 4 h on an IFN- -stimulated
HUVEC monolayer in the presence of anti-4C8 (1 µg/ml). The culture
was washed, fixed, and prepared for scanning electron microscopy.
Tightly adherent cells extending pseudopods into the junction could be
seen. Bar, 5 µm.
|
|
Anti-4C8 mAb Impregnated into Collagen Gels Predominantly
Stimulates Chemokinetic Migration of CD26hi T Cells.
Activation of cell motility is a critical event in the transmigration process of T cells through the EC junctions. If the 4C8 antigen plays an essential role in the process, stimulation of T cells via the antigen should promote cell motility
and migration. To examine this hypothesis, we used collagen gels in which anti-4C8 had been impregnated to substitute for the 4C8 ligand. The impregnated anti-4C8 IgG
increased T cell migration in a dose-dependent manner, whereas anti-4C8 Fab fragments, as well as control IgG3
and anti-CD11a, had no such effect (Table I). In the
checkerboard analysis, when soluble anti-4C8 was directly
added to T cells above plain gels, migration was significantly enhanced only at the high dose of 10 µg/ml but not
at doses
1 µg/ml. When the dose of the antibody was the
same above and in the gels, the degree of migration was almost equivalent to that induced by anti-4C8 impregnated
in the gels, irrespective of the presence of a gradient. Thus,
stimulation via the 4C8 antigen appeared to induce migration with increased random motility resembling chemokinesis but not chemotaxis. In our transmigration assay system using HUVEC monolayers, most T cells that do not
adhere to HUVECs express low CD26 (CD26lo), whereas
adherent (but not migrating) cells are predominantly CD26
and migrating cells are CD26hi (Fig. 8 A). If the
4C8 antigen is involved in the transmigration, its chemokinetic action should result in migration of CD26hi cells. We
therefore examined whether anti-4C8 stimulation actually causes selective migration of CD26hi cells. After T cells
were incubated for 5 h on collagen gels, with or without
impregnated anti-4C8 (10 µg/ml), cells that migrated were
isolated and analyzed for CD26 expression. The CD26
profiles of migrated T cells with or without anti-4C8 were
different from profiles of the initial T cells (Fig. 8 B). The
proportion of CD26
cells increased among cells that had
spontaneously migrated without anti-4C8, whereas the
proportion of CD26hi cells substantially increased among
cells that migrated after stimulation with anti-4C8. The
CD26 profile of cells that migrated in response to anti-4C8
stimulation was similar to the profile of cells that migrated
through HUVEC monolayers.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 8.
The profile of CD26 expression on T cells that migrated
through HUVEC monolayers (A) and into collagen gels with impregnated anti-4C8 mAb (B). (A) After T cells were incubated for 5 h with
resting HUVEC monolayers, unbound, adherent (but not migrating), and
migrated T cells were isolated as described in Materials and Methods. (B)
T cells were incubated for 5 h on collagen gels with impregnated anti-4C8 (10 µg/ml) or control IgG3 (10 µg/ml). After unbound cells and
cells adhering to the apical surface of collagen gels were removed with
EDTA treatment, migrated cells were released from the gels by treatment
with collagenase. The isolated cells were stained with fluorescein-conjugated anti-CD26 and anti-CD3 mAbs. The CD26 expression of the cells
was analyzed by flow cytometry with gating on CD3+ cells.
|
|
Anti-4C8 Stimulation Induces Formation of Pseudopods Rich
in F-actin and Increases F-actin Content in CD26hi T Cells.
Polarization of F-actin content and changes in cell shape
are essential events in cell movement (23). Therefore,
these changes should occur selectively in CD26hi cells with
cell movement induced by anti-4C8. When T cells were incubated for 1 h on the substrate with immobilized anti-4C8, the majority of T cells attached by the mAb displayed
cell flattening and irregularities in cell shape. With fluorescence microscopy using TRITC-conjugated phalloidin staining, ~15% of the cells revealed extreme cell polarization
with lamellipodia or filopodia rich in F-actin, whereas with
immobilized control IgG3, almost all T cells maintained
spherical morphologies (Fig. 9). We determined whether
the anti-4C8-induced increase in F-actin content is characteristic of CD26hi T cells. After stimulation for 3 h with antibodies in solution, T cells were fixed and double-stained
with FITC-conjugated phalloidin and rhodamine-conjugated anti-CD26 mAb. Flow cytometry revealed that a
definite increase in F-actin content was induced in CD26hi
cells, and to a lesser extent in CD26lo cells, by 10 µg/ml of
anti-4C8 but not by control IgG3 at the same dose (Fig.
10). The small increase of F-actin in CD26lo cells was consistent with the observation that anti-4C8 stimulation also
induced minimal migration of CD26lo cells as described
above. However, 1 µg/ml of anti-4C8, which strongly
blocked transmigration, had no such effect on CD26hi cells.
These data suggest that signaling via the 4C8 antigen induces an increase in F-actin content and morphologic polarization most strongly in CD26hi T cells, resulting in activated motility and migration of the subset.

View larger version (10K):
[in this window]
[in a new window]

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 9.
Morphologic changes of T cells and redistribution of F-actin
induced by immobilized anti-4C8 mAb. T cells were incubated for 2 h
on glass slides precoated with anti-4C8 (10 µg/ml) or control IgG3 (10 µg/ml). After staining with TRITC-conjugated phalloidin, microscopic
observation was performed (×1,000). Extreme cell polarization and large
pseudopods rich in F-actin were noted with immobilized anti-4C8 (B)
but not IgG3 (A).
|
|

View larger version (77K):
[in this window]
[in a new window]
|
Fig. 10.
Soluble anti-4C8 mAb augments F-actin content in CD26hi
T cells. T cells were incubated for 3 h with anti-4C8 or control IgG3.
The cells were fixed, permeabilized, and stained with FITC-conjugated
phalloidin and rhodamine-conjugated anti-CD26 mAb. The stained cells
were analyzed by a flow cytometer. Increased F-actin content was observed in CD26hi T cells at 10 µg/ml, but not 1 µg/ml, of anti-4C8. The
staining shown is representative of three independent experiments.
|
|
G proteins Are Involved in Postadhesive T Cell Transmigration and Migration Induced by Anti-4C8 Impregnated in Collagen Gels.
In general, chemoattractant receptor signaling
is G protein linked and can be inhibited by PT (26, 27). G
proteins may mediate the signal via the 4C8 antigen, as
anti-4C8 stimulation induced chemokinetic migration into
collagen gels. Finally, we determined that PT-sensitive G
proteins are involved in T cell migration, as well as adhesion and transmigration with IFN-
-stimulated HUVEC
monolayers. The anti-4C8-induced migration and the
transendothelial migration were strongly inhibited by PT
pretreatment of T cells (Fig. 11). In contrast, spontaneous
T cell migration and adhesion to the monolayers were
insensitive to PT. The inhibitory effect of PT suggests
that both anti-4C8-induced chemokinetic migration and
transendothelial migration are mediated by signaling pathways that include G proteins.

View larger version (43K):
[in this window]
[in a new window]

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 11.
Inhibitory effect of PT on
T cell transmigration through HUVEC
monolayers and migration into collagen
gels impregnated with anti-4C8 mAb.
T cells were pretreated for 1 h with PT
(100 ng/ml), washed three times, and cultured for 2 h with IFN- -stimulated
HUVEC monolayers (A) or for 3 h on
collagen gels with or without impregnated anti-4C8 (10 µg/ml; B). Adhesion
and migration are expressed as the adhesion and the migration index, respectively. The data are presented as the mean ± SD of three independent experiments.
|
|
 |
Discussion |
In our culture system, a restricted subset, ~6%, of the
added T cells migrate through resting HUVEC monolayers. However, the migrating subset mainly consists of
CD26hi cells, despite adhesion of both CD26
and CD26hi
cells to the HUVECs (16; Fig. 8 A). This strongly suggests
that there are molecules (distinct from those mediating adhesion) that selectively stimulate the motility of CD26hi
cells, causing them to move toward the subendothelial
space. The present data indicate that the antigen recognized
by anti-4C8 mAb is one of the molecules, due to the following lines of evidence: first, anti-4C8 blocked postadhesive transmigration of T cells without interfering with adhesion and cell motility; second, anti-4C8 impregnated in
collagen gels induced chemokinetic migration of T cells,
predominantly CD26hi cells; and third, anti-4C8 stimulation induced formation of large pseudopods rich in F-actin
and increased F-actin content selectively in CD26hi cells.
These results suggest that the cellular interaction via the
4C8 antigen between T cells and HUVECs predominantly
stimulates cell motility and transmigration of CD26hi cells.
Previous studies by others have suggested the possibility
that adhesion molecules, such as integrins or ICAMs,
participate in transendothelial migration of adherent lymphocytes (28). For example, ICAM-2 peptide or cross-linking of LFA-1 (CD11a/CD18) receptors induces migration of NK cells and actin polymerization (28). In the
present study, we have shown that anti-CD11a mAb blocked T cell transmigration. However, this blockage is
due to the inhibitory effect of anti-CD11a on T cell adhesion, because the mAb did not inhibit subsequent transmigration of T cells after tight adhesion to HUVEC monolayers (Fig. 4). Based on this finding, although stimulation of
LFA-1 may be capable of triggering cell locomotion, the
molecule seems unlikely to directly mediate T cell transmigration in our system. This conclusion is in line with a recent report showing that activated T cells can not migrate through ICAM-1-transfected monolayers in the absence of
a chemotactic gradient (8). The authors speculated that the
diffuse distribution of ICAM-1 on the apical cell surface
may not support directed locomotion of T cells across the
monolayers. In this regard, the concentrated distribution of
CD31 at the EC junctions may be an appropriate stimulus
for transmigration of leukocytes, as it has been reported
that pretreatment of the EC junctions with anti-CD31 blocks transmigration of monocytes (10). A hypothesis is
now proposed that homophilic interactions of leukocyte
and EC CD31 mediate transmigration of adherent leukocytes, including T cells, monocytes, neutrophils, and NK
cells (3, 8). However, it should be noted that CD31+ T
cells display a naive phenotype characterized by CD45RA+
expression, whereas a majority of T cells that migrate through HUVEC monolayers express a memory type phenotype
characterized by CD45RO+ expression (15). Indeed,
CD31
CD45RO+ cells have been shown to be the predominant transmigrated cells (15, 18, and our unpublished data).
Thus, CD31 appears not to be required for transmigration of
memory T cells across HUVEC monolayers.
Soluble anti-4C8 inhibited T cell transmigration up to
90% at a dose as low as 1 µg/ml. In contrast, it also promoted cell migration into collagen gels when impregnated
in the gels at doses from 1 to 10 µg/ml or when used in solution at 10 µg/ml (Table I). Since Fab fragments of anti-4C8 did not stimulate migration into collagen gels, it seems
likely that anti-4C8, at the high dose of 10 µg/ml, cross-links the 4C8 antigen existing on T cells at high density
and generates a signal to increase F-actin content, consequently activating cell movement. Thus, the blockage of
transmigration might be due to random movement stimulated by anti-4C8, which is sufficient to overcome the
force to transmigrate generated on HUVEC monolayers.
However, soluble anti-4C8 at 1 µg/ml affected neither
F-actin content in T cells nor the spontaneous and chemotactic motility of resting and activated T cells. Rather, these
findings suggest that anti-4C8 at the low dose blocked the
cellular interaction through the 4C8 antigen (which mediates T cell transmigration) between T cells and HUVEC
monolayers without cross-linking of the antigen. Complete
inhibition of postadhesive transmigration by Fab fragments
strongly supports this notion. Microscopic observations imply that the interaction occurs at the EC junction and that the 4C8 ligand might be concentrated in the junctions like
endothelial CD31. More studies are needed to elucidate
the existence and the nature of the ligand.
CD26hi T cells actively transmigrate, but CD26
T cells
remain adherent to HUVEC. This finding strongly suggests
that additional cytoskeletal changes should occur to support
active cell movement of adherent CD26hi cells, but not
CD26
cells, directed toward the subendothelial space. To
migrate, cells must acquire the redistribution of actin-based
cytoskeleton from symmetry around the cell rim to concentration in a particular region, followed by morphological polarization characterized by extension of lamellipodia
and filopodia (23, 24, and 32). Interestingly, anti-4C8
caused these changes in resting T cells under culture conditions in which the mAb can stimulate T cells. Solid-phase- immobilized anti-4C8 induced changes in cell shape, including extreme morphological polarization and formation
of lamellipodia or filopodia rich in F-actin (Fig. 9). When
impregnated in collagen gels, anti-4C8 promotes migration
of T cells into the gels. The checkerboard analysis suggests
that although a gradient is established with the mAb, the
migration appears to depend upon the concentration of the
antibody itself but not the gradient. Moreover, these effects were observed preferentially in CD26hi cells (Figs. 8 and
10). The 4C8 antigen thus appears to transduce a signal
preferentially in CD26hi cells for cell polarization, with
protrusion of the membrane that is tightly coupled to polymerization of F-actin. A similar cellular polarization induced by mAbs other than anti-4C8 has been reported
with use of T lymphoblasts (33). Those reports showed
that, similar to chemokines, engagement of ICAM-3 or
CD43 with specific mAbs activates the integrin-mediated
adhesion and causes the development of a cytoplasmic
projection at the tailing edge, termed the uropod, and also
induces the redistribution of ICAM-1, -3, CD43, and
CD44 to the uropod. These morphological and functional
changes may contribute to lymphocyte locomotion and recruitment. However, the 4C8 antigen is definitely distinct from ICAM-3 and CD43 in structural characteristics and
tissue distribution. Whether the cellular events caused by
anti-4C8 are similar to those caused by anti-ICAM-3 and
anti-CD43 mAbs remains to be studied.
It is unclear why CD26hi T cells are preferentially stimulated by anti-4C8, despite the intense expression of the
4C8 antigen on all CD3+ T cells. CD26 is a widely distributed cell surface glycoprotein of 110 kD with multiple
functions (37, 38). On human T cells, its expression is preferentially restricted to the CD4+ memory subset and
strongly upregulated following cell activation. Recent
studies indicate that CD26 has dipeptidyl-peptidase IV activity, is not only a functional receptor for collagen but also a receptor for adenosine deaminase, and acts as a costimulatory molecule for T cell activation. More recently, the
chemokine RANTES (regulated on activation, normal T
cell expressed and secreted) has been shown to be a natural
substrate for CD26 (39, 40). These findings suggest that
CD26 plays an important role in immune system events
such as T cell activation and migration. However, two different anti-CD26 mAbs failed to inhibit T cell transmigration (our unpublished data), suggesting that CD26 is not
directly involved in the transmigration process. The avidity
of LFA-1 for ICAM-1 is enhanced by a conformational
change in the integrin upon activation (41). Similarly, the
activation state of T cells as related to CD26 expression
might regulate the affinity of the 4C8 antigen.
We have shown that PT pretreatment of T cells inhibited both anti-4C8-induced chemokinetic migration and
postadhesive transmigration. Thus, both of these types of
cell motility are mediated via PT-sensitive, G protein-coupled pathways. This is consistent with the idea that the 4C8
antigen is involved in the process of transmigration after adhesion of T cells. The result also raises the question of
whether the 4C8 antigen belongs to the chemokine receptor family, because chemoattractant signaling is generally
mediated by G proteins and inhibited by PT (26, 27). CC
chemokines have been shown to attract a subset of CD26hi
CD45RO+ T cells (42). More recently, it has been reported that transendothelial migration can be triggered by
an agonistic mAb against the CC chemokine receptor 2 (CCR2) via a PT-sensitive signaling pathway (45). Therefore, the 4C8 antigen might be a receptor for these chemokines. However, the 4C8 appears to be different
from chemokine receptors identified to date in the following ways: the molecular mass of the 4C8 antigen is 80 kD,
whereas the molecular mass of a chemokine receptor was
estimated to be ~35 kD (45, 46); in contrast to the 4C8
antigen, chemokine receptor levels on T cells are generally
much lower than those on neutrophils and eosinophils (43,
47); and the CCR2 expression is low on the CD26hi T
cells and undetectable on other T cells. Moreover, a novel membrane-bound chemokine with a CX3C motif that was
recently identified has potent adhesive and chemoattractant
activity for unstimulated lymphocytes, particularly CD16+
NK cells (48, 49). The chemokine is induced on activated endothelial cells and thus presumed to regulate leukocyte
trafficking. However, its receptor is expressed on only a
small population (up to 14%) of CD3+ T cells (49). Thus,
although chemokine receptors and the 4C8 antigen show
functional similarities, they appear to be different molecules
based on the differences described above. To our knowledge, the 4C8 antigen is a previously unknown molecule.
Gene cloning and identification will define the structural
nature of this molecule in the near future.
Finally, we propose herein a hypothesis that the interaction via the 4C8 antigen between a T cell and a HUVEC
transduces a signal to stimulate crawling locomotion of
CD26hi cells with profound changes in cell morphology
and cytoskeletal assembly, resulting in preferential migration of this T cell subset.
Address correspondence to J. Masuyama, Division of Rheumatology and Clinical Immunology, Jichi Medical School, Yakushiji, Minamikawachi-machi 329-04, Japan. Phone: 81-285-44-2111, Ext. 3463; Fax: 81-285-44-2779; E-mail: jmas{at}ms.jichi.ac.jp
We thank Dr. M. Miyasaka (Osaka University, Osaka, Japan) for helpful comments on the manuscript, Dr.
Y. Terano and Mr. A. Shimada (Gifu Research Laboratory, Immunology Division, JBC Inc., Gifu, Japan)
for preparing Fab fragments of anti-4C8 mAb, and Ms. Mamiko Semba for her technical assistance.
This work was supported by grants from the Japanese Ministry of Education and Ministry of Welfare.
1.
|
Springer, T.A..
1994.
Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm.
Cell.
76:
301-314
[Medline].
|
2.
|
Butcher, E.C., and
L.J. Picker.
1996.
Lymphocyte homing
and homeostasis.
Science.
272:
60-66
[Abstract].
|
3.
|
Bianchi, E.,
J.R. Bender,
F. Blasi, and
R. Pardi.
1997.
Through and beyond the wall: late steps in leukocyte
transendothelial migration.
Immunol. Today.
18:
586-591
[Medline].
|
4.
|
Muller, W.A..
1995.
The role of PECAM-1 (CD31) in leukocyte emigration: studies in vitro and in vivo.
J. Leukoc.
Biol.
57:
523-528
[Abstract].
|
5.
|
Allport, J.R.,
H. Ding,
T. Collins,
M.E. Gerristen, and
F.W. Luscinskas.
1997.
Endothelial-dependent mechanisms regulate
leukocyte transmigration: a process involving the proteasome
and disruption of the vascular endothelial-cadherin complex at
endothelial cell-to-cell junctions.
J. Exp. Med.
186:
517-527
[Abstract/Free Full Text].
|
6.
|
Del Maschio, A.,
A. Zanetti,
M. Corada,
Y. Rival,
L. Ruco,
M.G. Lampugnani, and
E. Dejana.
1996.
Polymorphonuclear
leukocyte adhesion triggers the disorganization of endothelial
cell-to-cell adherens junctions.
J. Cell Biol.
135:
497-510
[Abstract].
|
7.
|
Muller, W.A.,
C.M. Ratti,
S.L. McDonnell, and
Z.A. Cohn.
1989.
A human endothelial cell-restricted, externally disposed plasmalemmal protein enriched in intercellular junctions.
J. Exp. Med.
170:
399-414
[Abstract].
|
8.
|
Zocchi, M.R.,
E. Ferrero,
B.E. Leone,
P. Rovere,
E. Bianchi,
E. Toninelli, and
R. Pardi.
1996.
CD31/PECAM-1-driven chemokine-independent transmigration of human
T lymphocytes.
Eur. J. Immunol.
26:
759-767
[Medline].
|
9.
|
Muller, W.A., and
S.A. Weigl.
1992.
Monocyte-selective
transendothelial migration: dissection of the binding and
transmigration phases by an in vitro assay.
J. Exp. Med.
176:
819-828
[Abstract].
|
10.
|
Muller, W.A.,
S.A. Weigl,
X. Deng, and
D.M. Phillips.
1993.
PECAM-1 is required for transendothelial migration of
leukocytes.
J. Exp. Med.
178:
449-460
[Abstract].
|
11.
|
Berman, M.E.,
Y. Xie, and
W.A. Muller.
1996.
Roles of
platelet/endothelial cell adhesion molecule-1 (PECAM-1,
CD31) in natural killer cell transendothelial migration and 2
integrin activation.
J. Immunol.
156:
1515-1524
[Abstract].
|
12.
|
Liao, F.,
J. Ali,
T. Greene, and
W.A. Muller.
1997.
Soluble
domain 1 of platelet-endothelial cell adhesion molecule (PECAM) is sufficient to block transendothelial migration in
vitro and in vivo.
J. Exp. Med.
185:
1349-1357
[Abstract/Free Full Text].
|
13.
|
Vaporciyan, A.A.,
H.M. DeLisser,
H.-C. Yan,
I.I. Mendiguren,
S.R. Thom,
M.L. Jones,
P.A. Ward, and
S.M. Albelda.
1993.
Involvement of platelet-endothelial cell adhesion molecule-1 in
neutrophil recruitment in vivo.
Science.
262:
1580-1582
[Medline].
|
14.
|
Wakelin, M.W.,
M.-J. Sanz,
A. Dewar,
S.M. Albelda,
S.W. Larkin,
N. Boughton-Smith,
T.J. Williams, and
S. Nourshargh.
1996.
An anti-platelet-endothelial cell adhesion molecule-1 antibody inhibits leukocyte extravasation from mesenteric microvessels in vivo by blocking the passage through
the basement membrane.
J. Exp. Med.
184:
229-239
[Abstract].
|
15.
|
Bird, I.N.,
J.H. Spragg,
A. Ager, and
N. Matthews.
1993.
Studies of lymphocyte transendothelial migration: analysis of
migrated cell phenotypes with regard to CD31 (PECAM-1),
CD45RA and CD45RO.
Immunology.
80:
553-560
[Medline].
|
16.
|
Masuyama, J.,
J.S. Berman,
W.W. Cruikshank,
C. Morimoto, and
D.M. Center.
1992.
Evidence for recent as well as
long term activation of T cells migrating through endothelial
cell monolayers in vitro.
J. Immunol.
148:
1367-1374
[Abstract/Free Full Text].
|
17.
|
Berman, J.S.,
K. Mahorney,
J.J. Saukkonen, and
J. Masuyama.
1995.
Migration of distinct subsets of CD8+ blood
T cells through endothelial cell monolayers in vitro.
J. Leukoc. Biol.
58:
317-324
[Abstract].
|
18.
|
Brezinschek, R.I.,
P.E. Lipsky,
P. Galea,
R. Vita, and
N. Oppenheimer-Marks.
1995.
Phenotypic characterization of
CD4+ T cells that exhibit a transendothelial migratory capacity.
J. Immunol.
154:
3062-3077
[Abstract/Free Full Text].
|
19.
|
Takahashi, M.,
J. Masuyama,
U. Ikeda,
T. Kasahara,
Y. Takahashi,
S. Kiragawa,
Y. Takahashi,
K. Shimada, and
S. Kano.
1995.
Induction of monocyte chemoattractant protein-1 synthesis in human monocytes during transendothelial
migration in vitro.
Circ. Res.
76:
750-757
[Abstract/Free Full Text].
|
20.
|
Takahashi, M.,
S. Kitagawa,
J. Masuyama,
U. Ikeda,
T. Kasahara,
Y. Takahashi,
Y. Furukawa,
S. Kano, and
K. Shimada.
1996.
Human monocyte-endothelial cell interaction induces
synthesis of granulocyte-macrophage colony-stimulating factor.
Circulation.
93:
1185-1193
[Abstract/Free Full Text].
|
21.
|
Wilkinson, P.C..
1984.
A visual study of chemotaxis of human lymphocyte using a collagen gel assay.
J. Immunol. Methods.
76:
105-120
.
|
22.
|
Parsey, M.V., and
G.K. Lewis.
1993.
Actin polymerization
and pseudopod reorganization accompany anti-CD3-induced
growth arrest in Jurkat T cells.
J. Immunol.
151:
1881-1893
[Abstract/Free Full Text].
|
23.
|
Gumbiner, B.M..
1996.
Cell adhesion: the molecular basis of
tissue architecture and morphogenesis.
Cell.
84:
345-357
[Medline].
|
24.
|
Lauffenburger, D.A., and
A.F. Horwitz.
1996.
Cell migration: a physically integrated molecular process.
Cell.
84:
359-369
[Medline].
|
25.
|
Howard, T.H., and
W.H. Meyer.
1984.
Chemokine peptide
modulation of actin assembly and locomotion in neutrophils.
J. Cell Biol.
98:
1265-1271
[Abstract].
|
26.
|
Bokoch, G.M..
1995.
Chemoattractant signaling and leukocyte activation.
Blood.
86:
1649-1660
[Free Full Text].
|
27.
|
Premack, B.A., and
T.J. Schall.
1996.
Chemokine receptors:
gateways to inflammation and infection.
Nat. Med.
2:
1174-1178
[Medline].
|
28.
|
Somersalo, K.,
O. Carpen,
E. Saksela,
C.G. Gahmberg,
P. Nortamo, and
T. Timonen.
1995.
Activation of natural killer
cell migration by leukocyte integrin-binding peptide from
intracellular adhesion molecule-2 (ICAM-2).
J. Biol. Chem.
270:
8629-8636
[Abstract/Free Full Text].
|
29.
|
Van Epps, D.E.,
J. Potter,
M. Vachula,
C.W. Smith, and
D.A. Anderson.
1989.
Suppression of human lymphocyte
chemotaxis and transendothelial migration by anti-LFA-1 antibody.
J. Immunol.
143:
3207-3210
[Abstract/Free Full Text].
|
30.
|
Dustin, M.L.,
O. Carpen, and
T.A. Springer.
1992.
Regulation of locomotion and cell-cell contact area by the LFA-1 and
ICAM-1 adhesion receptors.
J. Immunol.
148:
2654-2663
[Abstract/Free Full Text].
|
31.
|
Poggi, A.,
P. Costa,
M.R. Zocchi, and
L. Moretta.
1997.
Phenotypic and functional analysis of CD4+ NKRP1A+
human T lymphocytes. Direct evidence that the molecule is
involved in transendothelial migration.
Eur. J. Immunol.
27:
2345-2350
[Medline].
|
32.
|
Haston, W.S.,
J.M. Shield, and
P.C. Wilkinson.
1982.
Lymphocyte locomotion and attachment on two-dimensional
surface and in three-dimensional matrices.
J. Cell Biol.
92:
747-752
[Abstract/Free Full Text].
|
33.
|
del Pozo, M.A.,
P. Sánchez-Mateos,
M. Nieto, and
F. Sánchez-Madrid.
1995.
Chemokines regulate cellular polarization and adhesion receptor redistribution during lymphocyte interaction with endothelium and extracellular matrix.
Involvement of cAMP signaling pathway.
J. Cell Biol.
131:
495-508
[Abstract].
|
34.
|
Campanero, M.R.,
P. Sánchez-Mateos,
M.A. del Pozo, and
F. Sánchez-Madrid.
1994.
ICAM-3 regulates lymphocyte
morphology and integrin-mediated T cell interaction with
endothelial cell and extracellular matrix ligands.
J. Cell Biol.
127:
867-878
[Abstract].
|
35.
|
del Pozo, M.A.,
C. Cabanas,
M.C. Montoya,
A. Ager,
P. Sánchez-Mateos, and
F. Sánchez-Madrid.
1997.
ICAMs redistributed by chemokines to cellular uropods as a mechanism
for recruitment of T lymphocytes.
J. Cell Biol.
137:
493-508
[Abstract/Free Full Text].
|
36.
|
Sánchez-Mateos, P.,
M.R. Campanero,
M.A. del Pozo, and
F. Sánchez-Madrid.
1995.
Regulatory role of CD43 leukosialin on integrin-mediated T-cell adhesion to endothelial
and extracellular ligands and its polar redistribution to a cellular uropod.
Blood.
86:
2228-2239
[Abstract/Free Full Text].
|
37.
|
Morimoto, C., and
S.F. Schlossman.
1998.
The structure and
function of CD26 in the T-cell immune response.
Immunol.
Rev.
161:
55-70
[Medline].
|
38.
|
von Bonin, A.,
J. Huhn, and
B. Fleischer.
1998.
Dipeptidyl-peptidase IV/CD26 on T cells: analysis of an alternative T-cell
activation pathway.
Immunol. Rev.
161:
43-53
[Medline].
|
39.
|
Oravecz, T.,
M. Pall,
G. Roderiquez,
M.D. Gorrell,
M. Ditto,
N.Y. Nguyen,
R. Boykins,
E. Unsworth, and
M.A. Norcross.
1997.
Regulation of the receptor specificity and
function of the chemokine RANTES (regulated on activation, normal T cell expressed and secreted) by dipeptidyl
peptidase IV (CD26)-mediated cleavage.
J. Exp. Med.
186:
1865-1872
[Abstract/Free Full Text].
|
40.
|
Proost, P.,
I. De Meester,
D. Schols,
S. Struyf,
A.M. Lambeir,
A. Wuyts,
G. Opdenakker,
E. De Clercq,
S. Scharpe, and
J. Van Damme.
1998.
Amino-terminal truncation of
chemokines by CD26/dipeptidyl-peptidase IV. Conversion
of RANTES into a potent inhibitor of monocyte chemotaxis
and HIV-1-infection.
J. Biol. Chem.
273:
7222-7227
[Abstract/Free Full Text].
|
41.
|
Springer, T.A..
1994.
Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm.
Cell.
76:
301-314
[Medline].
|
42.
|
Roth, S.J.,
M.W. Carr, and
T.A. Springer.
1995.
C-C
chemokines, but not the C-X-C chemokines interleukin-8
and interferon- inducible protein-10, stimulate transendothelial chemotaxis of T lymphocytes.
Eur. J. Immunol.
25:
3482-3488
[Medline].
|
43.
|
Qin, S.,
G. LaRosa,
J.J. Campbell,
H. Smith-Heath,
N. Kassam,
X. Shi,
L. Zeng,
E.C. Butcher, and
C.R. Mackay.
1996.
Expression of monocyte chemoattractant protein-1
and interleukin-8 receptors on subsets of T cells: correlation
with transendothelial chemotactic potential.
Eur. J. Immunol.
26:
640-647
[Medline].
|
44.
|
Bleul, C.C.,
L. Wu,
J.A. Hoxie,
T.A. Springer, and
C.R. Mackay.
1997.
The HIV coreceptors CXCR4 and CCR5
are differentially expressed and regulated on human T lymphocytes.
Proc. Natl. Acad. Sci. USA.
94:
1925-1930
[Abstract/Free Full Text].
|
45.
|
Frade, J.M.R.,
M. Mellado,
G. del Real,
J.C. Gutierrez-Ramos,
P. Lind,
C. Martinez, and
-A.
1997.
Characterization
of the CCR2 chemokine receptor: functional CCR2 receptor expression in B cells.
J. Immunol.
159:
5576-5584
[Abstract].
|
46.
|
Murphy, P.M..
1994.
The molecular biology of leukocyte
chemoattractant receptors.
Annu. Rev. Immunol.
12:
593-633
[Medline].
|
47.
|
Mackay, C.R..
1996.
Chemokine receptors and T cell
chemotaxis.
J. Exp. Med.
184:
799-802
[Medline].
|
48.
|
Bazan, J.F.,
K.B. Bacon,
G. Hardiman,
W. Wang,
K. Soo,
D. Rossi,
D.R. Greaves,
A. Zlotnik, and
T. Schall.
1997.
A new
class of membrane-bound chemokine with a CX3C motif.
Nature.
385:
640-644
[Medline].
|
49.
|
Imai, T.,
K. Hieshima,
C. Haskell,
M. Baba,
M. Nagira,
M. Nishimura,
M. Kakizaki,
S. Takagi,
H. Nomiyama,
T.J. Schall, et al
.
1997.
Identification and molecular characterization of fractalkine receptor CX3CR1, which mediates both
leukocyte migration and adhesion.
Cell.
91:
521-530
[Medline].
|