* Basel Institute for Immunology, CH-4005 Basel, Switzerland; Department of Pathology, and § Department of Internal
Medicine, Washington University Medical Centre, St. Louis, Missouri 63110; and
Department of Pathology, Centre Médical
Universitaire, CH-1211 Geneva, Switzerland
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Abstract |
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The 2 integrins and intercellular adhesion
molecule-1 (ICAM-1) are important for monocyte migration through inflammatory endothelium. Here we
demonstrate that the integrin
v
3 is also a key player
in this process. In an in vitro transendothelial migration
assay, monocytes lacking
3 integrins revealed weak migratory ability, whereas monocytes expressing
3 integrins engaged in stronger migration. This migration
could be partially blocked by antibodies against the integrin chains
L,
2,
v, or IAP, a protein functionally
associated with
v
3 integrin. Transfection of
3 integrin chain cDNA into monocytes lacking
3 integrins resulted in expression of the
v
3 integrin and conferred on these cells an enhanced ability to transmigrate through cell monolayers expressing ICAM-1.
These monocytes also engaged in
L
2-dependent locomotion on recombinant ICAM-1 which was enhanced by
v
3 integrin occupancy. Antibodies against
IAP were able to revert this
v
3 integrin-dependent
cell locomotion to control levels. Finally, adhesion assays revealed that occupancy of
v
3 integrin could decrease monocyte binding to ICAM-1.
In conclusion, we show that v
3 integrin modulates
L
2 integrin-dependent monocyte adhesion to and migration on ICAM-1. This could represent a novel mechanism to promote monocyte motility on vascular ICAM-1
and initiate subsequent transendothelial migration.
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Introduction |
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MONOCYTES are among the first leukocytes to enter inflamed tissue where they play a vital role in
the healing process. These cells, like other leukocytes, leave the circulation by crossing the vascular endothelium. The dynamic process of transendothelial migration (TEM)1 in vivo is a multistep mechanism. It
includes initial tethering of leukocytes to the vessel wall,
followed by rolling along the endothelium, tight adhesion
to the endothelial surface, and ultimately movement of the
leukocyte through the intercellular junctions into the underlying tissue (9, 20, 66). The selectin family of adhesion
molecules and their ligands have been implicated in the
initial tethering of leukocytes to the vessel wall through weak adhesions that permit leukocytes to roll in the direction of flow (40). Another class of adhesion molecules, the
integrins, of which 1 and
2 are key players in TEM (1,
34, 65, 70), mediate arrest, tight adhesion, and spreading of
leukocytes on the endothelium (2). Cellular activation precedes integrin-mediated adhesion and chemoattractants
are potent activators in vivo (34, 68).
Monocytes express a selection of adhesion molecules including selectins, 1,
2, and
v integrins (28, 45). The
1
integrin
4
1 present on monocytes promotes arrest and
adhesion to vascular cell adhesion molecule-1 (VCAM-1) on
the vascular endothelium (1). The
2 integrins
L
2 and
M
2 (CD11a/CD18 and CD11b/CD18, respectively) also
present on monocytes (60), bind to the endothelial ligand
ICAM-1 (CD54) (17, 65), and mediate tight adhesion to
the endothelium (70). However, this presents a paradox: if
2 integrins mediate tight adhesion of a leukocyte to
ICAM-1, how does the cell initiate the motility necessary
for subsequent diapedesis? The cell must be able to modulate adhesions at the cell surface in order to move forward.
It was recently shown that
L
2 on lymphocytes can
downregulate
4
1 integrin activity and enhance cell motility on fibronectin (52). We have previously demonstrated that the
v
3 integrin can regulate lymphocyte
motility on VCAM-1 by modulating the function of
4
1
(32).
The v
3 integrin can bind to multiple ligands in an
Arg-Gly-Asp-dependent manner (22, 23). The integrin
per se mediates cell locomotion and is involved in cell migration on components of the extracellular matrix (ECM)
(11, 41). It can also modulate the activity of other integrins, such as phagocytosis mediated by
5
1 (3) and adhesion through
M
2 (33, 71). The
v
3 integrin has been
shown to be physically and functionally associated with integrin-associated protein (IAP, CD47) (7), a 50-kD membrane protein found on a variety of different cell types
(55), as antibodies against IAP can block some
v
3 integrin-mediated functions (7, 44). IAP on its own is a receptor for the carboxy-terminal domain of thrombospondin-1
(25), and anti-IAP antibodies can block TEM of leukocytes at a step subsequent to tight adhesion (12). It was recently shown that certain forms of platelet endothelial cell
adhesion molecule (PECAM-1)/CD31 are heterotypic ligands for
v
3 integrin (8, 51). Interestingly, several
groups have shown that antibodies against PECAM-1 are
also able to block TEM (49, 72). Therefore, there is some
evidence to suggest that the
v
3 integrin might be involved in TEM.
We looked specifically at the role of v
3 integrin in
monocyte migration. A
3 integrin-deficient monocytic
cell line displayed poor migratory ability compared with a
3 integrin-positive monocytic cell line in TEM assays.
Antibodies against
v or IAP inhibited transmigration of
3-positive monocytes. Moreover, transfection of the
3
chain into
3-deficient cells with subsequent expression of
3 integrins conferred on these cells an enhanced ability to transmigrate. In the process of elucidating the mechanism
of this enhanced transmigration, we found that
3 integrin-positive monocytic cells preferentially transmigrated
through ICAM-1-expressing cell monolayers. Subsequent
studies of monocyte locomotion on recombinant ICAM-1
and adhesion assays revealed a cross talk mechanism between
v
3 integrin and
L
2 integrin on monocytes
which affects monocyte binding to and migration on
ICAM-1.
Our results point to a role for the v
3 integrin in
2 integrin-dependent migration of monocytes on ICAM-1,
which could be a mechanism that enables monocytes to
overcome tight adhesion to endothelial ICAM-1 under inflammatory conditions and engage in subsequent TEM.
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Materials and Methods |
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Cell Lines
J774.2 and WEHI-3 murine monocytic cell lines were obtained from American Type Culture Collection (Rockville, MD). The mouse endothelioma cell line e.end2 was from W. Risau (Max-Planck, Bad Neuheim, Germany). Untransfected L cells and L cells transfected with full-length CD31 were obtained from S. Albelda (The Wistar Institute, Philadelphia, PA) and have previously been described (14). The L cells transfected with ICAM-1 were obtained from C. Figdor (University Hospital, Nijmengen, The Netherlands). The THP-1 human monocytic cell line was obtained from the lab of A. Lanzavecchia (The Basel Institute for Immunology, Basel, Switzerland).
Medium and Reagents
J774.2, e.end2, untransfected L cells, and L cells transfected with CD31
were grown in DME media (GIBCO BRL, Paisley, Scotland) supplemented with 10% FCS (GIBCO BRL, Auckland, New Zealand), nonessential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 mg/ml
streptomycin (all from GIBCO BRL, Auckland) and 5 × 105 M 2-mercaptoethanol (Fluka, Buchs, Switzerland). WEHI-3 cells were grown in Iscove's
modified Dulbecco's (IMDM) media (GIBCO BRL, Paisley) supplemented
as above. Murine
3-transfected WEHI-3 cells were cultured in IMDM
with 0.5 g/liter geneticin, (G418 sulfate from Calbiochem-Novabiochem
Corp., La Jolla, CA). L cells transfected with ICAM-1 were cultured in
IMDM with 1 g/liter of G418. The human monocytic cell line THP-1 was
cultured in RPMI (GIBCO BRL, Paisley) with 10% FCS. Human unbilical
vein endothelial cells (HUVEC) cells were obtained from U. Vischer (Centre
Médical Universitaire, Geneva, Switzerland) at first or second passage.
The mouse soluble recombinant adhesion molecules ICAM-1, PECAM-1, and VCAM-1 have been previously described (51). Soluble recombinant human ICAM-1 was obtained from J.E. Meritt (Roche Products Ltd., Herts, UK). The mouse and human chemokine MCP-1 used in
the transmigration assays were from R&D Systems, Inc. (Abingdon, UK).
Mouse and human TNF- and mouse laminin were all from GIBCO BRL
(Paisley). Human plasma fibronectin and human plasma vitronectin were
from Collaborative Research (Bedford, MA). BSA was from Sigma
Chemical Co. (Buchs, Switzerland).
Other Reagents and Antibodies
For FACS® analysis the following antibodies were used: anti-3, anti-
M,
anti
4, anti-CD31 (all from PharMingen, San Diego, CA), anti-MHC
class II, anti-
v, anti-IAP, anti-
6 (EA-1) (57) and anti-
L (see below).
For TEM and migration assays on ICAM-1, only affinity-purified preservative-free antibodies were used. The anti-mouse antibodies were anti-
v
integrin (RMV-7 from H. Yagita [Juntendo University, Tokyo, Japan]),
anti-
L (FD441.8) (61), anti-
4 (PS/2) (48), anti-IAP (MIAP 301) (43),
anti MHC class II (M5/114) ATCC TIB 120, and anti-
6 (GoH3) (53). The anti-human antibodies directed against the integrins
1 (JB1a),
2
(P489-A11),
v
3 (LM609),
v
5 (P1F6), and
v (CLB-706), and anti-MHC class I were all from Chemicon (Temecula, CA). Anti-human IAP
(B6H12) has previously been described (7, 26). The anti-
L subunit function blocking antibody (mAb 38) was from the lab of N. Hogg (Imperial
Cancer Research Fund, London, UK) (52).. For cross-linking experiments,
the following secondary affinity-purified preservative-free polyclonal antibodies were used: Fc fragment-specific goat anti-rat IgG and Fc fragment-specific goat anti-mouse IgG (Chemicon). Rabbit antibodies against human fibronectin (Sigma Chemical Co., St. Louis, MO) or against human
fibrinogen (Dako A/S, Copenhagen, Denmark), both cross-reactive with
the mouse proteins, were used in the immunofluorescent studies. The secondary reagent was a FITC-labeled goat anti-rabbit antibody (Southern Biotechnologies, Birmingham, AL).
Isolation of Human Peripheral Blood Monocytes
Human blood from healthy donors was collected with heparin (Liquemin; Roche). Peripheral blood mononuclear cells were separated from whole blood by density gradient centrifugation using Ficoll-hypaque (Pharmacia Biotech., Inc., Dübendorf, Switzerland). Monocytes were then separated from the lymphocytes using a Percoll gradient (Pharmacia Biotech., Inc.). The isolated monocytes were used within 48 h for TEM assays or FACS® analysis, and cultured in RPMI medium with 10% FCS (Boehringer Mannheim, Mannheim, Germany).
Stable Transfection of the 3 Integrin Chain into
WEHI-3 Cells
A 2.6-kb cDNA fragment containing the entire mouse 3 integrin coding
region was excised from the pBluescript II KS
vector with BamHI and
XhoI and then inserted into the pcDNA3 vector (Invitrogen, Leek, The
Netherlands). WEHI-3 cells were transfected using the lipofectamine
method. Briefly, 12 µg of DNA in a 50-µl volume was mixed with 30 µl of
lipofectamine (GIBCO BRL, Basel, Switzerland) in a total volume of 100 µl
with distilled water. After a 15-min incubation at room temperature, this
was added dropwise to 5 × 106 WEHI-3 cells in 3 ml Opti-MEM (GIBCO
BRL, Paisley). After 24 h at 37°C without serum, 3 ml of medium containing 20% FCS was added and cells were left for another 24 h at 37°C. Cells
were then harvested and cultured in medium with 500 mg/ml geneticin and seeded at limiting dilution into 96-well plates. Colonies were picked
14 d later. Cell clones were expanded individually and clones expressing
the
3 integrin chain were selected by FACS® analysis. These were then
expanded further.
Flow Cytometry
Suspension and trypsinized adherent cells were collected and resuspended in Dulbecco's PBS with 1% BSA. Cells (105 per sample) were washed twice in this medium and then resuspended in DPBS/BSA with saturating amounts of mAbs. After a 30-min incubation at 4°C, cells were washed twice in DPBS/BSA and then resuspended in staining solution containing FITC-labeled goat anti-rat IgG (Jackson ImmunoResearch, Milan, Italy and Analytica, La Roche, Switzerland) for rat monoclonals, FITC-labeled goat anti-hamster IgG for hamster monoclonals, or FITC-labeled goat anti-rabbit IgG for antibodies raised in rabbit (Southern Biotechnologies, Birmingham, Alabama). After another 30-min incubation at 4°C, cells were washed twice, resuspended in the staining solution containing 0.1% propidium iodide, and then analyzed by flow cytometry (FACScan®; Becton Dickinson Co., Mountain View, CA). Control cell suspensions were incubated with secondary antibody alone.
Transendothelial Migration Assay
Transwell culture inserts of 24-well tissue culture plates (6.5-mm-diam
polycarbonate membranes with 5 µm diameter pores [Costar Corp., Cambridge, MA]) were coated with 50 mg/ml laminin in Earle's balanced salt
solution for 30 min. Excess laminin was removed from the inserts and
e.end2 cells at 106/ml were seeded on the inserts in 100 µl of medium.
Cells were allowed to grow to confluence on filters for 48 h. Cell confluence was checked by staining some filters with May-Grunwald-Wright- Giemsa solution (Fluka) followed by microscopic control. The cultures were then washed once in DME with 5% FCS and then preincubated in
medium with or without 20 ng/ml of TNF- for 5 h at 37°C, after which the
cultures were washed twice with medium.
Cells of the J774.2 or WEHI-3 line were washed once in medium and then adjusted to 106 cells/ml. 100 µl of cell suspension were added per insert. Thereafter, 300 µl of medium were placed into the lower chambers of the Transwells with 125 ng/ml of monocyte chemoattractant protein-1 (MCP-1). The inserts were carefully placed into the lower chambers to avoid air bubbles forming at the interface between the underside of the insert and the medium. Migration was allowed to proceed for 4 h at 37°C. The assay was stopped by removing the medium from the upper well and rinsing the upper surface of the insert twice with 0.2% EDTA in PBS. The number of cells which had migrated into the lower chamber was determined by light microscopy at a magnification of 10. Alternatively for the human cell experiments, HUVECs were cultured for 48 h on filters precoated with 50 µg/ml fibronectin. The rest of the assay was as described before. However, freshly isolated human monocytes were allowed to transmigrate for 2 h.
For antibody-blocking studies, 300 µl of monocytic cells at 106 cells/ml were spun down and re-suspended in 100 µl of medium in an Eppendorf tube (Hamburg, Germany). The antibodies were added to a final concentration of 50 µg/ml. The tubes were then incubated on a shaker for 40 min at 4°C. Before the assay, cells were spun down, washed once in medium, and then resuspended in 300 µl of fresh medium. This was then added to three wells per condition. For each experiment, the number of cells which had transmigrated was expressed as the mean value of cells counted in three wells.
Transmigration through L Cells
Basically, the same method as for endothelial cells was used. Untransfected L cells, L cells expressing PECAM-1, or L cells expressing ICAM-1 were seeded at a concentration of 105 cells per well on laminin-precoated inserts and then allowed to grow to confluence for 48 h. Cultures were washed once in medium and monocytic cells were added in a 100-µl volume to the upper well. Chemokines were used at the same concentration as in the TEM assay. Cell confluence was checked as before. For this experiment, the mean value of transmigrated cells counted in three wells was expressed as a percentage of the total number of cells added per well.
Cell Migration Assay
Single wells of a 96-well plate (Serocluster; Costar Corp.) were coated with 2.4 µM of recombinant soluble adhesion molecules. This concentration has been shown to be the saturating concentration for a single well of a 96-well plate (32). A typical saturating coating consisted of either 100% mouse or human ICAM-1. In the case of mixes, the saturating coating consisted of 99% ICAM-1 and 1% CD31, obtained by mixing 2.376 µM of ICAM-1 and 0.024 µM of CD31. The total protein concentration remained at 2.4 µM. For the control experiments ~1% vitronectin or ~1% laminin was used. Wells were coated for 1 h with protein and then blocked for 1 h with 20% BSA, all at room temperature, after which wells were washed three times with serum-free medium. 105 cells in the exponential growth phase were washed once with 500 µl of serum-free medium, resuspended in 100 µl, added to a coated well, and then allowed to adhere at 37°C for 5 min. Nonadherent cells were washed away by gently adding 100 µl of medium to the well and exchanging this volume twice. The plate was then placed under an Axiovert 100 television microscope (Carl Zeiss AG, Jena, Germany) equipped with an incubator chamber. The temperature of the air and the microscope plate was maintained at 37°C by a TRZ 3700 unit and the CO2 level (10 or 5%) was controlled by a CTI controller 3700 (all from Carl Zeiss AG). Continuous recording of cell migration was performed using a 20× objective with video time-lapse equipment.
During incubation, antibodies were added manually in a volume of 10 µl
using a Gilson pipette and a curved multiprecision tip (Sorenson Bioscience,
Inc., Salt Lake City, Utah). Before the addition of antibody, cells were allowed
to migrate on the substrate for 40 min in order to record locomotion in the
absence of antibody. After antibody had been added, cell locomotion was recorded for another 40 min. To block molecules on the cell surface, anti-L
or anti-IAP antibodies were added at a final concentration of 50 µg/ml. To
cross-link molecules, anti-
v, anti-
2, anti-
6 or anti-major histocompatibility complex (MHC) antibodies were used at a final concentration of 10 µg/ml, together with 10 µg/ml of secondary anti-Fc-specific antibody.
Data Analysis
After completion of the assay, the video was played 60 times faster on a Sony color video television. The displacement of individual cells was traced on transparent write-on films. A minimum of 10 tracks were followed for 30 min in each experiment. Migration distance was measured in centimeters for each track using a curvimeter. Results are expressed in µm/h by using the conversion factor 8 cm = 100 µm.
Immunofluorescent Studies
L cells expressing ICAM-1 were trypsinized and cultured on eight chamber glass slides (Nunc, Inc., Naperville, IL) for 24 h. Cells were fixed in ice-cold acetone for 7 min and then allowed to air dry. Wells were blocked with 10% FCS in PBS for 30 min after which primary antibody was added at a 1:50 dilution in PBS/BSA. This was left at room temperature for 30 min, after which the secondary FITC-labeled antibody was added and left for another 30 min. Each step was followed by a washing step in PBS and distilled water.
Cell Bead Attachment Assay
Ligand-coated beads were prepared as described previously (52). Briefly, 200 µl (108) of 3.2-µm polystyrene beads (Sigma Chemical Co.) were washed twice in distilled water followed by two further washes and resuspension in 0.1 M bicarbonate buffer, pH 9. ICAM-1 or fibronectin (control) was added to the beads at a final concentration of 10 µg/ml. To prepare BSA-coated control beads, they were incubated with 2% BSA. The beads were rotated for 1 h, washed once in PBS and blocked with 2% BSA for 2 h, all at room temperature. Finally, the beads were washed twice in 20 mM Hepes, 140 mM NaCl, and 2 mg/ml glucose, pH 7.4 (assay buffer).
Multiwell Lab-Tek chamber slides (Nunc, Inc.) were coated overnight
at 4°C with the following molecules: recombinant ICAM-1; vitronectin;
anti-v
3, anti-
v
5, anti-
6, anti-
2, and anti-MHC class I antibodies; all
at 50 µg/ml or BSA. The next day, the wells were washed twice with PBS
and nonspecific binding sites were blocked with 2% BSA at room temperature for 2 h. THP-1 monocytic cells (150 µl of 2 × 106/ml) in assay buffer
were added to the wells and allowed to settle on ice for 15 min. Freshly
prepared ligand-coated beads were then added to the wells at a 100:1
bead/cell ratio in 50 µl of assay buffer. After 30 min at 37°C, unbound
beads and cells were removed by washing the wells four times in prewarmed assay buffer. Bound cells were fixed with 1% formaldehyde in
PBS for 20 min and the cells were then stained with haematoxylin for 10 min. 100 cells were counted under the microscope (40× oil immersion objective; Carl Zeiss AG, Jena, Germany) and the number of beads which
had bound to these cells was determined (attachment index). For antibody-blocking studies, anti-
L (mAb38), anti-
1, or anti-
6 was added to
the cells at a final concentration of 50 µg/ml and left for 15 min at 4°C before the addition of beads.
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Results |
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The v
3 Integrin Is Involved in Monocyte
Transendothelial Migraton
To study molecules involved in TEM we set up an in vitro
assay. A murine endothelial cell line was grown to confluence on laminin-precoated polycarbonate filters with defined 5-µm-diam pores. Several murine monocytic cell
lines were screened for their ability to transmigrate. In
vivo, monocytes preferentially home to acute inflammatory tissue. Inflammation is accompanied by increased expression of both ICAM-1 and VCAM-1 on the endothelium, which are essential molecules for leukocyte TEM
(47). We therefore treated the endothelial monolayer with
the inflammatory cytokine TNF-, which led to increased
expression levels of ICAM-1 and VCAM-1 as determined
by FACS® analysis (data not shown). As a soluble gradient of endogenous chemokine promotes the TEM of
monocytes in vitro (54), we included the chemokine MCP-1
in our assay (69). An optimal concentration of 125 ng/ml
was chosen because MCP-1 has been shown to be maximally chemotactic at around this concentration (56). As
expected, transmigration of monocytes was more efficient
through the TNF-
-activated endothelial monolayer. The
J774.2 monocytic cell line was able to selectively migrate
through the endothelial monolayer, but not through plain
filters or filters coated with ECM molecules alone (Fig. 1
and data not shown). In comparison, the WEHI-3 monocytic cell line transmigrated threefold less efficiently
through the endothelial monolayer. We performed FACS®
analysis on the J774.2 and WEHI-3 cells to quantitate the
expression levels of different adhesion molecules known
to play a role in leukocyte migration. Although both
J774.2 and WEHI-3 cells expressed
L,
M,
4,
6 and
v
integrin chains, and IAP,
3 integrin chain expression was
markedly low on WEHI-3 cells (Fig. 2). There was no differential expression of PECAM-1 on the two murine cell
lines (data not shown).
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3 integrins have not previously been described to play
a role in monocyte TEM. However, previous experiments
have shown that IAP plays a role in the TEM of some leukocyte subsets, whereas others have shown that IAP is
necessary for some
v
3-mediated functions (7). The adhesion molecule PECAM-1, found on circulating leukocytes and endothelial cells, is another molecule involved in
TEM (49, 72). We previously demonstrated that some
forms of PECAM-1 can interact with the
v
3 integrin.
We investigated the effect of antibodies against the v
integrin chain in the in vitro assay. These antibodies were
able to block TEM of J774.2 cells by 50% through TNF-
-activated endothelium as shown in Fig. 3 a. Antibodies
against IAP,
L, and
4 integrins but not against
6 or
MHC class II also blocked TEM under inflammatory conditions. Although these experiments reinstated the importance of IAP,
L, and
4 for monocyte TEM, they also indicated that
v integrins were involved in the process. In a
subsequent TEM assay using primary HUVEC cell cultures as the endothelial monolayer, we tested the ability of
human peripheral blood monocytes to transmigrate under
normal and inflammatory conditions. Antibodies against
2 and
v integrins were able to inhibit TEM across nonactivated endothelium by 50%, whereas anti-
1 had no effect (Fig. 3 b). This was consistent with the fact that nonactivated endothelium does not express the
4
1 ligand
VCAM-1. As a result of TNF-
treatment, HUVECs express VCAM-1, and ICAM-1 levels are increased (data
not shown). This resulted in a twofold enhancement of
TEM which could be inhibited by anti-
1, anti-
2, and
anti-
v antibodies (Fig. 3 b). Control anti-MHC class I antibodies had no effect on TEM under either condition.
Freshly isolated human monocytes express the
v
3 integrin albeit at lower levels than
2 integrins (Fig. 3 c).
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Enhanced TEM of WEHI-3 Cells Transfected with
Full-length 3 cDNA
To clarify the importance of the v
3 integrin in TEM,
3-deficient WEHI-3 cells were transfected with full-length
mouse
3 integrin cDNA. WEHI-3 clone 1D10 expressed
3 integrin chains and also showed increased expression
levels of
v as compared with clone 3E9 which did not express
3 integrin chains (Fig. 4). A further WEHI-3 clone
(1C10) expressing similar levels of the
3 integrin chain to
clone 1D10 (data not shown), was also selected for subsequent TEM assays. The WEHI-3
3+ clones 1D10 and
1C10 exhibited an enhanced ability to transmigrate under
inflammatory conditions as compared with the
3
clone
3E9, and also surpassed J774.2 cells (Fig. 5 a). Furthermore, antibody-blocking studies on clone 1D10 and clone
3E9 cells showed that anti-
L and anti-
v integrin antibodies could inhibit TEM of clone 1D10 cells (Fig. 5 b).
An antibody against
6 integrin or MHC class II, had no
effect on TEM.
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To compare the transmigratory capacity of the WEHI-3
parental cell line with clone 1D10, we compared their ability to transmigrate through an inflammatory endothelium
versus plain laminin-coated filters. WEHI-3 and clone
1D10 cells transmigrated at comparable levels through
laminin-coated filters. However, only clone 1D10 cells
transmigrated significantly through inflammatory endothelium (Fig. 5 c). These experiments demonstrated the
importance of the v
3 integrin in monocyte TEM.
Transmigration Is Increased through L Cells Expressing ICAM-1
Expression of the v
3 integrin is known to be required
for cell migration on ECM substrates (11, 22, 58, 59). To examine how the
v
3 integrin increases cell migration in
our studies, we used a modified transmigration assay. Fibroblast type L cells, which express the
v
3 ligand fibronectin (data not shown) were used in lieu of the endothelial monolayers and grown to confluence on nucleopore
filters. Neither J774.2 nor WEHI-3
3+ clone 1D10 cells
were able to transmigrate efficiently through these L cells
(Fig. 6). We then used L cells expressing PECAM-1. Again, we could not detect significant transmigration of
monocytic cells. In contrast, when L cells expressing
ICAM-1 were used in the assay, we found that both J774.2
and WEHI-3
3+ cells were able to transmigrate very efficiently, though ICAM-1 is not a known ligand for
v
3
(2% of added J774.2 cells and 6% of added WEHI-3
3+
cells transmigrated). L cells transfected with ICAM-1 also
expressed fibronectin but not fibrinogen (Fig. 7), the latter
can act as a bridging molecule between ICAM-1 and the
M
2 integrin (38). Therefore, it was conceivable that the
binding of fibronectin to
v
3 potentiated the transmigration of monocytes across ICAM-1. However, this implied
the existence of a cross talk mechanism between
v
3 integrin and
2 integrins on the monocyte.
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Monocyte Migration on Recombinant Molecules
To investigate a potential cross talk mechanism, we analyzed the migratory behavior of WEHI-3 3+ cells on recombinant ICAM-1. Recombinant ICAM-1 was coated on plastic at a concentration of 2.4 µM, which has been determined to be the saturating protein concentration for these
assays (32). Furthermore, this concentration is consistent
with the expression levels of ICAM-1 on cytokine activated
e.end2 monolayers as determined by ELISA (data not
shown). Monocytes are able to migrate on recombinant ICAM-1 and this migration could be decreased by antibodies against the
L integrin chain but not with antibodies
against MHC class II molecules (Fig. 8 a). In the first 10 min after the addition of anti-
L, the cells begin to lose
their adherent morphology and start to round up. They reduce their velocity of migration and reach a stationary
phase 50 min after the addition of antibody. Cell migration
was recorded during the first 40 min of the experiment.
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When a low concentration of PECAM-1 or vitronectin,
also a ligand for v
3, was coated together with ICAM-1,
the speed of cell locomotion increased (Fig. 8 b). In contrast,
coating the same concentration of laminin together with
ICAM-1 had no effect (Fig. 8 b). Subsequently, cell locomotion on a mixture of ICAM-1 and PECAM-1 could be inhibited by antibodies against IAP, the protein associated with
some functions of the
v
3 integrin. In contrast, anti-IAP did
not decrease the speed of monocyte locomotion on ICAM-1
alone (Fig. 8 c). In these experiments, whereas ICAM-1 coating was at 99% saturation, coating of PECAM-1, vitronectin, or laminin was at 1% saturation. The 1% saturation of different proteins alone does not support cell adhesion or migration on surfaces (data not shown).
In addition, we observed a 1.4-fold increase in cell migration on ICAM-1 alone when v was cross-linked on the
cell surface by antibodies (Fig. 9 a). Cross-linking antibodies against MHC class II had no effect. To determine
whether the effect of
v cross-linking on monocytes was
specific for
2 integrins, or if it could also influence the activity of other integrins important in TEM such as
4
1,
we looked at monocyte migration on recombinant VCAM-1.
As can be seen in Fig. 9 b, cross-linking
v on monocytes migrating on VCAM-1 failed to increase their speed of locomotion and cross-linking the
6 integrin chain as a control also had no effect. Surface molecule cross-linking was
done in the presence of a low concentration of primary
antibody (10 µg/ml) plus a secondary anti-Fc antibody
(10 µg/ml), to ensure capping of the integrin/MHC on the
cells for lateral migration. This is contrary to the effect of antibodies used in the TEM-blocking studies. There, 50 µg/ml of primary antibody alone was used, to ensure blocking
and not capping of cell surface molecules.
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Finally, we compared locomotion of cells of the WEHI-3
3+ clone 1D10 with cells of the WEHI-3
3
clone 3E9,
on recombinant ICAM-1. Clone 1D10 and clone 3E9 cells migrated at comparable levels on ICAM-1 alone. However, after
v cross-linking, locomotion was enhanced only
with cells of clone 1D10 (Fig. 9 c). Clone 3E9 cells did not
respond to cross-linking of the
v integrin chain.
These experiments were repeated with the human
monocytic cell line THP-1, which expresses L
2 and
v
3 integrins (tested by FACS®, data not shown). From
Fig. 10 a, it is clear that locomotion of THP-1 monocytic
cells on recombinant ICAM-1 was increased 2.5-fold upon
cross-linking of the
v integrin chain, but cross-linking the
6 or
2 integrin chains had no effect. (The anti-mouse
6 antibody recognizes
6 integrins on THP-1 cells as detected by FACS®; data not shown). In a further experiment, the effect of blocking
L
2 on monocytic cells after
enhancing their migration on ICAM-1 by cross-linking
v,
was assessed by adding an anti-
L antibody. As can be
seen in Fig. 10 b, cell motility on ICAM-1 returned to control levels after addition of anti-
L, an indication that
modulation of cell migration on ICAM-1 by
v is dependent on the function of the
L
2 integrin. THP-1 cell migration was also enhanced twofold on a mixture of ICAM-1/vitronectin which could be decreased by antibodies
against IAP. Again, anti-IAP had no effect on monocyte migration on ICAM-1 alone (Fig. 10 c). Finally, as a control for integrin cross talk on monocytic cells, we looked at
the effect of cross-linking
v on THP-1 cells migrating on
laminin to determine whether
v integrins could influence
6 integrins. As can be seen from Fig. 10 d, background
migration on laminin was low. However, there was no increase in cell locomotion of monocytes on laminin after
cross-linking the
v integrin chain.
|
Effect of v
3 Integrin Occupancy on ICAM-1 Binding
To determine the effect of v
3 occupancy on the function of
2 integrins, we investigated the ability of THP-1
monocytic cells to bind beads coated with ICAM-1 upon
adherence to immobilized BSA, anti-MHC class I, ICAM-1,
vitronectin, or antibodies against the integrins
v
5 (THP-1
cells express
v
5; data not shown),
v
3,
6, and
2. The
data summarized in Fig. 11 a show that monocytes adherent on anti-MHC class I, anti-
v
5, anti-
6, and anti-
2 antibodies or ICAM-1, bound ICAM-1-coated beads at
comparable levels. However, if the cells were allowed to
interact with an anti-
v
3 antibody or vitronectin, significantly fewer ICAM-1-coated beads were bound. The
epitope for the anti-
v
3 antibody used here (LM609), is
near the Arg-Gly-Asp binding site of the integrin (4).
Therefore, binding of the antibody to the integrin could mimic integrin occupancy. As a control, the ability of
THP-1 cells to bind fibronectin-coated beads under similar
conditions was assessed. Cells immobilized on anti-
v
3
or vitronectin bound similar numbers of fibronectin coated
beads as compared with cells immobilized on other substrates (Fig. 11 b). Hardly any monocytes adhered to BSA
(data not shown) and there was only negligible binding of
BSA-coated beads to monocytes immobilized on the different substrates (Fig. 11 b).
|
Last but not least, we tested whether anti-L integrin
antibodies could block the binding of ICAM-1-coated
beads to THP-1 cells adherent on ICAM-1. As can be seen
from Fig. 12 b, addition of 50 µg/ml of this antibody dramatically reduced binding of ICAM-1-coated beads to the
cells. On the other hand, addition of a control antibody against
6 integrin had no effect (Fig. 12 a). Moreover, an
anti-
1 integrin antibody reduced binding of fibronectin
coated beads to THP-1 cells immobilized on ICAM-1 (Fig.
12 d), whereas the anti-
6 antibody again had no effect
(Fig. 12 c).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although much is known about the rolling and tight adhesion steps before TEM, little is known about the events
that lead to transition from tight adhesion to migration of
a leukocyte on the apical surface of the endothelium and
subsequent diapedesis between the endothelial cells to the
basal side of the blood vessel wall. The 1 and
2 integrins
mediate tight adhesion of the leukocyte to inflammatory
vascular endothelium. However, induction of TEM requires a dynamic regulation of adhesion of these integrins
to their respective ligands. Our results indicate that occupancy of
v
3 integrin on monocytes can modulate
2 integrin-dependent adhesion to and migration on ICAM-1.
This could be a mechanism which enables monocytes to
overcome tight adhesion to endothelial ICAM-1 under inflammatory conditions and engage in subsequent TEM.
J774.2 monocytic cells expressing the v
3 integrin transmigrated through TNF-
-activated endothelium, whereas
WEHI-3 cells deficient in this integrin were hampered in
the process. TEM of J774.2 cells could be partially blocked
under inflammatory conditions by antibodies against IAP,
4
1,
L
2 and
v integrins. TEM assays carried out with
primary human monocytes reinstated that
2 and
v integrins are important in this process. Transfection of
3 integrin chain cDNA into WEHI-3 cells resulted in expression
of the
v
3 integrin on the cell surface. These cells were
then able to engage in enhanced TEM through TNF-
-activated endothelium which could be inhibited by antibodies
against
L or
v. Although these experiments demonstrate the importance of the
v
3 integrin in monocyte
TEM, they do not reveal how the integrin is involved in
the process. The integrin
v
3 can mediate cell spreading
and migration on immobilized vitronectin (41, 42), and is a
molecule involved in tumor metastasis (63). The integrin is
also upregulated on proliferating endothelial cells (24),
and initiates a Ca+2-dependent signaling pathway that
leads to endothelial cell migration and the process of angiogenesis (6, 42). To study how the
v
3 integrin is involved in TEM, we modified the transmigration assay by
using L cells instead of e.end2 cells. Neither e.end2 cells
nor L cells form tight junctions, but grow to confluence on
laminin-coated filters in 48 h. L cells express fibronectin, an
v
3 integrin ligand. However, transmigration of
v
3
integrin-positive monocytic cells was low through untransfected L cells or L cells expressing PECAM-1. This demonstrated that simply the presence of
v
3 ligands could
not ensure efficient transmigration. Surprisingly, however,
3+ monocytic cells were able to transmigrate effectively
through ICAM-1-expressing L cells which also expressed
fibronectin. ICAM-1 is not a known ligand for
v
3 but
can bind fibrinogen which in turn can interact with
M
2
on leukocytes (15, 39). This
M
2-fibrinogen-ICAM-1 association is able to mediate leukocyte TEM (38). However, since our L cells did not express fibrinogen, we ruled
out this mechanism. We speculated instead that perhaps
the binding of
v
3 to fibronectin was enhancing
2 integrin-mediated migration of the monocytes on ICAM-1.
We used time-lapse video microscopy studies to test this
hypothesis. Both murine and human monocytic cells engage
in L
2-dependent migration on recombinant ICAM-1.
Cocoating a ligand for
v
3 or cross-linking the
v integrin to mimic
v
3 integrin occupancy increased the speed
of monocyte locomotion on ICAM-1. The
v chain can associate with other
chains such as
1,
5, and
8 (16, 31,
68). However, cross-linking the
v integrin chain on
3 integrin-deficient WEHI-3 monocytic cells failed to enhance
their locomotion on ICAM-1, indicating that
3 is the essential partner chain for
v in
v-mediated monocyte motility on ICAM-1. The
v
3 integrin is functionally associated
with IAP (7), since IAP has been shown to be necessary for
some
3 integrin-dependent functions (43). The effect of
anti-IAP antibodies on the migration of monocytes on the
mix of ICAM-1 and PECAM-1 or ICAM-1 and vitronectin suggests that IAP is also involved in
v
3 integrin-
mediated locomotion on ICAM-1. However, as anti-IAP
antibodies were able to block the TEM of monocytes to a
greater degree than anti-
v antibodies alone, it is likely
that IAP may also have an
v
3-independent function in
leukocyte TEM.
We previously showed that cross-linking the v integrin
chain on T lymphocytes regulates
4
1 function and cell
migration on VCAM-1 (32). However, in the present study
cross-linking
v on monocytes migrating on VCAM-1 did
not affect their speed of locomotion, indicating that the
activity of
4
1 on monocytes is not influenced by occupancy of the
v
3 integrin. We also examined whether
v
integrins could influence
6 integrins to rule out a nonspecific cross talk mechanism between
v integrins and other integrins on the cell. Cross-linking
v on THP-1 cells
migrating on laminin had no effect on their speed of locomotion. Finally, to prove that the increase in monocyte
locomotion on ICAM-1 is not an artifact of integrin cross-linking, we cross-linked MHC class II on murine monocytic cells and chains from two other integrins on THP-1
cells. Cross-linking of MHC,
6, or
2 failed to have any
effect on the migration of monocytic cells on ICAM-1.
Thus, cross-linking specifically the
v integrin chain on
monocytic cells enhanced their migration on ICAM-1, and
although the
v
3 integrin modulated
2 integrin function, it did not modulate the function of either
4
1 or
6
integrins on these cells.
The 2 integrins
M
2 and
L
2 are both expressed on
the monocytic cells used in our assays. Does
v
3 modulate one or both of these integrins? The focus of our
present study was the
L
2 integrin. ICAM-1 has five tandemly repeated Ig-like domains (21, 27), and whereas the
binding site for
L
2 is on the first two Ig-like domains
(67), the binding site for
M
2 is on the third Ig-like domain (18). The recombinant murine ICAM-1 used in our experiments consists of just the first two Ig-like domains
which lack the
M
2 binding site but supports murine
monocyte migration, which could be decreased by anti-
L
antibodies. Furthermore, anti-
L antibodies also decreased
the enhanced migration of human THP-1 cells on ICAM-1
brought about by cross-linking the
v integrin chain.
Therefore, we concluded that
L
2 is a candidate
2 integrin that responds to occupancy of
v
3. This does not
rule out that
v
3 integrin occupancy may also affect the
activity of
M
2 in vivo.
The integrin L
2 forms tight interactions with endothelial ICAM-1. Cell adhesion is regulated both by the affinity of the extracellular regions of integrins for their
ligands and by intracellular integrin-cytoskeletal associations (29). The strength of adhesion between cell surface
receptors and the substrate is therefore a key factor in the
migration process (30). Previous studies have indicated an
inverse correlation between adhesion and cell migration
(19). Studies on the
IIb
3 integrin revealed that high-
affinity states of the receptor results in a decrease in the
migration rate of the cell (29), or locking
1 integrins in a
state of high avidity by using activating
1 mAb inhibits leukocyte extravasation (35). Thus, tight adhesion of receptors to their substrates is detrimental for cell locomotion. Therefore, it seemed likely that if
v
3 occupancy
could modulate monocyte locomotion on ICAM-1, this occupancy must lead to a deadhesion between
L
2 and
ICAM-1. Monocytes adherent on anti-
v
3 or vitronectin were less efficient in binding ICAM-1-coated beads than
monocytes adherent on ICAM-1, anti-
v
5 or other control substrates. Furthermore, monocytes adherent to anti-
v
3 or vitronectin do not display differential ability to
bind fibronectin-coated beads. This demonstrated that occupancy of
v
3 integrin on the monocyte can decrease
the cell's binding capacity to ICAM-1. ICAM-1-coated
bead binding to THP-1 cells could be blocked with antibodies against
L. But occupancy of
v
3 did not reduce
ICAM-1-coated bead binding to the same extent as the
anti-
L antibodies. However, if
v
3 occupancy reduced
the interactions between
L
2 and ICAM-1 totally, the cell would not be able to migrate on the surface of the endothelium, but would detach instead from the vessel wall.
Modulation of integrin function is therefore a key concept
for cell locomotion. A cell can continuously move forward
only if there is a dynamic regulation of integrin mediated
adhesion and deadhesion. Chemokines can differentially regulate the avidity of
4
1 integrins by rapidly activating
and deactivating them on monocytes and eosinophils (73,
74). No doubt this mechanism contributes to monocyte
motility on VCAM-1. We previously showed that the
v
3
integrin can modulate the activity of
4
1 on T lymphocytes and enhance their migration on VCAM-1 (32). Now
we demonstrate that
v
3 can modulate the function of
L
2 integrins on monocytes and favor their migration on
ICAM-1.
How do integrins communicate with each other? Integrins lack intrinsic enzymatic activity to trigger signaling,
but several groups have shown that integrin cytoplasmic
tails can bind to structural cytoskeletal proteins which in
turn interact with components of the intracellular signaling machinery en route to other cell surface receptors (36,
62). Integrins can also interact directly to form cis-acting
complexes on the cell surface. The 2 integrins serve as
signaling partners for leukocyte receptors in this way. The
urokinase plasminogen activator receptor (CD87) and
M
2 form a functional unit on monocytic cells (64). Interestingly, it has recently been shown that the urokinase
plasminogen activator receptor (uPAR) is necessary for
L
2-mediated leukocyte migration under inflammatory
conditions, and monocyte recruitment to sites of inflammation is impaired in the absence of uPAR (46). The urokinase receptor can also associate with
1 and
3 integrins on tumor cells adherent on vitronectin which may
regulate tumor cell migration (75). Further work will address the mechanism by which the
v
3 integrin regulates
L
2 function on the same cell in monocyte transmigration.
Peripheral blood monocytes express v
3 albeit at
lower levels than
2 integrins. This level is probably sufficient to mediate the signal required to initiate cell motility
on ICAM-1. However, cell motility on the ECM requires
high expression levels of the integrin (5, 76). The cytokine
granulocyte macrophage colony-stimulating factor (GM-CSF) can upregulate the expression levels of
v
3 on monocytes (13), and is produced by inflammatory endothelium (50). Interestingly, it has been shown that mice
transgenic for the GM-CSF gene develop accumulations
of macrophages in tissues (37). Therefore, it is likely that
levels of
v
3 on monocytes immobilized to inflammatory
endothelium in vivo is upregulated by the influence of
GM-CSF released by the endothelium, which in turn
would promote monocyte locomotion on the endothelium
and the underlying ECM.
Previous studies have emphasized the requirement for
an integrin hierarchy to facilitate the coordinated migration of leukocytes across the endothelium into tissues. The
4
1 integrin is involved in the arrest and initial adhesion
of rolling leukocytes to inflammatory endothelium via
VCAM-1. Subsequently,
L
2 mediates tight adhesion of
the leukocyte to vascular ICAM-1 after cellular activation (10). The
L
2 integrin is then able to downregulate
4
1
and cell adhesion to VCAM-1 (52). In the next step of the
integrin hierarchy, the
v
3 integrin downregulates
L
2
activity, modulating leukocyte adhesion to ICAM-1, and
enabling the cell to migrate effectively across the endothelium.
In summary, we show that the v
3 integrin is involved
in the transition between tight adhesion of monocytes to
the vascular endothelium and subsequent diapedesis. This
may be an important mechanism not only for the TEM of
monocytes but also for other leukocyte subsets that use
2
integrins during transendothelial diapedesis.
![]() |
Footnotes |
---|
Received for publication 11 September 1997 and in revised form 18 June 1998.
Address all correspondence to Beat A. Imhof, Professor of Pathology, Geneva University, Department of Pathology, 1, rue Michel-Servet, CH-1211 Geneva, Switzerland. Tel.: (41) 22-702-57-47. Fax: (41) 22-702-57-46. E-mail: beat.imhof{at}medecine.unige.chWe would like to thank P. Hammel, G. Wiedle, J.-P. Dangy, B. Ecabert, M. Dessing, S. Meyer, and S. Cooper (all from Basel Institute for Immunology, Basel, Switzerland, except Wiedle from Céntre Medicale Universitaire, Geneva, Switzerland) for their technical assistance. We would also like to thank K. Campbell and R. Torres (both from Basel Institute for Immunology) for reviewing and improving the manuscript, and H. Stahlberger, H. Spalinger, and B. Pfeiffer (all three from Basel Institute for Immunology) for artwork and photography. Finally, we thank U. Vischer for the HUVEC cultures, B. Sinha (both from Basel Institute for Immunology) for his help with experimental protocols, K. Willimann (Theador Kocher Institute, Bern, Switzerland) for reagents, and B. Englehardt (Max Planck Institute, Bad Nauheim, Germany) for advice with the transmigration assays.
This work was supported in part by grants from the Swiss National Science Foundation (3100-049241.96) and the Recherche Suisse contre le Cancer (KFS 412-1-97). The Basel Institute for Immunology was founded and is supported by F. Hoffmann-La Roche AG, CH-4005 Basel, Switzerland.
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Abbreviations used in this paper |
---|
ECM, extracellular matrix; GM-CSF, granulocyte macrophage colony-stimulating factor; HUVEC, human umbilical vein endothelial cells; IAP, integrin-associated protein; ICAM-1, intercellular adhesion molecule-1; IMDM, Iscove's modified Dulbecco's medium; MCP-1, monocyte chemoattractant protein-1; MHC, major histocompatibility complex; PECAM-1, platelet endothelial cell adhesion molecule-1; TEM, transendothelial migration; VCAM-1, vascular cell adhesion molecule-1.
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