This information is current as
of June 17, 2017.
Lymphocyte Migration Through Brain
Endothelial Cell Monolayers Involves
Signaling Through Endothelial ICAM-1 Via a
Rho-Dependent Pathway
Peter Adamson, Sandrine Etienne, Pierre-Olivier Couraud,
Virginia Calder and John Greenwood
J Immunol 1999; 162:2964-2973; ;
http://www.jimmunol.org/content/162/5/2964
Subscription
Permissions
Email Alerts
This article cites 40 articles, 16 of which you can access for free at:
http://www.jimmunol.org/content/162/5/2964.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 1999 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
References
Lymphocyte Migration Through Brain Endothelial Cell
Monolayers Involves Signaling Through Endothelial ICAM-1
Via a Rho-Dependent Pathway1
Peter Adamson,2* Sandrine Etienne,† Pierre-Olivier Couraud,† Virginia Calder,* and
John Greenwood*
T
o fulfill their role in tissue surveillance, lymphocytes must
be able to leave the circulation and traffic through the
solid tissues of the body. Thus, the migration of lymphocytes across the vascular endothelial cell (EC)3 wall is a prerequisite in the implementation of lymphocyte function. Lymphocyte
transendothelial migration is dependent on lymphocyte activation
via signaling through the T cell and IL-2 receptor (1). The in vivo
correlate of these observations is that only Ag-specfic, IL-2-dependent T cells are capable of trafficking to the central nervous
system (CNS) (2). The vascular recruitment of circulating lymphocytes and their transvascular migration has been the subject of
substantial investigation and has led to a greater understanding of
the role of lymphocytes under normal and inflammatory condi-
*Department of Clinical Ophthalmology, Institute of Ophthalmology, University College London, London, United Kingdom; and †Laboratoire d’Immuno-Pharmacologie
Moléculaire, Institut Cochin de Génétique Moléculaire, Paris, France
Received for publication September 8, 1998. Accepted for publication November
23, 1998.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by the Wellcome Trust (U.K.), Centre National de la
Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale,
Association pour le Developpment de la Recherche sur le Cancer, Ligue Nationale
Francaise contre le Cancer, Université de Paris, and the Ministere de la Recherche et
de l’Enseignement Superieur.
2
Address correspondence and reprint requests to Dr. Peter Adamson, Department of
Clinical Ophthalmology, Institute of Ophthalmology, University College London,
Bath Street, London, EC1V 9EL, U.K. E-mail address: [email protected]
3
Abbreviations used in this paper: EC, endothelial cell(s); CNS, central nervous
system; FAK, focal adhesion kinase; PLNC, peripheral lymph node lymphocytes;
RAM, rabbit anti-mouse; LPA, lysophosphatidic acid; GTP, guanosine triphosphate;
GDP, guanosine diphosphate; ECL, enhanced chemiluminescence; BCA, bicinchoninic acid; AEBSF, 4-(2-aminoethyl) benzenesulfonyl fluoride; ADP, [32P]adenosine
59-diphosphate; HRP, horseradish peroxidase; NAD, nicotinamide adenine
dinucleotide.
Copyright © 1999 by The American Association of Immunologists
tions. Unlike most vascular endothelia, which are connected together by incomplete, permeable junctions, those of the brain and
retina are joined together by impermeable tight junctions forming
the blood-brain and inner blood-retinal barriers, respectively. Despite these barriers, a low level of leukocyte traffic into the CNS
occurs (2), and this can be dramatically up-regulated during the
development of immune-mediated diseases. To achieve entry,
lymphocytes must either penetrate the tight junctions or migrate
through the body of the CNS EC through the formation of pores.
Whichever path is utilized during leukocyte diapedesis within the
CNS, it is highly likely that this process can only operate with the
active participation of the EC. Although it is clear that the activation state of lymphocytes is central in controlling transvascular
migration (1, 3, 4), additional control may be provided by the CNS
endothelia to further facilitate the passage of migratory cells across
the impermeable vessel wall.
The mechanisms by which vascular EC capture circulating lymphocytes are now well characterized and many of the ligands that
contribute to their adhesion and subsequent transvascular migration identified (5, 6). Previous studies have shown that initial capture of immune cells from the circulation occurs via endothelial
selectin molecules that result in weak attachment and, under conditions of flow, result in leukocyte rolling along the vessel wall (6).
Tighter binding and subsequent migration through the EC wall is
mediated predominantly by the LFA-1/ICAM-1 (and possibly
ICAM-2) pairing. However, on cytokine-activated endothelia, both
the LFA-1/ICAM-1 and the very late Ag-4/VCAM-1 interaction
appear to play a part in T lymphocyte migration (3, 7–9). Recent
studies indicate that the molecules employed during lymphocyte
adhesion to and migration through CNS endothelium are essentially the same as those governing recruitment at other vascular
beds, with ICAM-1 being the predominant endothelial molecule
involved in migration (1, 10, 11). However, the active participation
0022-1767/99/$02.00
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Lymphocyte extravasation into the brain is mediated largely by the Ig superfamily molecule ICAM-1. Several lines of evidence
indicate that at the tight vascular barriers of the central nervous system (CNS), endothelial cell (EC) ICAM-1 not only acts as a
docking molecule for circulating lymphocytes, but is also involved in transducing signals to the EC. In this paper, we examine the
signaling pathways in brain EC following Ab ligation of endothelial ICAM-1, which mimics adhesion of lymphocytes to CNS
endothelia. ICAM-1 cross-linking results in a reorganization of the endothelial actin cytoskeleton to form stress fibers and activation of the small guanosine triphosphate (GTP)-binding protein Rho. ICAM-1-stimulated tyrosine phosphorylation of the
actin-associated molecule cortactin and ICAM-1-mediated, Ag/IL-2-stimulated T lymphocyte migration through EC monolayers
were inhibited following pretreatment of EC with cytochalasin D. Pretreatment of EC with C3 transferase, a specific inhibitor of
Rho proteins, significantly inhibited the transmonolayer migration of T lymphocytes, endothelial Rho-GTP loading, and endothelial actin reorganization, without affecting either lymphocyte adhesion to EC or cortactin phosphorylation. These data show
that brain vascular EC are actively involved in facilitating T lymphocyte migration through the tight blood-brain barrier of the
CNS and that this process involves ICAM-1-stimulated rearrangement of the endothelial actin cytoskeleton and functional EC Rho
proteins. The Journal of Immunology, 1999, 162: 2964 –2973.
The Journal of Immunology
Materials and Methods
Materials
[a- P]Nicotinamide adenine dinucleotide (NAD) was obtained from New
England Nuclear (Herts, U.K.). Na[51Cr207], [32P]orthophosphate, peroxidase coupled rabbit anti-mouse (RAM) IgG, and enhanced chemiluminescence (ECL) reagents were obtained from Amersham International (Bucks,
U.K.). Recombinant epidermal growth factor was obtained from R&D Systems (Oxon, U.K.). Polyclonal anti-Rho Ab was obtained from Autogenbioclear (Wilts, U.K.). Anti-transferin receptor mAb (MAB451) was obtained from Chemicon (London, U.K.). Mouse anti-rat ICAM-1 (1A29)
was obtained azide-free from Serotec (Oxon, U.K.). The mouse mAb to
p80/85 cortactin was a generous gift from J. T. Parsons (University of
Virginia, Charlottesville, VA). The pGEX-2T-C3 construct was a generous
gift from L. A. Fieg (Tufts University, Boston, MA). Unless otherwise
stated, all chemicals used were obtained from Sigma (Dorset, U.K.).
32
Endothelial cells
Primary rat brain endothelium. Cultures were isolated as previously described (24, 25), and these methods routinely produced primary cultures of
.95% purity. Briefly, rat cerebral cortex was dispersed by enzymatic di-
gestion, microvessel fragments separated from other material and single
cells by density-dependent centrifugation and plated onto collagen-coated
plastic. Cultures were maintained in Ham’s F-10 medium supplemented
with 17.5% plasma-derived serum (First Link, West Midlands, U.K.), 7.5
mg/ml of EC growth supplement (First Link), 80 mg/ml of heparin, 2 mM
glutamine, 0.5 mg/ml of vitamin C, 100 U/ml of penicillin, and 100 mg/ml
of streptomycin at 37°C in 5% CO2. Medium was replaced every 3 days.
Immortalized EC. The immortalized Lewis rat brain EC line GP8.3 (22)
was maintained in Ham’s F-10 medium supplemented with 17.5% FCS, 7.5
mg/ml of EC growth supplement, 80 mg/ml of heparin, 2 mM glutamine,
0.5 mg/ml of vitamin C, 100 U/ml of penicillin, and 100 mg/ml of streptomycin. The immortalized Lewis rat brain EC line RBE4 was maintained
in 1:1 Hams F-10/aMEM (Life Technologies, Paisley, U.K.) supplemented
with 10% FCS (Life Technologies), as previously described (21). Cultures
were maintained at 37°C in 5% CO2, and medium was replaced every 3
days until the formation of monolayers.
Fluorescence microscopy
Actin localization. Cells were fixed in 3.7% paraformaldehyde in PBS for
10 min, followed by 50 mM Tris-HCl (pH 7.5)/PBS for 10 min. Cells were
subsequently permeabilized in 0.5% Triton X-100/PBS and incubated with
0.1 mg/ml of FITC-phalloidin for 1 h. Cells were exhaustively washed in
PBS and viewed on a Leica (Milton Keynes, U.K.) confocal fluorescence
microscope.
ICAM-1 localization. For ICAM-1 localization, cells were fixed as described above and incubated with 10% normal goat serum/PBS. Cells were
subsequently exposed to 1 mg/ml of anti-rat ICAM-1 (1A29) for 1 h at
room temperature, exhaustively washed in PBS, and incubated with antimouse-FITC or anti-mouse-Cy3 (1:50; Jackson ImmunoResearch, West
Grove, PA) for 1 h. Cells were exhaustively washed in PBS and viewed on
a Leica confocal fluorescence microscope.
Tyrosine phosphorylation of cortactin
Cell cultures (equivalent to 1 3 105 cells) were lysed with 250 ml of
SDS-PAGE buffer (10 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol,
0.4% dithithreitol, 1 mM sodium orthovanadate, and 0.1% bromophenol
blue), boiled for 10 min, and subject to 7.5% SDS-PAGE. Proteins were
electrophoretically transferred to nitrocellulose membranes (Amersham)
and immunoblotted using either 2.5 mg/ml of mouse anti-phosphotyrosine
(Upstate Biotechnology, Lake Placid, NY) or 1 mg/ml of anti-cortactin
followed by peroxidase-conjugated RAM IgG. Blots were subsequently
developed using the ECL system (Amersham) and exposed to x-ray film.
Immunoprecipitation of p80/85 cortactin was achieved following lysis of
cells for 30 min in buffer (10 mM Tris-HCl (pH 7.5), 140 mM NaCl, 1%
digitonin, 1 mM phenylmethylsulfonyl fluoride, 1 mM orthovanadate, 50
U/ml of aprotinin, 1 mM EDTA, 2 mg/ml of pepstatin, and 2 mg/ml of
leupeptin), followed by removal of nuclei (13,000 3 g for 30 min) and
incubation with 1 mg/ml of anti-cortactin mAb for 1 h at 4°C. Cell lysates
were preincubated with 50 ml of RAM-coated pansorbin (Calbiochem,
Notts, U.K.) for an additional 30 min at 4°C, and immune complexes were
collected by centrifugation. Immune complexes were extensively washed
in lysis buffer and further analyzed by Western blot analysis. p80/85 cortactin phosphorylation was quantitated by densitometry and normalized to
levels of immunoprecipitated cortactin.
Soluble (S)-Ag specific CD41 T cell lines
Lewis rat T lymphocyte cell lines specific for purified bovine retinal S-Ag
were prepared as previously described (26). Briefly, lymph nodes were
collected from bovine S-Ag immunized rats, and the T lymphocytes were
propagated by periodically alternating Ag activation with IL-2 stimulation.
The cell lines express the marker of the CD41 T cell subset, are
CD45Rclow, and recognize S-Ag in the molecular context of MHC class II
determinants (26). These cells have previously been shown to be highly
migratory across monolayers of primary cultured brain and retinal endothelia (1, 4, 11) and represent Ag-stimulated lymphocytes.
Adhesion of peripheral lymph node cells (PLNC) to endothelia
Adhesion assays were conducted as previously described (27, 28) using
cells harvested from Lewis rat peripheral lymph nodes. Briefly, PLNC
were isolated, and T lymphocytes were obtained after purification on nylon
wool columns. These cells that represent non-Ag-activated T lymphocytes
are therefore nonmigratory but highly adhesive when activated with the
mitogen Con A (1, 4, 11). PLNC were activated for 24 h with type V Con
A, washed twice in HBSS, and cells labeled with 3 mCi [51Cr] per 106 cells
in HBSS for 90 min at 37°C. After washing the cells three times with
HBSS, they were resuspended in RPMI 1640 medium containing 10%
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
of CNS EC in leukocyte extravasation, above that of the provision
of adhesion molecules, has remained largely speculative.
If CNS endothelia play an active part in facilitating lymphocyte
diapedesis, it is likely that they receive external signals from adherent lymphocytes. This has led to the possibility that molecules
intimately involved in lymphocyte diapedesis, such as EC
ICAM-1, may also be involved in the transduction of extracellular
signals. Proteins of the Ig superfamily, including CD2, CD4, MHC
molecules and Fcg are well-documented as signal transducers in
both lymphocytes (12) and U937 cells (13). In addition, neural cell
adhesion molecule has also been shown to be capable of signal
transduction in PC12 cells (14). Although it has been demonstrated
in leukocytes that ICAM-1 is capable of generating intracellular
signals (15–17), and more recently, signaling via VCAM-1 and
platelet endothelial call adhesion molecule-1 has been demonstrated in platelets (18) and EC (19), the functional effects of signaling via Ig superfamily molecules in EC remain unresolved.
Studies aimed at exploring the signal transduction pathways in
CNS EC that may lead to lymphocyte extravasation have been
greatly expedited by the recent development of two immortalized
Lewis rat brain microvessel EC lines that have been immortalized
with the E1A adenovirus protein (RBE4 cell line; 20, 21) and
SV40 large T Ag (GP8.3 cell line; 22) and that retain in culture the
differentiated phenotype of brain endothelia. Previous studies with
RBE4 cells have shown that Ab cross-linking of endothelial
ICAM-1 molecules (used to mimic lymphocyte binding to EC via
LFA-1) or coculture of EC with lymphocytes triggers p60src activity, which appears to be responsible for the enhanced tyrosine
phosphorylation of the actin-binding protein cortactin (20). The
enhanced tyrosine phosphorylation of cortactin following crosslinking of EC with ICAM-1 or coculture with encephalitogenic T
lymphocytes was the first indication of an active endothelial involvement in controlling transvascular lymphocyte migration following T cell binding. Furthermore, it also served to demonstrate
that T lymphocyte binding can be mimicked by ligation of EC
ICAM-1. However, more recently, additional actin cytoskeletalassociated proteins, such as focal adhesion kinase (FAK), paxillin,
and p130cas, have also been shown to be tyrosine phosphorylated
in response to ICAM-1 cross-linking (23). In this paper, we have
further extended this work and present evidence for the first time
that CNS vascular EC are intimately involved in the processes
controlling the transendothelial migration of T lymphocytes across
cultures of rat brain endothelial monolayers. These processes require an ICAM-1-stimulated rearrangement of the endothelial actin cytoskeleton and functional endothelial Rho proteins.
2965
2966
LYMPHOCYTE MIGRATION THROUGH BRAIN EC REQUIRES Rho
FCS. Endothelial monolayers grown on 96-well plates were prepared by
removing the culture medium and washing the cells four times with HBSS.
[51Cr]-labeled PLNC (200 ml) at a concentration of 1 3 106/ml was then
added to each well and incubated at 37°C for 1.5 h. In each assay, g
emissions from each of six replicate blank wells were determined to provide a value for the total amount of radioactivity added per well and to
allow calculation of the specific activity of the cells. After incubation,
nonadherent cells were removed with four separate washes from the four
poles of the well with 37°C HBSS as previously described (27, 29). Adherent PLNC were lysed with 2% SDS, the lysate removed, and g emissions quantitated by spectrometry. Results are expressed as the fractional
adhesion of PLNC to untreated EC. Results were obtained from at least
three independent experiments using a minimum of four separate wells per
treatment. The results are expressed as the means 6 SEM, and significant
differences between groups were determined by Student’s t test.
T lymphocyte transendothelial migration
Guanine-nucleotide, immunoprecipitation, and immunoblot
analysis of endothelial Rho proteins
Serum-starved GP8.3 (5 3 106) cells were incubated in phosphate-free
DMEM overnight in the presence of 0.2 mCi/ml of [32P]orthophosphate.
Cells were washed in HBSS and recultured in serum-free DMEM. After
addition of stimuli, cells were lysed in 100 mM HEPES (pH 7.4), 2%
Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 300 mM NaCl, 10 mM
MgCl2, 2 mM EGTA, 2 mg/ml of BSA, and protease inhibitors (20 mM
benzamidine, 20 mg/ml of leupeptin, 20 mg/ml of pepstatin, 20 mg/ml of
aprotinin, 20 mg/ml of soybean trypsin inhibitor, and 2 mM AEBSF). Lysates were centrifuged at 14,000 3 g for 5 min to sediment nuclei, and
supernatants were adjusted to 500 mM NaCl. Lysates were immunoprecipitated by incubation with 4 mg rabbit polyclonal anti-Rho (Autogenbioclear) for 2 h at 4°C followed by incubation with 50 ml of 50% protein
G-Sepharose (Pharmacia, Oxon, U.K.) for 2 h at 4°C. Immunoprecipitates
were washed eight times with 1 ml of 50 mM HEPES (pH 7.4), 0.005%
SDS, 500 mM NaCl, and 0.1% Triton X-100. Immunoprecipitates were
subsequently heated to 68°C for 20 min in the presence of 5 mM EDTA,
2 mM DTT, 0.2% SDS, 0.5 mM GTP, and 0.5 mM guanosine diphosphate
(GDP) to elute [32P]phosphate-labeled nucleotides. Nucleotides were separated by thin-layer chromatography on 0.1-mm polyethyleneimine-cellulose plates (Machery-Nagel Beds, U.K.) in 1.2 M ammonium formate/0.8
M HCl (30) and autoradiographed at 270°C using Fuji-RX film. Autoradiographs were subsequently used as templates and radioactive areas that
comigrated with GTP and GDP standards, scraped, and radioactivity determined by b-scintillation spectrometry. EC lysates were also resolved on
15% SDS-PAGE gels and electrotransferred to nitrocellulose. Membranes
were blocked with 10% dried milk protein for 1 h at room temperature and
incubated in 0.1 mg/ml of polyclonal anti-Rho Ab (Autogenbioclear) for
1 h at room temperature. Endothelial Rho proteins were visualized following incubation with horseradish peroxidase (HRP)-conjugated goat antirabbit (1:15,000; Promega, Hants, U.K.) and subsequent development by
ECL (Amersham). [32P]-nucleotides were normalized to levels of immunoprecipitated Rho proteins.
Protein determination
Protein concentration in cell lysates was determined using BCA reagent
(Pierce) with BSA as standard.
Expression and purification of C3 transferase
Glutathione S-transferase-C3 was expressed in Escherichia Coli for 5 h
using 1 mM isopropyl b-D-thiogalactoside (Life Technologies/BRL). Cells
were harvested by centrifugation at 4000 3 g for 15 min and sonicated
three times for 5 min in lysis buffer (50 mM Tris-HCl (pH 8), 50 mM NaCl,
5 mM MgCl2, 1 mM DTT, and 1 mM 4-(2-aminoethyl) benzenesulfonyl
fluoride (AEBSF)). Bacterial lysates were then centrifuged at 10,000 3 g
for 30 min, and the supernatant was chromatographed over glutathioneagarose. The column was washed with 5 vol of AEBSF-free lysis buffer
and 3 vol of thrombin cleavage buffer (50 mM Tris-HCl (pH 8), 150 mM
NaCl, 5 mM MgCl2, 2.5 mM CaCl2, and 1 mM DTT). Bovine plasma
thrombin (10 U/ml gel) was then added to the column for 16 h at 4°C. The
eluate from the column was collected and subsequently washed with 3 vol
of PBS. Thrombin was removed from C3 transferase protein released from
the column by chromatography over p-aminobezamidine-agarose and dialysed into PBS. C3 transferase was then concentrated in ultrafiltration units
(Amicon, Beverley, MA). This procedure produced pure C3 transferase
protein as assessed by SDS-PAGE. Protein concentration was assessed
using bicinchoninic acid (BCA) reagent (Pierce, Chester, U.K.).
[32P]adenosine 59-diphosphate (ADP)-ribosylation of endothelial
and lymphocyte lysates with C3 transferase
Cells were lysed in 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 2 mM MgCl2,
and 1 mM AEBSF, and protein concentrations were determined using BCA
reagent (Pierce). Lysate protein (25 mg) was then added to reactions containing 50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 2 mM MgCl2, 0.5 mM
ATP, 0.3 mM guanosine triphosphate (GTP), 1 mM AEBSF, 5 mCi/ml of
[a-32P]NAD, and 250 ng/ml of recombinant C3 transferase and incubated
at 37°C for 1 h. Reactions were stopped by the addition of 2 vol of 30% w/v
trichloroacetic acid on ice for 30 min. Precipitated proteins were separated
by centrifugation at 14,000 3 g for 10 min, and pellets were washed three
times with ethanol at 220°C. Pellets were dried, solubilized in 10 mM
Results
Cross-linking of endothelial ICAM-1 results in clustering of
ICAM-1 and actin stress-fiber formation
Ligation of the external domain of endothelial ICAM-1 molecules
with mouse anti-rat ICAM-1 (1A29; 20 mg/ml for 30 min) followed by cross-linking with RAM IgG (60 mg/ml for 15 min) led
to clustering of endothelial ICAM-1 molecules and a redistribution
of the endothelial actin cytoskeleton in both serum-starved RBE4
(Fig. 1, c and d) and GP8.3 cells (data not shown), which had
previously been incubated with 50 U/ml of IFN-g for 48 h to
increase ICAM-1 expression. In EC, under identical conditions but
in the absence of ICAM-1 cross-linking, FITC-phalloidin staining
showed the actin cytoskeleton to be diffuse with few stress fibers
and a cortical concentration of actin in both RBE4 (Fig. 1b), GP8.3
(data not shown), and primary culture cells (data not shown).
ICAM-1 staining was uniformly distributed within the plasma
membrane (Fig. 1a). However, following ICAM-1 cross-linking
for 15 min, there was a dramatic increase in the number of actin
stress fibers that was not observed after treatment of cells with
either RAM (Fig. 1b) or anti-ICAM-1 alone (data not shown).
Identical results were observed in both RBE4 and GP8.3 cells (data
not shown). Exposure of RBE4 (Fig. 1c and j) or GP8.3 (data not
shown) cells to 10 mM lysophosphatidic acid (LPA) also resulted
in a similar induction of actin stress fibers (Fig. 1j). Cross-linking
of the transferin receptor did not induce stress fibers in either
GP8.3 or RBE4 cells and were identical to control or RAM-treated
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
The ability of the immortalized cells to support the transendothelial migration of Ag-specific T lymphocytes was determined using a well-characterized assay as previously described (1, 4, 11). Briefly, T lymphocytes
were added (2 3 105 cells/well) to 24-well plates containing EC monolayers. Lymphocytes were allowed to settle and migrate over a 4-h period.
To evaluate the level of migration, cocultures were placed on the stage of
a phase-contrast inverted microscope housed in a temperature controlled
(37°C), 5% CO2 gassed chamber (Zeiss, Herts, U.K.). A 200 3 200-mm
field was randomly chosen and recorded for 10 min spanning the 4-h time
point using a camera linked to a time-lapse video recorder. Recordings
were replayed at 1603 normal speed, and lymphocytes were identified and
counted that had either adhered to the surface of the monolayer or that had
migrated through the monolayer. Lymphocytes on the surface of the monolayer were identified by their highly refractive morphology (phase-bright)
and rounded or partially spread appearance. In contrast, cells that had migrated through the monolayer were phase-dark, highly attenuated, and were
seen to probe under the EC in a distinctive manner (1, 4, 11). Treatment of
EC with cytochalasin D or C3 transferase was conducted before addition of
lymphocytes, following extensive washing and replacement into new medium. Control data were expressed as the percentage of total lymphocytes
within a field that had migrated through the monolayer. All other data were
expressed as a percentage of the control migrations. A minimum of three
independent experiments using a minimum of wells per assay was performed. The results are expressed as the means 6 SEM, and significant
differences between groups are determined by Student’s t test.
Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 0.4% dithithreitol, and 0.1%
bromophenol blue, and proteins resolved on 15% SDS-PAGE.
The Journal of Immunology
2967
cells (data not shown). Similar effects on actin stress-fiber formation were also observed in both RBE4 and GP8.3 cells following
ICAM-1 cross-linking to basal levels of ICAM-1 (data not shown).
Cross-linking of endothelial ICAM-1 or coculture with T
lymphocytes results in GTP-loaded endothelial Rho
Serum-starved GP8.3 cells induced with 50 U/ml of IFN-g for 48 h
were labeled with 0.2 mCi/ml of [32P]-orthophosphate in phosphate-free growth medium for 20 h. Cells were washed in phos-
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 1. Effect of ICAM-1 cross-linking Abs on
ICAM-1 localization and actin distribution: effect of cytochalasin D and C3 transferase. RBE4 cells were preincubated for 48 h with 50 U/ml of rat IFN-g and exposed to 20
mg/ml of mouse anti-rat ICAM-1 (1A29) for 30 min, followed by RAM (60 mg/ml) for 15 min. Cells were then subsequently fixed in 3.7% paraformaldehyde for 10 min, followed by 50 mM Tris-HCl (pH 7.5) for 10 min and stained
for actin and ICAM-1 localization. Cells were either incubated with 1 mg/ml of anti-rat ICAM-1 (1A29) or permeabilized in 0.5% Triton X-100 and incubated in 0.1 mg/ml of
FITC-phalloidin for 1 h. For ICAM-1 staining, cells were
washed six times in phosphate buffered saline A and incubated with anti-mouse-Cy3 (1:50) for 1 h. Cells were then
exhaustively washed in phosphate buffered saline A and visualized by confocal fluorescence microscopy. Cells were
serum- and growth factor-deprived 48 h before addition of
cross-linking Abs. Identical fields were stained for ICAM-1
and actin. a and b, RAM (60 mg/ml) only. c and d, ICAM-1
(20 mg/ml) followed by RAM (60 mg/ml). e and f, ICAM-1
(20 mg/ml) followed by RAM (60 mg/ml) in presense of 2
mM cytochalasin D. g and h, ICAM-1 (20 mg/ml) followed
by RAM (60 mg/ml) following 8 h pretreatment with 50
mg/ml of C3 transferase. i and j, 10 mM LPA. a, c, e, g, and
i, stained for ICAM-1. b, d, f, h, and j, stained for actin. Scale
bar 5 25 mm. ICAM-1 stains are shown as projections of all
optical sections. A single identical optical section is shown
for phalloidin staining.
phate containing serum-free growth media and stimulated with
RAM (60 mg/ml) alone, anti-ICAM-1 (20 mg/ml) followed by
RAM (60 mg/ml), anti-transferin receptor (20 mg/ml) followed by
RAM (60 mg/ml), or a 10:1 ratio of syngeneic peripheral node
lymphocytes. Rho proteins were subsequently immunoprecipitated
from cell lysates, bound nucleotides eluted, and analyzed by TLC.
Analysis of GTP:GDP ratios of Rho proteins immunoprecipitated
from GP8.3 EC showed that following cross-linking of EC
ICAM-1 for 10 min, or coculture with T lymphocytes for 10 min
2968
LYMPHOCYTE MIGRATION THROUGH BRAIN EC REQUIRES Rho
(following initial settling and adhesion of T lymphocytes), there
was an 8.0-fold and 9.5-fold increase in the GTP:GDP ratio, respectively, as compared with either unstimulated cells or cells
treated with RAM only (Fig. 2A). The increase in GTP:GDPbound Rho was transitory following ICAM-1 cross-linking, returning to control levels at 30 min. C3 transferase pretreatment of EC
showed an 85% and 77% inhibition of ICAM-stimulated and lymphocyte-stimulated Rho-GTP loading in EC, respectively. Immunoprecipitation of transferin receptor from ICAM-1/RAM-treated
cells followed by GTP/GDP analysis revealed no labeled guanine
nucleotides demonstrating that 32P-labeled nucleotides were derived from immunoprecipitated Rho proteins. In addition, immunoblot analysis of cell lysates demonstrated that similar amounts of
EC Rho proteins were immunoprecipitated following each treatment (Fig. 2B).
Cytochalasin D inhibits ICAM-1-mediated stress-fiber formation
and cortactin phosphorylation in EC
Since ICAM-1 cross-linking induced the formation of actin stress
fibers in EC, the potential role of the actin cytoskeleton in regulating ICAM-1-mediated signals was assessed. Therefore, ICAM-1
was cross-linked on RBE4 EC as described above in the presence
of 2 mM cytochalasin D or vehicle (0.5% DMSO). p80/85 cortactin was immunoprecipitated from EC lysates obtained from either
control or treated cells. Immunoprecipitated proteins were resolved
on 7.5% SDS-PAGE and immunoblotted with anti-phosphotyrosine Ab. p80/85 cortactin (20) showed a 2.7-fold increase in
tyrosine phosphorylation following cross-linking of ICAM-1,
which was abolished (inhibited by 100%) in the presence of 2 mM
cytochalasin D (Fig. 3). Reprobing of immunoblots with mouse
anti-cortactin mAb revealed that equal amounts of p85 cortactin
were immunoprecipitated following each treatment. Treatment of
RBE4 cells with 2 mM cytochalasin D for 1 h resulted in the
disruption of all polymerized actin that exhibited a punctate distribution (Fig. 1f). Under these conditions, cross-linking ICAM-1
FIGURE 3. Effect of cytochalasin D on ICAM-1-stimulated cortactin
phosphorylation. Serum-starved RBE4 cells cultured in the presence of 50
U/ml of rat IFN-g for 48 h were stimulated with either RAM (60 mg/ml)
alone (2) or anti-ICAM-1 (20 mg/ml), followed by RAM (60 mg/ml) (1)
in the presence of 2 mM cytochalasin D (in 0.5% DMSO) or vehicle
(DMSO alone). Cells were lysed and immunoprecipitated with anticortactin Ab, and immunoprecipitates were resolved on 7.5% SDS-PAGE.
Proteins were transferred to nitrocellulose and immunoblotted with antiphosphotyrosine Ab (A), followed by stripping and reprobing with anticortactin Ab (B). Immunoblots were exposed to anti-mouse HRP-conjugated secondary Ab and developed using ECL.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 2. ICAM-1 cross-linking induces an increase in GTP-loaded Rho proteins. A, Serum-starved GP8.3 cells were cultured in the presence of 50
U/ml of rat IFN-g for 48 h and in phosphate-free growth medium supplemented with 0.2 mCi/ml of [32P]orthophosphate for 20 h. Cells were washed in
normal growth medium and were stimulated with either RAM (60 mg/ml) alone for 10 min, anti-ICAM-1 (20 mg/ml) for 30 min followed by RAM (60
mg/ml) for 10 min, anti-transferin receptor (20 mg/ml) followed by RAM (60 mg/ml), or a 10:1 ratio of lymphocytes. In C3 transferase experiments, cells
were treated for 4 h with 50 mg/ml of C3 transferase before incubation with Abs or coculture with PLNC. Cells were lysed on ice in 0.5 ml of 100 mM
HEPES (pH 7.4), 2% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 300 mM NaCl, 10 mM MgCl2, 2 mM EGTA, 2 mg/ml of BSA, and protease
inhibitors. Nuclei were removed and lysates adjusted to 500 mM NaCl. Anti-Rho Ab (4 mg) (1) or anti-transferin receptor (control) (1) was added and
incubated with lysates for 2 h at 4°C followed by 50 ml of 50% protein-G Sepharose. Immunoprecipitates were washed with 50 mM HEPES (pH 7.4),
0.005% SDS, 500 mM NaCl, 0.1% Triton X-100 and heated to 68°C for 20 min in the presence of 5 mM EDTA, 2 mM DTT, 0.2% SDS, 0.5 mM GTP,
and 0.5 mM GDP. Labeled nucleotides were separated by TLC on polyethyleneimine-cellulose plates in 1.2 M ammonium formate/0.8 M HCl and
autoradiographed. Areas containing GTP and GDP as assessed by comigration with GTP and GDP standards were scraped and radioactivity determined
by b-scintillation spectrometry. 1, presence of ICAM-1 mAb, RAM transferin-receptor mAb, C3 transferase, or lymphocytes. 2, no mAb, C3 transferase,
or lymphocytes added. Time points involving the addition of lymphocytes were taken following an initial coculture period of 30 min that was necessary
to allow the majority of the T lymphocytes to settle on the EC monolayer.
The Journal of Immunology
2969
FIGURE 4. Time course of C3-mediated ADP ribosylation of Rho proteins in EC and lymphocytes. Confluent cultures of EC were pretreated for
specified times (0 – 8 h) with 50 mg/ml of recombinant C3 transferase. In separate experiments, GP8.3 cells, RBE4 cells, or lymphocytes cocultured with
C3-treated GP8.3 EC were washed, lysed, and ADP ribosylated with 250 ng/ml of C3 transferase in the presence of 5 mCi/ml of [a-32P]NAD, 50 mM
Tris-HCl (pH 7.5), 1 mM EDTA, 2 mM MgCl2, 1 mM AEBSF, 300 mM GTP, and 0.5 mM ATP. Lysate proteins were then resolved on 15% SDS-PAGE
and autoradiographed.
Abs were unable to induce the appearance of actin stress fibers.
Pretreatment of EC with cytochalasin D also appeared to alter the
cellular localization of ICAM-1 (Fig. 1e).
In a variety of other cell types, the regulation of actin stress fibers
is regulated by members of the Rho family of small GTP-binding
proteins (31). Therefore, to assess the potential role of these proteins in ICAM-1-mediated signaling, EC were treated with the
bacterial toxin C3 transferase, which is capable of entering cells
and inactivating Rho proteins. Treatment of EC with 50 mg/ml of
C3 transferase followed by in vitro ribosylation of EC lysates with
C3 transferase and [a-32P]NAD showed a time-dependent inactivation of EC Rho proteins (Fig. 4). Inactivation of EC Rho proteins is apparent as a reduction in the availability of substrate for
subsequent in vitro [a-32P]ADP ribosylation in EC lysates. Only
one major C3 transferase substrate was observed in EC or lymphocyte lysates with an apparent m.w. of ;25 kDa, which is consistent with Rho proteins that have been correctly posttranslationally modified (32). C3 transferase treatment of both RBE4 (Fig.
1h) and GP8.3 cells (data not shown) significantly reduced the
number of actin stress fibers and abolished the induction of stressfiber formation following subsequent ICAM-1 cross-linking. In a
similar manner to cytochalasin D, C3 transferase also resulted in a
redistibution of EC ICAM-1 (Fig. 1g). Immunoprecipitation and
Western blot analysis demonstrated that preincubation of either
RBE4 (Fig. 4) or GP8.3 (data not shown) EC for 8 h with 50 mg/ml
of C3 transferase, under which conditions all EC Rho protein was
inactivated, was ineffective in inhibiting the ICAM-1-mediated enhanced tyrosine phosphorylation of p80/85 cortactin (Fig. 5).
Cross-linking of RBE4 cells with ICAM-1/RAM led to a 3.7-fold
increase in tyrosine phosphorylated cortactin, which compared with a
3.6-fold induction following preincubation with C3 transferase.
Cytochalasin D inhibits lymphocyte migration through, but not
adhesion to, EC monolayers
Rat brain EC monolayers were able to support the ICAM-1-dependent transendothelial migration of Ag-specific lymphocytes
over a 4-h period with 43 6 5.5% (n 5 22) of the lymphocytes
migrating through the EC monolayer. The ICAM-1-dependency of
Ag-specific T cell migration through CNS EC has previously been
demonstrated in Ab blockade studies (1, 11). Pretreatment of
GP8.3 monolayers with 2 mM cytochalasin D for 1 h followed by
removal, exhaustive washing, and replacement with fresh medium
before coculture with lymphocytes dramatically reduced transendothelial migration of Ag-specific lymphocytes to 61.8 6 14.3%
( p , 0.0001; n 5 6) of the control value (Fig. 6A). Increasing the
FIGURE 5. Effect of C3 transferase on ICAM-1-stimulated cortactin
phosphorylation. RBE4 cells cultured in the presence of 50 U/ml of IFN-g
for 48 h and serum-starved for 24 h were stimulated with either RAM alone
or anti-ICAM-1 (20 mg/ml), followed by RAM (60 mg/ml) (1) in either
untreated cells or cells previously treated with 50 mg/ml of recombinant C3
transferase for 8 h. Cells were lysed and immunoprecipitated with anticortactin Ab, and immunoprecipitates were resolved on 7.5% SDS-PAGE.
Proteins were transferred to nitrocellulose and immunoblotted with antiphosphotyrosine Ab (A), followed by stripping and reprobing with anticortactin Ab (B). Immunoblots were exposed to anti-mouse HRP-conjugated secondary Ab and developed using ECL.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
ICAM-1-stimulated stress-fiber formation, but not cortactin
phosphorylation, is inhibited by pretreatment of EC with C3
transferase
pretreatment time of GP8.3 monolayers with 2 mM cytochalasin D
to either 3 h or 24 h brought about a further decrease in migration
to 39.7 6 12.3.% and 35.8 6 14.8% of control values, respectively. Similar results were obtained from identical experiments
using both primary cultures (data not shown) and RBE4 cells (data
not shown). Increased pretreatment times of EC with cytochalasin
D were found to be necessary since cytochalasin D was not present
during the 4-h lymphocyte coculture, and the effects of cytochalasin D treatment on EC stress-fiber formation was observed to be
partially reversible under these conditions. Basal adhesion of Con
A-stimulated PLNC, which adhere in an identical manner to Agspecific T cell lines, but are not capable of transendothelial migration, (4) to GP8.3 EC was 18.0 6 0.7%. Basal adhesion of PLNC
to primary cultures and RBE4 cells was similar to that observed for
GP8.3. Pretreatment of EC with either 2–20 mM cytochalasin D
did not affect the adhesion of PLNC to GP8.3 (Fig. 6B), RBE4, or
primary cultures of EC (data not shown). These findings demonstrate that the effects of cytochalasin D in inhibiting lymphocyte
migration is due to an effect on the endothelial support of lymphocyte migration and not to prevention of lymphocyte binding to
endothelia. In addition, it was also observed that treatment of EC
with cytochalasin D did not affect the adhesion of cocultured Agspecific lymphocytes since, during time-lapse video microscopy,
lymphocytes displayed normal spreading and motile behavior on
the EC surface. All EC monolayers used were able to exclude
trypan blue following pretreatment with cytochalasin D and coculture with lymphocytes.
2970
LYMPHOCYTE MIGRATION THROUGH BRAIN EC REQUIRES Rho
Transendothelial lymphocyte migration but not adhesion is
inhibited by pretreatment of EC with C3 transferase
Pretreatment of both GP8.3 and RBE4 EC with 50 mg/ml of C3
transferase resulted in the inhibition of transendothelial lymphocyte migration (Fig. 7, A and B). Lymphocyte migration through
monolayers of GP8.3 cells after incubation with C3 transferase for
4 h and 8 h was reduced to 60.1 6 4.2% ( p , 0.005) and 18.4 6
4.1% ( p , 0.005) of control values, respectively. Lymphocyte
migration across cultures of RBE4 cells was only significantly inhibited after preincubation with C3 transferase for 12 h before
coculture with lymphocytes, which resulted in a reduction to
23.4 6 4.6% ( p , 0.0001) of control lymphocyte migration. The
time of C3 transferase incubation necessary for significant inhibition of lymphocyte migration corresponded to total inactivation of
RBE4 cell Rho protein as assessed by in vitro ADP ribosylation
(Fig. 4).
ADP ribosylation of lymphocyte lysates obtained from cells previously cocultured for 4 h with C3 treated EC showed no inactivation of lymphocyte Rho proteins (Fig. 4). The ability of lymphocytes to migrate across untreated endothelial monolayers was
also not affected by their pre-exposure to C3-treated EC, since
lymphocytes that had previously been cocultured with C3-treated
endothelial monolayers were found to migrate normally when subsequently cocultured with untreated EC (data not shown). The
binding of Con A-activated PLNC to GP8.3 or RBE4 cells was
unaffected by preincubation of EC with 50 mg/ml of C3 transferase
for up to 24 h (Fig. 7, C and D). In addition, treatments that are
known to activate Rho function, such as exposure of cells to LPA
(10 mM) (31) or epidermal growth factor (100 nM), which is capable of up-regulating RhoB mRNA in GP8.3 cells (P.A. and J.G.,
unpublished observations) and rat fibroblasts (33), did not affect
the extent of lymphocyte/EC adhesion in either GP8.3 or RBE4
cells (Fig. 7, C and D). Ag-specific lymphocytes cocultured with
C3 transferase-treated EC displayed normal surface spreading and
motility on the EC surface. C3 treatment of EC had no affect on the
integrity of the EC monolayer, and the EC retained their ability to
exclude trypan blue. Con A-stimulated PLNC were used in adhesion assays to demonstrate that the effects of both cytochalasin D
and C3 transferase were due to effects on EC mechanisms responsible for facilitating T lymphocyte migration, and not due to inhibition of lymphocyte binding to EC. Con A-stimulated PLNC and
Ag-specific T lymphocytes have the same adhesive capacity, but
PLNC do not have the ability to migrate due to the absence of Ag
and IL-2 stimulation (1). This approach allows independent assessment of factors affecting the separate steps of adhesion and
migration. Using time-lapse video microscopy, no differences were
observed in the adhesion of Ag-specific T lymphocytes to EC following treatment of cells with either cytochalasin D or C3 transferase (data not shown)
Discussion
The activation state of a circulating T lymphocyte is of paramount
importance in determining its ability to migrate out of the circulation (2– 4, 7). Nevertheless, using functional assays, our studies
have shown for the first time that brain EC are intimately and
actively involved in facilitating lymphocyte diapedesis. It has previously been demonstrated that the expression of the adhesion
molecule ICAM-1 on EC is pivotal in supporting lymphocyte migration across vascular endothelium (1, 3, 7, 11). To date, its role
in this process has been generally interpreted as the provision of an
extracellular docking molecule for leukocyte attachment and locomotion via ligation with receptor LFA-1. However, despite a
lack of direct evidence, it has been proposed that the efficient transvascular migration of lymphocytes require intracellular signals to
be generated within the EC. This paper demonstrates that the migration of lymphocytes through CNS vascular barriers can only
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 6. Effect of cytochalasin D on lymphocyte adhesion to and migration through brain endothelial monolayers. GP8.3 cells were treated with either
vehicle (0.5% DMSO) or 2 mM cytochalasin D for various times. After removal and vigorous washing, 51Cr-labeled Con A-activated (5 mg/ml) rat PLNC
(adhesion) or Ag-specific T cells (migration) were added and allowed to adhere for 90 min or migrate over a 4-h period, respectively. A, Migration of
Ag-specific lymphocytes through GP8.3 cells. B, Adhesion of Con A-stimulated PLNC to GP8.3 cells. Observations are a minimum of three independent
experiments using a minimum of four wells per assay. Data is expressed as mean 6 SEM percent of control migration. Significant differences between
groups were determined by Student’s t test. p, p , 0.02. Identical data was also obtained with both primary cultures and RBE4 cells.
The Journal of Immunology
2971
proceed through the active involvement of intracellular processes
within the EC, in addition to those elicited within the lymphocyte
following Ag stimulation. In support of this thesis, we have recently shown that brain EC ICAM-1 is capable of eliciting signal
transduction events following ICAM-1 cross-linking or coculture
with T lymphocytes, demonstrating that EC actively respond to
leukocyte adhesion (20). Mimicking lymphocyte attachment
through cross-linking EC ICAM-1 molecules in vitro, we have
confirmed in both GP8.3 and RBE4 rat brain EC lines that there is
an enhanced tyrosine phosphorylation of cortactin (20), FAK, paxillin, and p130cas (23). Following ligation of EC ICAM-1, which
was up-regulated by prior exposure to IFN-g, there is a reorganization of the EC actin cytoskeleton that results in stress-fiber formation. However, similar effects were also observed following ligation to basal ICAM-1 expressed on EC, suggesting that
pretreatment of cells with IFN-g was not involved in ICAM-1induced signals. Pretreatment of EC with cytochalasin D inhibited
both ICAM-1-stimulated cortactin phosphorylation and lymphocyte transendothelial migration, which supports the proposal that
the endothelial actin cytoskeleton plays a vital role in orchestrating
these events.
The observation that both ICAM-1 cross-linking and coculture
of EC with Con A-stimulated PLNC results in increased levels of
GTP-loaded endothelial Rho proteins strongly suggests that endo-
thelial Rho proteins are involved in ICAM-1-mediated signaling
events. As previously reported (34), Rho itself may be activated
through cell-surface signals propagated through the actin cytoskeleton. In support of this, pretreatment of EC with C3 transferase,
which is able to specifically ADP ribosylate (35) and inhibit Rho
proteins, resulted in a substantial inhibition of transendothelial
lymphocyte migration, inhibition of both ICAM-1 and lymphocyte-stimulated Rho-GTP loading, and inhibition of ICAM-1-stimulated stress-fiber formation, but was unable to inhibit ICAM-1stimulated cortactin phosphorylation. Other studies from these
laboratories have also demonstrated that ICAM-1-induced increases in the tyrosine phosphorylation of FAK, paxillin, and
p130cas and increases in the activity of Jun-kinase, are all effectively inhibited following pretreatment of GP8.3 and RBE4 cells
with C3 transferase (23). Treatment of EC monolayers with 50
mg/ml of recombinant C3 transferase for up to 8 h resulted in the
complete inactivation of EC Rho proteins, as assessed by the inability of C3 transferase to incorporate [a-32P]ADP-ribose into
lysates of EC monolayers previously exposed to C3 transferase in
culture (Fig. 4). It was also observed that the inactivation of Rho
proteins with C3 transferase was more rapid in GP8.3 cells than in
RBE4 cells, which may reflect either a reduced uptake of C3 transferase into RBE4 cells or, as suggested in Fig. 4, increased levels
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 7. Effect of C3 transferase on lymphocyte adhesion to and migration through EC monolayers. Endothelial monolayers were pretreated with
50 mg/ml of recombinant C3 transferase for specified times between 0 –12 h. After removal and vigorous washing, 51Cr-labeled Con A-activated (5 mg/
ml) rat PLNC (adhesion) or Ag-specific T cells
(migration) were added and allowed to adhere for 90
min or migrate over a 4-h period, respectively. A,
Migration of Ag-specific lymphocytes through
GP8.3 cells. B, Migration of Ag-specific lymphocytes through RBE4 cells. C, Adhesion of Con Astimulated PLNC to GP8.3 cells. D, Adhesion of
Con A-stimulated PLNC to RBE4 cells. Observations are a minimum of three independent experiments using a minimum of four wells per assay.
Data is expressed as mean 6 SEM percent of control migration. Significant differences between
groups were determined by Student’s t test. p, p ,
0.02.
2972
LYMPHOCYTE MIGRATION THROUGH BRAIN EC REQUIRES Rho
penetrate the tight vascular barriers of the CNS. In conclusion, we
propose that T lymphocyte diapedesis through CNS microvessels
is actively facilitated by mechanisms involving adhesion-dependent signaling within the endothelium by a process that requires an
intact endothelial actin cytoskeleton and functional endothelial
Rho proteins.
Acknowledgments
We thank L. Fieg for the pGEX-2T-C3 construct, J. T. Parsons for anticortactin mAb, and O. Durieu-Trautmann for her contribution to the initial
phase of this study.
References
1. Pryce, G., D. K. Male, I. Campbell, and J. Greenwood. 1997. Factors controlling
T-cell migration across rat cerebral endothelium in vitro. J. Neuroimmunol. 75:
84.
2. Hickey, W. F., B. L. Hsu, and H. Kimura. 1991. T-lymphocyte entry into the
central nervous system. J. Neurosci. Res. 28:254.
3. Oppenheimer-Marks, N., L. S. Davis, and P. E. Lipsky. 1990. Human T-lymphocyte adhesion to endothelial cells and transendothelial cell migration: alteration of receptor use relates to the activation status of both the T-cell and the
endothelial cell. J. Immunol. 145:140.
4. Greenwood, J., and V. Calder. 1993. Lymphocyte migration through cultured
endothelial cell monolayers derived from the blood-retinal barrier. Immunology
80:401.
5. Osborn, L. 1990. Leukocyte adhesion to endothelium in inflammation. Cell 62:3.
6. Springer, T. A. 1994. Traffic signals for lymphocyte recirculation and leukocyte
emigration: the multistep paradigm. Cell 76:301.
7. Oppenheimer-Marks, N., L. S. Davis, D. T. Bogue, J. Ramberg, and P. E. Lipsky.
1991. Differential utilization of ICAM-1 and VCAM-1 during the adhesion and
transendothelial migration of human T lymphocytes. J. Immunol. 147:2913.
8. Kavanaugh, A. F., E. Lightfoot, P. E. Lipsky, and N. Oppenheimer-Marks. 1991.
Role of CD11/CD18 in adhesion and transendothelial migration of T cells. J. Immunol. 146:4149.
9. Yednock, T .A., C. Cannon, I. C. Fritz, F. Sanchez-Madrid, L. Steinman, and
N. Karin. 1992. Prevention of experimental autoimmune encephalitis by antibodies against a4b1 integrin. Nature 356:63.
10. Male, D., J. Rahman, G. Pryce, T. Tamatani, and M. Miyasaka. 1994. Lymphocyte migration into the CNS modeled in vitro: roles of LFA-1, ICAM-1 and
VLA-4. Immunology 81:366.
11. Greenwood, J., Y. Wang, and V. Calder. 1995. Lymphocyte adhesion and transendothelial migration in the CNS: the role of LFA-1, ICAM-1, VLA-4 and
VCAM-1. Immunology 86:408.
12. Veillette, A., M. A. Bookman, E. M. Horak, and J. B. Bolan. 1988. The CD4 and
CD8 T cell surface antigens are associated with the internal membrane tyrosine
protein kinase p56lck. Cell 55:301.
13. Liao, F., H. S. Shin, and S. G. Rhee. 1992. Tyrosine phosphorylation of phospholipase C-g 1 induced by cross-linking the high affinity or low affinity Fc
receptor for IgG in U937 cells. Proc. Natl. Acad. Sci. USA 89:3659.
14. Doherty, P., and F. S. Walsh. 1992. Cell adhesion molecules, second messengers
and axonal growth. Curr. Opin. Neurobiol. 2:595.
15. Rothlein, R., T. K. Kishimoto, and E. Mainolfi. 1994. Cross-linking of ICAM-1
induces co-signaling of an oxidative burst from mononuclear leukocytes. J. Immunol. 152:2488.
16. Chintana, C., S. A. Tibbetts, M. A. Chan, and S. H. Benedict. 1995. Cross-linking
of ICAM-1 on T-cells induces transient tyrosine phosphorylation and inactivation
of cdc2 kinase. J. Immunol. 155:5479.
17. van Horssen, M., S. Loman, G. T. Rijkers, S. E. Bloom, and A. C. Bloem. 1995.
Co-ligation of ICAM-1 (CD54) and membrane IgM negatively affects B-cell
receptor signaling. Eur. J. Immunol. 25:154.
18. Jackson, D. E., C. M. Ward, R. Wang, and P. J. Mewman. 1997. The protein
tyrosine phosphatase SHP-2 binds platelet/endothelial cell adhesion molecule-1
(PECAM-1) and forms a distinct signaling complex during platelet aggregation.
J. Biol. Chem. 272:6986.
19. Ricard, I., M. D. Payet, and G. Dupuis. 1997. Clustering the adhesion molecules
VLA-4 (CD49d)/(CD29) in Jurkat T-cells or VCAM-1 (CD106) in endothelial
cells (ECV304) activates the phosphoinositide pathway and triggers Ca21 mobilization. Eur. J. Immunol. 27:1530.
20. Durieu-Trautmann, O., N. Chaverot, S. Cazaubon, A. D. Strosberg, and
P-O. Couraud. 1994. Intercellular adhesion molecule 1 activation induces tyrosine phosphorylation of the cytoskeleton-associated protein cortactin in brain
microvessel endothelial cells. J. Biol. Chem. 269:12536.
21. Roux, F., O. Durieu-Trautmann, N. Foignant-Chaverot, M. Claire, P. Mailly,
J. M. Bourre, A. D. Strosberg, and P-O. Couraud. 1994. Regulation of g-glutamyl
transpeptidase and alkaline phosphatase activities in immortalized rat brain microvessel endothelial cells. J. Cell. Physiol. 159:101.
22. Greenwood, J., G. Pryce, L. Devine, D. K. Male, W. L. C. dos Santos,
V. L. Calder, and P. Adamson. 1996. SV40 large T immortalized cell lines of the
rat blood-brain and blood-retinal barriers retain their phenotypic and immunological characteristics. J. Neuroimmunol. 71:51.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
of C3 transferase substrate. The reduced efficiency of C3 transferase in ribosylating Rho proteins in RBE4 cells, compared with
GP8.3 cells, correlated with the reduced effectiveness of C3 transferase in inhibiting transendothelial lymphocyte migration through
monolayers of RBE4 cells. These studies have also demonstrated
that the reduction in the ability of lymphocytes to migrate through
EC monolayers is not due to C3-mediated inactivation of lymphocyte Rho proteins, since cells cocultured with C3-treated EC
monolayers are able to migrate normally when transferred to untreated EC monolayers (data not shown). In addition, previous
studies have shown that pretreatment of leukocytes with C3 transferase prevents their adhesion to vascular endothelia (36), which
was not observed following treatment of EC with C3 transferase.
Taken together, these data suggest the effect of C3 transferase
treatment is mediated through inactivation of EC Rho proteins.
The inability of C3 transferase to inhibit tyrosine phosphorylation of cortactin, which is effectively inhibited following pretreatment of cells with cytochalasin D, suggests that the role of both the
actin cytoskeleton and cortactin phosphorylation may be upstream
of endothelial Rho proteins. Thus, it would appear that ICAM-1mediated signals within EC are propagated via the actin cytoskeleton, which results in the subsequent activation of Rho proteins.
This scheme has previously been suggested in studies on fibroblasts, in which constitutively activated Rho, when introduced directly into cells, was able to restore Rho mediated focal adhesions
in the presence of cytochalasin D (33). Recent studies have demonstrated that tyrosine phosphorylation of cortactin, which we
have shown to be phosphorylated by p60src (20), is responsible for
an increase in organized actin (37). The observation that increased
cortactin phosphorylation following ICAM-1 cross-linking is insensitive to pretreatment of EC with C3 transferase implies that
cortactin may be intimately associated with the propagation of
ICAM-1-induced signals within EC, which results in the subsequent activation of Rho proteins and is therefore upstream of Rho
activation. Alternatively, tyrosine phosphorylation of cortactin
may lie on an independent ICAM-1-stimulated pathway.
The ability of Con A-stimulated PLNC to stimulate endothelial
Rho-GTP loading also demonstrates that the transduction of signaling events within the EC is not dependent on the ability of the
lymphocytes to migrate through the EC monolayer and is therefore
likely to occur through cell-cell adhesion. As previously suggested, the ability of T lymphocytes to migrate through EC monolayers therefore resides in an ability to effectively signal to EC
coupled with signal transduction events elicited within T lymphocytes via Ag stimulation (1). The finding that preincubation of T
lymphocytes with anti-LFA-1 Abs is able to effectively inhibit
both T lymphocyte adhesion and transendothelial migration (11)
also supports the view that signals induced following coculture of
T cells with EC are mediated through endothelial ICAM
molecules.
The physiological mechanisms within EC that allow lymphocytes to migrate through the physical barrier afforded by CNS EC,
and that lead to either pore formation or disaggregation of tight
junctions, remains unresolved. However, in polarized epithelial
cells, RhoA has been shown to be present in the cytosol of Madin
Darby canine kidney (MDCK) cells where cell-cell contacts are
disrupted, but translocate to cell margins when cell-cell contacts
are restored (38). In addition, Rho has been reported to regulate
both perijunctional actin organization and tight junctions in both
T84 and Caco2 epithelial cells (39). Alternatively, Rho proteins
have been shown to regulate cell membrane invaginations in Xenopus oocytes (40). A similar role for Rho proteins in regulating
junctional organization or pore formation within CNS EC may
therefore provide a mechanism by which lymphocytes are able to
The Journal of Immunology
33. Jahner, D., and T. Hunter. 1991. The ras related gene rhoB is an immediate early
gene inducible by v-fps, epidermal growth factor and platelet derived growth
factor in rat fibroblasts. Mol. Cell. Biol. 11:3682.
34. Nobes, C. D., and A. Hall. 1995. Rho, rac and cdc42 regulate the assembly of
multi-molecular focal complexes associated with actin stress fibers, lamellipodia
and filopodia. Cell 81:53.
35. Aktories, K., S. Braun, S. Rosener, I. Just, and A. Hall. 1989. The rho gene
product expressed in E. Coli is a substrate for botulinum ADP-ribosyltransferase
C3. Biochem. Biophys. Res. Commun. 158:209.
36. Laudanna, C., J. J. Campbell, and E. C. Butcher. 1996. The role of rho in
chemoattractant-activated leukocyte adhesion through integrins. Science 271:
981.
37. Huang C., Y. Ni, T. Wang, Y. Gao, G. C. Haudenschild, and X. Zhan. 1997.
Down regulation of the filamentous actin cross-linking activity of cortactin by
c-src mediated tyrosine phosphorylation. J. Biol. Chem. 272:13911.
38. Takaishi, K., T. Sasaki, T. Kameyama, S. Tsukita, S. Tsukita, and Y. Takai. 1995.
Translocation of activated rho from the cytoplasm to membrane ruffling area,
cell-cell adhesion sites and cleavage furrows. Oncogene 11:39.
39. Nusrat, A., M. Giry, J. R. Turner, S. P. Colgan, C.A. Parkos, D. Carnes,
E. Lemichez, P. Boquet, and J. L. Madara. 1995. Rho protein regulates tight
junctions and peripheral actin organization in polarized epithelia. Proc. Natl.
Acad. Sci. USA 92:10629.
40. Schmalzing, G., H. P. Richter, A. Hansen, W. Schwartz, I. Just, and K. Aktories.
1995. Involvement of the GTP-binding protein rho in constitutive endocytosis in
Xenopus laevia oocytes. J. Cell. Biol. 130:1319.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
23. Etienne S., P. Adamson, J. Greenwood, A. D. Strosberg, S. Cazaubon, and
P-O. Couraud. 1998. Rho-dependent signaling pathways coupled to ICAM-1 in
microvascular brain endothelial cells. J. Immunol. 161:5755.
24. Abbott, N. J., C. C. W. Hughes, P. A. Revest, and J. Greenwood. 1992. Development and characterization of a rat brain capillary endothelial cell culture: towards an in vitro blood-brain barrier. J. Cell Sci. 103:23.
25. Greenwood, J. 1992. Characterization of a rat retinal endothelial cell culture and
the expression of P-glycoprotein in brain and retinal endothelium. J. Neuroimmunol. 39:123.
26. Zhao, Z-S., V. L. Calder, M. McLauchlan, and S. L. Lightman. 1994. Differential
lymphokine expression by rat antigen-specific CD41 T cell lines with antigen and
mitogen. Cell. Immunol. 159:220.
27. Hughes, C. C. W., D. K. Male, and P. L. Lantos. 1988. Adhesion of lymphocytes
to cerebral microvascular cells: effects of interferon-g, tumor necrosis factor and
interleukin-1. Immunology 64:677.
28. Male, D., G. Pryce, C. Hughes, and P. Lantos. 1990. Lymphocyte migration into
brain modeled in vitro: control by lymphocyte activation, cytokines, and antigen.
Cell. Immunol. 127:1.
29. Wang, Y., V. Calder, J. Greenwood, and S. Lightman. 1993. Lymphocyte adhesion to cultured endothelial cells of the blood-retinal barrier. J. Neuroimmunol.
48:161.
30. Downward, J., J. D. Graved, P. H. Warne, S. Rayter, and D. A. Cantrell. 1990.
Stimulation of p21ras upon T-cell activation. Nature 346:719.
31. Ridley, A. J., and A. Hall. 1992. The small GTP-binding protein Rho regulates
the assembly of focal adhesions and actin stress fibers in response to growth
factors. Cell 70:389.
32. Adamson, P., C. J. Marshall, A. Hall, and P. A. Tilbrook. 1992. Post-translational
modifications of the p21rho proteins. J. Biol. Chem. 267:20033.
2973