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*
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
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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, 8, 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 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 Fc
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, 16, 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 cross-linking 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 cytoskeletal-associated 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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[
-32P]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.).
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 digestion, 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 µg/ml of EC growth supplement (First Link), 80 µg/ml of heparin, 2 mM glutamine, 0.5 µg/ml of vitamin C, 100 U/ml of penicillin, and 100 µg/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 µg/ml of EC
growth supplement, 80 µg/ml of heparin, 2 mM glutamine, 0.5 µg/ml
of vitamin C, 100 U/ml of penicillin, and 100 µg/ml of streptomycin.
The immortalized Lewis rat brain EC line RBE4 was maintained in 1:1
Hams F-10/MEM (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 µg/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 µg/ml of anti-rat ICAM-1 (1A29) for 1 h at room temperature, exhaustively washed in PBS, and incubated with anti-mouse-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 x 105 cells) were lysed with 250 µl 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 µg/ml of mouse anti-phosphotyrosine (Upstate Biotechnology, Lake Placid, NY) or 1 µg/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 µg/ml of pepstatin, and 2 µg/ml of leupeptin), followed by removal of nuclei (13,000 x g for 30 min) and incubation with 1 µg/ml of anti-cortactin mAb for 1 h at 4°C. Cell lysates were preincubated with 50 µl 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 CD4+ 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 CD4+ 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 µCi [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% 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 µl) at a concentration of
1 x 106/ml was then added to each well and incubated
at 37°C for 1.5 h. In each assay, 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 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 ± SEM, and
significant differences between groups were determined by Student’s
t test.
T lymphocyte transendothelial migration
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 x 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 x 200-µm 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 160x 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 ± SEM, and significant differences between groups are determined by Student’s t test.
Expression and purification of C3 transferase
Glutathione S-transferase-C3 was expressed in Escherichia Coli for 5 h using 1 mM isopropyl ß-D-thiogalactoside (Life Technologies/BRL). Cells were harvested by centrifugation at 4000 x 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 x g for 30 min, and the supernatant was chromatographed over glutathione-agarose. 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 5'-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 µg) 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 µCi/ml of [-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 x g for 10 min,
and pellets were washed three times with ethanol at -20°C. Pellets
were dried, solubilized in 10 mM Tris-HCl (pH 6.8), 4% SDS, 20%
glycerol, 0.4% dithithreitol, and 0.1% bromophenol blue, and proteins
resolved on 15% SDS-PAGE.
Guanine-nucleotide, immunoprecipitation, and immunoblot analysis of endothelial Rho proteins
Serum-starved GP8.3 (5 x 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 µg/ml of leupeptin, 20 µg/ml of pepstatin, 20 µg/ml of aprotinin, 20 µg/ml of soybean trypsin inhibitor, and 2 mM AEBSF). Lysates were centrifuged at 14,000 x g for 5 min to sediment nuclei, and supernatants were adjusted to 500 mM NaCl. Lysates were immunoprecipitated by incubation with 4 µg rabbit polyclonal anti-Rho (Autogenbioclear) for 2 h at 4°C followed by incubation with 50 µl 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 -70°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 ß-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 µg/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 anti-rabbit (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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ligation of the external domain of endothelial ICAM-1 molecules
with mouse anti-rat ICAM-1 (1A29; 20 µg/ml for 30 min) followed
by cross-linking with RAM IgG (60 µg/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- 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 µM
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 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).
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Serum-starved GP8.3 cells induced with 50 U/ml of IFN- 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 phosphate containing serum-free growth
media and stimulated with RAM (60 µg/ml) alone, anti-ICAM-1 (20
µg/ml) followed by RAM (60 µg/ml), anti-transferin receptor (20
µg/ml) followed by RAM (60 µg/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
(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:GDP-bound 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).
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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 µM
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 µM 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 µM cytochalasin D for 1 h resulted in the
disruption of all polymerized actin that exhibited a punctate
distribution (Fig. 1
f). Under these conditions,
cross-linking ICAM-1 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).
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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 µg/ml of C3
transferase followed by in vitro ribosylation of EC lysates with C3
transferase and [-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
[-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 stress-fiber 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 µg/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.
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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 ± 5.5% (n = 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 µM 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 ± 14.3%
(p < 0.0001; n = 6) of the
control value (Fig. 6A).
Increasing the pretreatment time of GP8.3 monolayers with 2 µM
cytochalasin D to either 3 h or 24 h brought about a further
decrease in migration to 39.7 ± 12.3.% and 35.8 ± 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 Ag-specific T
cell lines, but are not capable of transendothelial migration, 4 to
GP8.3 EC was 18.0 ± 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 µM 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 Ag-specific 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.
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Pretreatment of both GP8.3 and RBE4 EC with 50 µg/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 ± 4.2% (p < 0.005) and
18.4 ± 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 ± 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).
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). 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 µg/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 µM) 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 |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, 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- was not involved in ICAM-1-induced
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 endothelial 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-1-stimulated 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 µg/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 [-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 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-1-mediated 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 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.
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Acknowledgments |
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Footnotes |
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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:
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 5'-diphosphate; HRP, horseradish peroxidase; NAD, nicotinamide adenine dinucleotide.
Received for publication September 8, 1998. Accepted for publication November 23, 1998.
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 K. A. Harkness, P. Adamson, J. D. Sussman, G. A. B. Davies-Jones, J. Greenwood, and M. N. Woodroofe Dexamethasone regulation of matrix metalloproteinase expression in CNS vascular endothelium Brain, April 1, 2000; 123(4): 698 - 709. [Abstract] [Full Text] [PDF]
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