Lymphocyte Migration Through Brain Endothelial Cell Monolayers Involves Signaling Through Endothelial ICAM-1 Via a Rho-Dependent Pathway

Materials

[α-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

Fluorescence microscopy

Tyrosine phosphorylation of cortactin

Cell cultures (equivalent to 1 × 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 × 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 CD45Rc^low, 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 × 10⁶/ml was then added to each well and incubated at 37°C for 1.5 h. In each assay, γ 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 γ 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 × 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 × 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 160× 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 β-d-thiogalactoside (Life Technologies/BRL). Cells were harvested by centrifugation at 4000 × 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 × 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 [α-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 × 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 × 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 × 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 β-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.

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 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-γ 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. 1⇓b), GP8.3 (data not shown), and primary culture cells (data not shown). ICAM-1 staining was uniformly distributed within the plasma membrane (Fig. 1⇓a). 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. 1⇓b) 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. 1⇓c 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. 1⇓j). 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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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-γ and exposed to 20 μg/ml of mouse anti-rat ICAM-1 (1A29) for 30 min, followed by RAM (60 μg/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 μg/ml of anti-rat ICAM-1 (1A29) or permeabilized in 0.5% Triton X-100 and incubated in 0.1 μg/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 μg/ml) only. c and d, ICAM-1 (20 μg/ml) followed by RAM (60 μg/ml). e and f, ICAM-1 (20 μg/ml) followed by RAM (60 μg/ml) in presense of 2 μM cytochalasin D. g and h, ICAM-1 (20 μg/ml) followed by RAM (60 μg/ml) following 8 h pretreatment with 50 μg/ml of C3 transferase. i and j, 10 μM LPA. a, c, e, g, and i, stained for ICAM-1. b, d, f, h, and j, stained for actin. Scale bar = 25 μm. ICAM-1 stains are shown as projections of all optical sections. A single identical optical section is shown for phalloidin staining.

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-γ 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. 2⇓A). 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. 2⇓B).

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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-γ 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 μg/ml) alone for 10 min, anti-ICAM-1 (20 μg/ml) for 30 min followed by RAM (60 μg/ml) for 10 min, anti-transferin receptor (20 μg/ml) followed by RAM (60 μg/ml), or a 10:1 ratio of lymphocytes. In C3 transferase experiments, cells were treated for 4 h with 50 μg/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 μg) (+) or anti-transferin receptor (control) (+) was added and incubated with lysates for 2 h at 4°C followed by 50 μl 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 β-scintillation spectrometry. +, presence of ICAM-1 mAb, RAM transferin-receptor mAb, C3 transferase, or lymphocytes. −, 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.

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 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. 1⇑e).

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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-γ for 48 h were stimulated with either RAM (60 μg/ml) alone (−) or anti-ICAM-1 (20 μg/ml), followed by RAM (60 μg/ml) (+) in the presence of 2 μM cytochalasin D (in 0.5% DMSO) or vehicle (DMSO alone). Cells were lysed and immunoprecipitated with anti-cortactin Ab, and immunoprecipitates were resolved on 7.5% SDS-PAGE. Proteins were transferred to nitrocellulose and immunoblotted with anti-phosphotyrosine Ab (A), followed by stripping and reprobing with anti-cortactin Ab (B). Immunoblots were exposed to anti-mouse HRP-conjugated secondary Ab and developed using ECL.

ICAM-1-stimulated stress-fiber formation, but not cortactin phosphorylation, is inhibited by pretreatment of EC with C3 transferase

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 [α-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 [α-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. 1⇑h) 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. 1⇑g). 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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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 μg/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 μCi/ml of [α-³²P]NAD, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 2 mM MgCl₂, 1 mM AEBSF, 300 μM GTP, and 0.5 mM ATP. Lysate proteins were then resolved on 15% SDS-PAGE and autoradiographed.

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FIGURE 5.

Effect of C3 transferase on ICAM-1-stimulated cortactin phosphorylation. RBE4 cells cultured in the presence of 50 U/ml of IFN-γ for 48 h and serum-starved for 24 h were stimulated with either RAM alone or anti-ICAM-1 (20 μg/ml), followed by RAM (60 μg/ml) (+) in either untreated cells or cells previously treated with 50 μg/ml of recombinant C3 transferase for 8 h. Cells were lysed and immunoprecipitated with anti-cortactin Ab, and immunoprecipitates were resolved on 7.5% SDS-PAGE. Proteins were transferred to nitrocellulose and immunoblotted with anti-phosphotyrosine Ab (A), followed by stripping and reprobing with anti-cortactin Ab (B). Immunoblots were exposed to anti-mouse HRP-conjugated secondary Ab and developed using ECL.

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 ± 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. 6⇓A). 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. 6⇓B), 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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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 μM cytochalasin D for various times. After removal and vigorous washing, ⁵¹Cr-labeled Con A-activated (5 μg/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 ± SEM percent of control migration. Significant differences between groups were determined by Student’s t test. ∗, p < 0.02. Identical data was also obtained with both primary cultures and RBE4 cells.

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 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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FIGURE 7.

Effect of C3 transferase on lymphocyte adhesion to and migration through EC monolayers. Endothelial monolayers were pretreated with 50 μg/ml of recombinant C3 transferase for specified times between 0–12 h. After removal and vigorous washing, ⁵¹Cr-labeled Con A-activated (5 μg/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 A-stimulated 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 ± SEM percent of control migration. Significant differences between groups were determined by Student’s t test. ∗, p < 0.02.

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 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)