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(Journal of Leukocyte Biology. 2000;68:233-242.)
© 2000 by Society for Leukocyte Biology

Resting and activated T cells induce expression of E-selectin and VCAM-1 by vascular endothelial cells through a contact-dependent but CD40 ligand-independent mechanism

Helen Yarwood, Justin C. Mason, Danuta Mahiouz, Katharine Sugars and Dorian O. Haskard

The BHF Cardiovascular Medicine Unit, National Heart and Lung Institute, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London W12 ONN, UK

Correspondence: Dr. Helen Yarwood, Department of Biology, Sir Alexander Fleming Building, Imperial College of Science, Technology and Medicine, Imperial College Road, London SW7 2AZ, UK. E-mail: h.yarwood{at}ic.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study explored the effect on endothelial cell (EC) activation of contact with T lymphocytes, which occurs during lymphocyte emigration into inflamed tissues. Addition of T cells to umbilical vein or dermal microvascular EC monolayers stimulated expression of EC E-selectin and VCAM-1. This response required direct cell:cell contact, but not T-cell activation. The capacity of resting CD4+ T cells to activate EC was restricted to the CD45RO+ subset and could be enhanced by 6 h prestimulation of T cells with PMA and ionomycin. The EC-stimulating capacity of resting or activated T cells was independent of CD40 ligand. Furthermore, inhibition of TNF-{alpha}/ß and IL-1{alpha}/ß, together with CD40 ligand, failed to inhibit EC activation by resting T cells and only inhibited the response to PMA- and ionomycin-activated T cells by 40 ± 18%. Our data suggest that T-cell-EC interactions can lead to EC activation through a novel contact-dependent, but CD40 ligand-independent, mechanism.

Key Words: T lymphocytes • endothelial cells • adhesion molecules • inflammation • cell-to-cell interactions


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It is well accepted that the activation of vascular endothelial cells (EC) and the upregulation of EC adhesion-molecule expression is a critical component of the recruitment of leukocytes at sites of inflammation [1 ]. The regulation of EC activation by soluble inflammatory mediators, including tumor necrosis factor (TNF) and interleukin (IL)-1, has been studied extensively [2 ], and such mediators are undoubtedly important in the initial induction of EC adhesion-molecule expression and thereby of leukocyte adhesion to endothelium. However, there is relatively little knowledge about the consequences that contact between leukocytes and endothelial cells may have on ongoing endothelial cell function.

Previous studies have proposed the concept that T lymphocytes may influence EC activation through cell:cell contact. [3 4 5 6 ]. Moreover, other work has shown that T-cell contact can activate neutrophils [7 , 8 ], monocytes [8 9 10 11 ], and synovial fibroblasts [12 ]. In this study, we set out to characterize further the capacity of T cells to stimulate EC adhesion-molecule expression and to investigate the mechanisms underlying contact-mediated signals. In particular and in contrast to previous studies, we have focused on the effects of resting human T cells and have used large vessel and microvascular EC. Our approach has been to coincubate T cells with EC monolayers for varying intervals, using this as a model of the effect of ongoing exposure of endothelium to emigrating T cells.

In recent reports, the cell-surface glycoprotein CD40, a member of the TNF receptor family [13 ], was shown to be expressed by EC in vitro and on endothelium in situ [14 15 16 ]. Furthermore, it was reported that ligating CD40 on EC led to signaling events that resulted in the upregulation of expression of EC adhesion molecules, including E-selectin and vascular cell adhesion molecule (VCAM)-1. In light of these reports, we investigated the possibility that the interaction between EC CD40 and the CD40 ligand (CD40L, gp39) expressed on T lymphocytes [17 , 18 ] may underlie the ability of T-cell contact with EC to regulate E-selectin and VCAM-1 expression. We present data that indicate that CD40-CD40L interactions are not necessary for contact-mediated induction of EC adhesion-molecule expression by T cells and that a separate mechanism is likely to be involved.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies and other reagents
Polyclonal goat anti-human immunoglobulin (Ig) was a kind gift from Mr. Nick Davey (Department of Immunology, Imperial College School of Medicine, Hammersmith Hospital, London, UK). Mouse anti-human Ig (Fab-specific) was from Sigma Chemical Company (Poole, Dorset, UK). The mouse monoclonal antibodies (mAbs) used were: mAb 1.2B6 (anti-E-selectin, CD62E) [19 ]; mAbs 1.4C3 and 1G11 (anti-VCAM-1, CD106) [19 , 20 ]; mAb 15.2 [anti-intercellular adhesion molecule (ICAM)-1, CD54, a kind gift from Dr. Nancy Hogg, Imperial Cancer Research Fund, London, UK; ref.21]; mAb TS2/9 [anti-lymphocyte function-associated antigen (LFA)-3, CD58, American Type Culture Collection (ATCC), Rockville, MD]; mAb L243 (anti-HLA-DR{alpha}, ATCC); mAb OKT4 (anti-CD4, ATCC); mAb OKT8 (anti-CD8, ATCC); mAb Leu 19 (anti-CD56, Becton Dickinson, San Jose, CA); mAb UCHL1 (anti-CD45RO, a kind gift from Professor Peter Beverley, The Jenner Institute, Compton, UK) [22 ]; mAb SN130 (anti-CD45RA, a kind gift from Professor George Janossy, The Royal Free Hospital, London, UK) [23 ]; mAbs TRAP-1 and TRAP-2 (anti-CD40L, gifts from Dr. Daniel Graf, MRC Clinical Science Centre, London, UK) [24 ]; mAbs 357-101-4 (anti-TNF-{alpha}) and 359-81-11 (anti-TNF-ß, both gifts from Dr. Tony Meagher, National Institute for Biological Standards and Control, Potters Bar, UK); neutralizing anti-TNF- receptor p55 (TNFR p55) mAb (kindly supplied by Dr. David Wallach, The Weizmann Institute of Sciences, Rehovot, Israel) [25 ]; mAb HEC-7 (anti-PECAM-1, CD31, a kind gift from Dr. William Muller, Rockefeller University, New York) [26 ]; mAb MCA 1442 (anti-CD69, Serotec, Oxford, UK); and mAbs Leu-12 (anti-CD19, Becton Dickinson) and Leu-M4 (anti-CD14, Becton Dickinson), used to look for potential contaminating B cells and monocytes, respectively. Rabbit anti-human IL-1{alpha} and rabbit anti-human IL-1ß antisera were a gift from Dr. Jean-Jacques Mermot (Glaxo Institute for Molecular Biology, Switzerland). The mAbs were purified from tissue culture supernatant on protein G and diluted in phosphate-buffered saline (PBS) before use in experiments. Recombinant human TNF-{alpha} (rhTNF-{alpha}) was a kind gift from Dr. Martyn Robinson (Celltech Ltd., Slough, UK). Recombinant human interferon-{gamma} (rhIFN-{gamma}) was a gift from Biogen, Geneva, Switzerland. RhIL-1 receptor antagonist (RA) was from R&D Systems, Minneapolis, MN. Unless otherwise stated, all other reagents were from Sigma.

Cell isolation and culture
Human umbilical vein endothelial cells (HUVEC) were isolated from umbilical cords by digestion with collagenase type II (Boehringer Mannheim, Lewes, UK), as described previously [19 ], and cultured in 1% gelatin-coated tissue culture flasks (Costar, Cambridge, MA) in HUVEC medium [Medium 199, Flow (ICN Biomedicals Int.), Costa Mesa, CA], supplemented with 20% fetal bovine serum (FBS) (Hyclone Laboratories Inc., Logan, UT), 100 IU/ml penicillin, 0.1 mg/ml streptomycin, 2 mM L-glutamine (all from Gibco-BRL Life Technologies, Paisley, UK), 10 U/ml heparin (Leo Laboratories, Prince Risborough, UK), and 30 µg/ml EC growth supplement. Each experiment was conducted with EC at passage three and from a single donor. Human dermal microvascular EC (DMEC) were isolated from human foreskins and cultured in fibronectin-coated flasks (Costar), as previously described in detail [27 ].

Peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood of volunteers by density-gradient centrifugation on 1.077 g/ml Ficoll-Hypaque (Lymphoprep, Nycomed Pharma AS, Oslo, Norway). PBMC banded at the plasma-Ficoll interface were washed in RPMI-1640 (Gibco-BRL) with 2.5% FBS, following which residual erythrocytes were lysed by incubation with 0.2% sodium chloride for 1 min at 4°C. T lymphocytes were obtained from PBMC by negative selection involving two sequential depletion steps. First, monocytes, B lymphocytes, and platelets were removed by panning on Petri dishes precoated with goat anti-human Ig and mAb L243. After 2 h at 37°C, nonadherent cells were collected and incubated with a cocktail of purified mAbs (mouse anti-human Ig, L243, and Leu-19) for 30 min at 4°C, followed by two rounds of magnetic immunodepletion using sheep anti-mouse Ig-coated magnetic beads (Dynal AS, Oslo, Norway) to further deplete monocytes, B cells, and natural killer (NK) cells. T-cell subpopulations (CD4/CD8; CD45RA/CD45RO) were obtained by depletion of the reciprocal populations during the second negative selection procedure, using appropriate mAbs.

In all experiments, unfractionated or fractionated T cells were >95% CD3+ with undetectable contamination with B cells (<1% CD19+), monocytes (<1% CD14+), and NK cells (<1% CD56+). Less than 1% of T cells were contaminated with platelets as shown by anti-integrin {alpha}IIb mAb binding. Furthermore, the purified T cells showed no proliferation to 2 µg/ml phytohemagglutinin (PHA) in the absence of added accessory cells in a 48-h assay [28 ]. Thus, in 10 experiments, the mean ± SD 3H-thymidine uptake of T cells following correction for background was 48 ± 10 without accessory cells, and 17,285 ± 549.5 after introduction of accessory cells. CD4+ cells were 95 ± 2% (mean±SD, 12 experiments) CD4 positive, as determined by flow cytometry. CD45RO+ CD4+ cells were 89 ± 3% (mean±SD, seven experiments) CD45RO positive, and CD45RA+ CD4+ cells were 94.1 ± 1% (mean±SD, nine experiments) CD45RA positive.

The resting nature of the T-cell preparations was determined by fluorescein-activated cell sorter (FACS) analysis using mAbs specific for the T-cell activation markers CD25, CD40L, HLA-DR, and TNF-{alpha}. Thus, in 17 representative experiments, mean ± SD mean fluorescence intensity (MFI) values were irrelevant mAb 0.31 ± 0.02, CD25 0.32 ± 0.02, CD40L 0.34 ± 0.02, HLA-DR 0.29 ± 0.02, and cell-surface TNF-{alpha} 0.34 ± 0.03. In addition, T cells prepared using this method were found to be negative for cell-surface CD69, as determined by FACS. (Mean±SD MFI values for four representative T-cell preparations were irrelevant mAb 0.24±0.02; anti-CD69 0.3±0.02).

Following their purification, T cells were suspended in RPMI-1640 with 10% heat-inactivated human AB serum and 2 mM L-glutamine at a concentration of 106/ml. Experiments routinely used T cells that had been stored overnight at 37°C in 5% CO2, because preliminary observations demonstrated that T cells stored in this way had an equivalent capacity to activate EC as freshly isolated T cells. In some experiments, T cells were activated by culture for 6 h at 37°C with phorbol myristate acetate (PMA) (10 ng/ml) and ionomycin (1 mg/ml) (Calbiochem-Novabiochem, San Diego, CA), following which the cells were washed three times before fixation.

Neutrophils were isolated from peripheral blood by sedimentation in 6% Dextran 70 in 0.9% saline, followed by isotonic discontinuous plasma-Percoll gradient centrifugation [29 ]. Neutrophils isolated in this way were >95% pure and were used immediately in coculture assays.

T-cell fixation
T cells were fixed by incubation on ice for 7 min with 2% paraformaldehyde lysine periodate (PLP) (100 mM L-lysine monohydrochloride and 2.1 mg/ml sodium metaperiodate) [24 ]. Following fixation, cells were washed twice in blocking solution consisting of 100 mM glycine, 1% bovine serum albumin (BSA) w/v in Hanks’ balanced saline solution (HBSS). They were then incubated for, at least, a further 30 min in the same solution to block remaining reactive aldehyde groups. Fixed T cells were then washed twice in RPMI-1640 with 5% FBS and incubated for 24 h at 37°C to allow any remaining paraformaldehyde to leach out. T cells were then washed twice before addition to EC. Fixed T cells were unable to proliferate in the presence of 2 µg/ml PHA and accessory cells.

Induction of EC activation by T cells
EC were grown to confluence in 24-well plates (Costar), after which the wells were washed, and T cells or recombinant cytokine were added at the stated concentration in EC growth medium without heparin or EC growth supplement. Following the coculture period, EC monolayers were washed to remove T cells and then harvested for flow cytometry by incubation with trypsin/ethylenediaminetetraacetate (EDTA) solution [Flow (ICN Biomedicals Int.)] for 1 min. In some experiments, EC were stimulated in 96-well plates (Costar), following which EC-antigen expression was quantified by enzyme-linked immunosorbent assay (ELISA). Before conducting the blocking experiments, we confirmed by ELISA the capacity of IL-1RA and each of the neutralizing mAb to inhibit completely the induction of EC-VCAM-1 expression by the appropriate recombinant cytokines. To test for effects on T-cell-mediated activation of EC, saturating concentrations of antibodies were added as appropriate to either T cells or EC monolayers for 30 min before addition of T cells to the EC monolayers, following which the antibodies remained throughout the period of coculture. In some experiments semipermeable Transwell inserts (Costar) were used to physically separate T cells from EC.

Flow cytometry
Cells were incubated with primary mAb for 30 min at 4°C. After washing in HBSS with 2.5% FBS, cells were incubated with fluorescein isothiocyanate (FITC)-labeled rabbit anti-mouse Ig (Dako, Glostrup, Denmark). Cells were then washed twice and fixed in 1% paraformaldehyde. In experiments involving the use of function-blocking mouse mAb, EC were incubated with directly biotinylated primary antibodies, washed, and then further incubated with FITC-avidin DCS (Vector Labs Inc., Burlingame, CA). Antibody binding was analyzed on an Epics XL-MCL flow cytometer (Coulter, Hialeah, FL) by counting 104 cells per sample. Endothelial cells were readily distinguished from T cells or neutrophils by light scatter. The values for MFI are presented following subtraction of the MFI obtained with irrelevant, isotype-matched, control Ig.

ELISA
Following stimulation, EC monolayers were washed, fixed for 7 min with 2% PLP, and then stored in blocking solution at 4°C. Following aspiration of blocking solution, EC were incubated with primary mAb for 1 h, washed twice in PBS, and then incubated with biotinylated rabbit anti-mouse Ig (Dako) for 1 h at room temperature. Binding of the biotinylated Ab was detected by incubation with a high mwt complex of streptavidin-biotin-peroxidase (Dako). The ELISA was then developed and quantified, as previously described [19 ]. Specific mAb binding was calculated by subtracting the background, as represented by the mean optical density (OD) of triplicate wells incubated with an irrelevant isotope-matched Ab in place of the primary mAb.

Semiquantitative reverse transcription polymerase chain reaction (RT-PCR)
Levels of steady-state mRNA were measured using a semiquantitative, competitive RT-PCR, as previously described [30 ]. EC were washed in HBSS and then lysed in guanidium isothiocyanate. RNA was extracted and stored at -70°C. For RT-PCR, RNA was diluted 1:1 with Diethyl pyrocarbonate-treated water and then mixed with a known concentration of mutant E-selectin or mutant VCAM-1 RNA. Following RT-PCR, products were electrophoresed in 2% agarose gels, and the bands were visualized under ultra-violet light. The quantity of wild-type mRNA that could be reverse-transcribed and amplified in a given sample could be obtained from the amount of mutant RNA necessary to give wild-type and mutant bands of equal intensity.

CD40 ligand-transfected cells
Myeloma cells stably transfected with human CD40L (P3xTB.A7 cells) and the parental P3 x 63.Ag8.653 cell line were a kind gift from Dr. Daniel Graf [24 ]. They were grown in RPMI-1640 with 10% FBS and 1 mg/ml G418.

Statistical analysis
Differences among the results of experimental treatments were evaluated by the Mann-Whitney U test. Differences were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
T-cell induction of E-selectin and VCAM-1 expression
In preliminary experiments aimed at testing the capacity of lymphocytes to stimulate adhesion-molecule expression by EC, we found that freshly isolated resting T cells were capable of stimulating E-selectin and VCAM-1 expression, as detected by ELISA. The degree of stimulation of E-selectin (Fig. 1 ) and VCAM-1 (unpublished results) expression was dependent on the number of added T cells. Significant (P<0.02) EC activation first became evident with as few as 5:1 T cells to EC, and optimal stimulation occurred at a ratio of 20:1. Consequently, this ratio of T cells to EC was chosen for performing subsequent experiments.



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  		Figure 1. Dose dependence of T-cell-induced EC E-selectin induction. Confluent HUVEC monolayers were cocultured with T lymphocytes at the T:EC ratios indicated for 4 h. Following washing to remove T cells, EC were fixed using 2% PLP, and cell-surface E-selectin was measured by ELISA using mAb 1.2B6. Results show the mean ± SD of triplicate wells in a representative experiment of three experiments using different T-cell donors and EC lines. *P < 0.02 compared with EC incubated in the absence of T cells.

 
Incubation of resting T cells with HUVEC led to induction of E-selectin and upregulation of VCAM-1 expression with kinetics that were similar to those seen after stimulation with an optimal concentration of rhTNF-{alpha} (10 ng/ml). Thus, E-selectin surface expression was maximal at 4 h (Fig. 2A ), and VCAM-1 expression was maximal at 8 h (Fig. 2B) . Although upregulation was reproducibly observed in all experiments, there was some variation between donors in the degree of response observed. Induction of E-selectin and VCAM-1 surface-protein expression in response to T cells was found to be associated with an increase in steady-state mRNA, with increased mRNA for E-selectin and VCAM-1 being detectable by 1 and 4 h, respectively (Fig. 2C and 2D) . Coincubation of resting T cells with DMEC also led to upregulation of VCAM-1 (Fig. 3 ) and E-selectin (unpublished results) expression, as detected by FACS.



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  		Figure 2. Induction of E-selectin and VCAM-1 on HUVEC following coculture with T cells. HUVEC were cocultured with T lymphocytes (at a ratio of 20 T cells:1 EC) or stimulated with TNF-{alpha} (10 ng/ml) for 1–24 h. EC monolayers were then washed to remove T cells, after which they were fixed with 2% PLP for measurement of E-selectin (A) and VCAM-1 (B) expression by ELISA or were lysed and RNA-extracted for measurement by competitive RT-PCR of E-selectin (C) and VCAM-1 (D) mRNA. ELISA was performed using mAbs 1.2B6 and 1G11 for E-selectin and VCAM-1, respectively. Results show the mean ± SD of triplicate wells in one experiment and are representative of three similar experiments using different T-cell donors and EC isolates.

 


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  		Figure 3. Upregulation of VCAM-1 on DMEC following coculture with resting T cells. DMEC were cocultured with TNF-{alpha} (10 ng/ml) (A) or resting T cells (B) at a T:EC ratio of 20:1. EC monolayers were then washed and harvested, after which cell-surface VCAM-1 expression was assessed by flow cytometry using mAb 1G11. In both panels, the dotted line shows the level of VCAM-1 expression on unstimulated EC, and this was similar to the level of background fluorescence in the presence of irrelevant IgG (unpublished results). The data are representative of three similar experiments using different T-cell donors and DMEC preparations.

 
Contact dependence of T-cell-mediated VCAM-1 induction
To explore the possibility that the capacity of resting T cells to stimulate EC adhesion-molecule expression was dependent on T-EC contact, we performed a coculture experiment in which the two cell types were incubated on different sides of a 0.4µ pore-size membrane. As shown in Figure 4 , T cells separated from EC by the membrane failed to induce VCAM-1 expression. In contrast, rhTNF-{alpha} was able to diffuse across the membrane and activate EC. We also found that conditioned media from T cells cultured with EC for up to 10 h at 37°C were unable to induce VCAM-1 or E-selectin expression by fresh EC monolayers (unpublished results). Taken together, these data indicate that the ability of T cells to stimulate VCAM-1 and E-selectin expression in this model requires direct T-EC contact. Furthermore, the contact interaction was not a nonspecific effect, because incubation of neutrophils with EC failed to induce VCAM-1 (Fig. 5 ) or E-selectin (unpublished results).



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  		Figure 4. Contact dependence of T-cell-induced EC-VCAM-1 induction. Upper and lower compartments were separated by a 0.4µ pore size, semipermeable membrane. HUVEC were cultured as a confluent monolayer in the lower compartment in all samples. (A) The background fluorescence on unstimulated HUVEC in the presence of irrelevant, control IgG (MOPC 21); (B) the level of VCAM-1 expression by unstimulated HUVEC in the presence of medium alone. TNF-{alpha} (10 ng/ml; C, D) or T cells (20 T cells:1 EC; E, F) were added to the upper (C, E) or lower (D, F) compartment as indicated. After 16 h, EC were then harvested, and cell-surface, VCAM-1 expression was assessed by flow cytometry using mAb 1G11 (anti-VCAM-1). The data are representative of four similar experiments using different T-cell donors and EC isolates.

 


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  		Figure 5. Coculture of EC with neutrophils fails to upregulate EC-VCAM-1. Confluent EC monolayers were cocultured in 96-well plates with neutrophils or T cells (both at a ratio of 20:1 EC), which had been isolated in parallel from the same donor. Unstimulated or TNF-{alpha} (10 ng/ml)-stimulated EC were included as controls. Following a coculture period of 10 h, EC monolayers were washed to remove leukocytes and fixed using 2% PLP. Cell-surface VCAM-1 was then measured by ELISA. Results show the mean ± SD of triplicate wells in one experiment and are representative of three similar experiments using different leukocyte donors and EC preparations.

 
T-cell activation is not a prerequisite for contact-dependent EC adhesion-molecule expression
To address further the possibility that surface factor(s) on resting T cells are able to activate adhesion-molecule expression by EC, we next tested the effects of T-cell fixation. Resting T cells fixed with 2% PLP for 7 min were still able to induce VCAM-1 expression by HUVEC (Fig. 6 ) and DMEC (unpublished results), although the capacity of fixed, resting T cells to upregulate VCAM-1 was not as great as that of unfixed, resting T cells for HUVEC (in four experiments, mean±SD MFI for VCAM-1: unstimulated, 0.62±0.07; unfixed T cells, 7.34±2.4; fixed T cells, 4.75±1.9; TNF-{alpha}, 10 ng/ml, 15.0±2.0) or DMEC (in five experiments, mean±SD MFI for VCAM-1: unstimulated, 0.3±0.03;, unfixed T cells, 1.42±0.1; fixed T cells, 0.97±0.27; TNF-{alpha}, 10 ng/ml, 3.8±0.7). Allogeneic, unfixed T cells have been shown to be nearly as effective as rhIFN-{gamma} (500 U/ml) in stimulating EC-HLA-DR expression, operating through a mechanism that is known to be mediated by T-cell recognition of HLA class I [31 , 32 ]. As shown in Figure 6 , fixation completely abrogated the ability of unfixed, resting T cells to induce HLA-DR expression by EC. The capacity of fixed, resting T cells to stimulate VCAM-1 expression was not a result of leaking of cytoplasmic material, because the media from fixed T cells that had been incubated overnight had no effect in the assay (unpublished results). These results indicate that T-cell surface molecule(s) are able to activate adhesion-molecule expression on contact with EC and suggest that the mechanism of this upregulation does not depend on an allogeneic response or other form of T-cell activation.



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  		Figure 6. Prefixed T cells upregulate VCAM-1 but not HLA-DR on HUVEC. Confluent EC monolayers were cocultured with unfixed or 2% PLP-fixed T cells (20 T cells:1 EC). Monolayers were also stimulated with TNF-{alpha} (10 ng/ml) or IFN-{gamma} (500 u/ml) as positive controls for induction of VCAM-1 and HLA-DR, respectively. Following washing to remove T cells, EC were harvested and analyzed for cell-surface VCAM-1 (at 16 h; column A) or HLA-DR (at 24 h; column B) expression by flow cytometry. In each section, the dotted line represents background fluorescence with FITC-labeled rabbit anti-mouse Ig alone. Results are representative of three similar experiments performed on different isolates of HUVEC.

 
T-cell contact-dependent activation enhances the EC response to TNF-{alpha}
To investigate whether T-cell, contact-dependent activation of EC could synergistically enhance the EC response to TNF-{alpha} at suboptimal concentrations of cytokine, HUVEC monolayers were cultured with increasing concentrations of TNF-{alpha} in the absence and presence of fixed T cells. As shown in Fig. 7 , T cells were able to additively enhance expression of E-selectin and VCAM-1 in response to TNF-{alpha}. The degree of enhancement of E-selectin and VCAM-1 expression as a result of T cells was similar at concentrations of TNF-{alpha} up to 0.1 ng/ml, without evidence of synergy.



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  		Figure 7. Effect of T cells on TNF-{alpha} induction of VCAM-1 and E-selectin. Confluent HUVEC monolayers were stimulated with TNF-{alpha} (at the concentrations indicated) in the absence (solid bars) and presence (hatched bars) of 2% PLP-fixed T cells (20 T cells:1 EC). Following washing to remove T cells, EC were harvested and analyzed for cell-surface VCAM-1 (at 16 h; A) or E-selectin (at 4 h; B) expression by flow cytometry. Results show the mean ± SD of triplicate wells in one experiment and are representative of three similar experiments using different T-cell donors and EC preparations.

 
CD4+ CD45RO+ T cells stimulate EC activation
The results described so far utilized unfractionated, resting T cells. To dissect the response further, we next investigated the ability of purified, resting CD4+ T-cell subsets to stimulate VCAM-1 expression. We found that unfixed, resting CD45RO+ CD4+ T cells were able to stimulate VCAM-1 expression to a similar degree as unfractionated T cells, whereas unfixed CD4+ CD45RA+ resting T cells had almost no activity in the assay (Fig. 8 ), even when a T:EC concentration of up to 40:1 was used (unpublished results).



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  		Figure 8. CD4+ CD45RO+ but not CD4+ CD45RA+ T cells upregulate EC-VCAM-1. Confluent HUVEC monolayers were cocultured with the total T-cell population (B), CD4+ CD45RO+ T-cell population (C), or CD4+ CD45RA+ T-cell population (D) (all at a ratio of 20:1 EC) for 16 h. EC monolayers were harvested, and cell-surface VCAM-1 expression was assessed by flow cytometry using mAb 1G11 (solid line). In each section the dotted line represents background fluorescence with FITC-labeled rabbit anti-mouse Ig alone. (A) The level of VCAM-1 expression by unstimulated HUVEC in the presence of medium alone. The results represent data from a single experiment in which the T-cell populations were isolated in parallel from a single donor. This experiment is representative of three similar experiments.

 
Role of CD40:CD40L in T-cell-induced stimulation of VCAM-1 expression
In light of recent literature reports indicating that ligation of CD40 on EC can upregulate adhesion-molecule expression [14 15 16 ], we considered the possibility that CD40-CD40L interactions might underlie the capacity of the CD4+ population of T cells to stimulate EC in our model. In a preliminary flow cytometry study, we found that it was not possible to detect CD40L on the surface of our resting CD4+ T-cell populations, and furthermore, we were not able to detect upregulation of CD40L following contact of CD4+ T cells with EC (unpublished results), confirming the observations of Karmann et al. [33 ]. However, CD40L expression could be induced by activation of CD4+ T cells with PMA and ionomycin for 6 h (in five experiments, mean±SD MFI for CD40L: resting T cells, 0.12±0.05; PMA- and ionomycin-stimulated T cells, 18.8±2.6). Whereas CD4+ T cells that had been activated by PMA and ionomycin and then fixed showed an enhanced capacity to upregulate EC-VCAM-1 expression compared with resting CD4+ T cells, mAbs capable of blocking CD40L function had no effect on the EC-stimulating capacity of resting or activated T cells (Fig. 9 ). In contrast, the same anti-CD40L mAb preparation was able to block the induction of VCAM-1 expression by fixed CD40L-transfected P3 x TB.A7 cells (Fig. 9 ). Notably, CD40L-transfected P3 x TB.A7 cells expressed CD40L at approximately the same surface density as PMA- and ionomycin-activated CD4+ T cells, as determined by flow cytometry (unpublished results). The inability of anti-CD40L mAbs to inhibit EC activation by T cells activated by PMA and ionomycin was not a result of contamination with residual PMA and ionomycin, because in parallel wells, control, untransfected, P3 x 63.Ag8.653 cells that had been incubated with PMA and ionomycin, and fixed and washed in the same way, were unable to stimulate EC. Furthermore, the fixation procedure did not alter CD40L expression on activated T cells.



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  		Figure 9. T-cell regulation of EC-VCAM-1 is mediated by mechanisms independent of CD40 ligand. CD4+ T cells were cultured in medium alone (resting T cells) or medium containing PMA (10 ng/ml) and ionomycin (1 mg/ml) for 6 h at 37°C (activated T cells), after which they were fixed with 2% PLP. The fixed T cells were then pretreated with medium alone (column A) or with a combination of anti-CD40L mAb TRAP 1 (10 ug/ml) and TRAP 2 (20 ug/ml) for 30 min at 37°C (column B) prior to coculture with EC monolayers in the continued presence of mAb. As a positive control for the potential of anti-CD40L mAb to block CD40-mediated EC activation, P3xTB.A7 cells expressing human CD40L were fixed using 2% PLP and then incubated with medium alone (A) or the combination of anti-CD40L mAb, as above (B), before addition to the EC monolayers. EC were harvested after 16 h of coculture, and surface expression of VCAM-1 was measured by flow cytometry using directly biotinylated, anti-VCAM-1 mAb 1.4C3 for detection. The dotted line represents the level of VCAM-1 expression on EC in the absence of T cells (upper two rows) or in the presence of untransfected P3x63.Ag8.653 cells (lower row). The solid line represents the level of VCAM-1 expression on EC following coculture with resting T cells (upper row), activated T cells (middle row), or CD40L transfectants (lower row). The results are representative of three similar experiments performed on different HUVEC isolates.

 
In view of the possibility that the capacity of fixed T cells to stimulate VCAM-1 expression might relate to other known pathways of EC activation, we performed several experiments using blocking reagents to cytokines, either alone or in combination with one another and/or anti-CD40L mAb. We were unable to show any inhibition of the upregulation of VCAM-1 expression by fixed or unfixed resting T cells in the presence of IL-1RA and a cocktail of antibodies against IL-1{alpha}, IL-1ß, TNF-{alpha}, TNF-ß (lymphotoxin), and CD40L (unpublished results). Likewise, we were able to only partially inhibit the upregulation of VCAM-1 by fixed PMA- and ionomycin-activated CD4+ T cells (mean±SD inhibition, 40±18% in four experiments) in the presence of these reagents. Similar results were obtained using the IL-1RA and the same cocktail of anticytokine antibodies together with anti-p55 TNF—R. The individual contribution of these factors was not explored further. We were also unable to inhibit the EC response to resting CD4+ T cells with mAb against ICAM-1 (CD54), PECAM-1 (CD31), or LFA-3 (CD58) (unpublished results).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we have cocultured freshly isolated, resting T cells with EC to characterize the capacity of contact interactions between the two cell types to activate E-selectin and VCAM-1 expression by EC. Our observation that DMEC respond in a similar manner to HUVEC emphasizes the relevance of this effect with respect to events that may take place in the microvasculature during inflammatory responses in vivo.

It seems most likely that the initiation of EC activation- and adhesion-molecule expression at the onset of an inflammatory response is mediated by local release of soluble mediators rather than by contact interactions with T cells. The reasons for this are two-fold. First, lymphocyte recruitment into evolving inflammatory lesions is characteristically delayed compared with the recruitment of neutrophils, although the recruitment of both cell types is dependent on the induction of E-selectin expression [34 ]. Secondly, lymphocytes are not found in contact with the endothelium of the microvasculature in noninflamed tissues, as has been demonstrated using models of intravital microscopy to directly visualize the microcirculation [35 36 37 ]. Our data do, however, raise the possibility that T cells in the process of adhering to and moving across vascular endothelium may, themselves, provide activating signals that serve to regulate EC adhesion-molecule expression further. Our data showing that fixed T cells have the capacity to activate EC suggest that transmigration does not appear to be necessary for the activation event. However, it remains possible that the enhanced EC-activating response obtained with unfixed T cells is mediated by the process of transmigration. The observation that T cells are able to additively enhance TNF-{alpha}-induced adhesion-molecule expression strengthens the possibility that contact-dependent mechanisms, together with soluble mediators, may be involved in the de novo activation of EC in the initial stages of an inflammatory response when cytokine levels are likely to be low. Such cooperative actions may lead to augmented leukocyte recruitment and, hence, to firm establishment of the inflammatory focus. Furthermore, such contact-mediated events may be important in perpetuating EC activation, thereby maintaining leukocyte recruitment beyond the initial phase of inflammation, as observed in delayed hypersensitivity responses [5 , 38 , 39 ].

A period of 2 to 4 h was required for T cells to activate expression of EC in our assays. Although it is unlikely that individual, emigrating T cells would remain in contact with EC for that duration in vivo, persistent recruitment of T cells can occur over many hours during a chronic inflammatory response, and implicit in this is the continual contact between migrating T cells and endothelium. Furthermore, transmigrated lymphocytes often remain in close association with the overlying endothelium. Endothelial cell activation by adherent lymphocytes has been demonstrated in other in vitro studies, most of which have focused on activated rather than resting T cells [3 4 5 6 ]. Although Doukas and Pober [3 ] did not investigate VCAM-1 or E-selectin, they found that coculture of resting T cells with EC led to marked upregulation of EC-ICAM-1 expression. In a similar study, Damle et al. [4 ] demonstrated a moderate upregulation of VCAM-1 and ICAM-1 in response to resting T cells, focusing instead on the effects of T cells activated by phorbol ester. It is difficult to directly compare our results with those of Sunderkotter et al. [5 ] and Lou et al. [6 ], because the former used mouse lymphocytes and a transformed mouse brain EC line, and the latter stimulated brain microvascular EC with a T-cell membrane preparation.

It is particularly relevant that resting CD4+ T cells are able to stimulate EC in this way, because many T cells trafficking into inflamed tissues are in a relatively quiescent state. Furthermore, the fact that resting CD4+ CD45RO+ T cells were more effective than resting CD4+ CD45RA+ T cells parallels the greater potential of CD45RO+ T cells to enter inflamed nonlymphoid tissues [40 ]. Although we do not know the reason for the variability in EC-stimulating capacity that we observed between T-cell preparations from different donors, it could be because of subtle differences within the CD45RO+ T-cell population, perhaps related to the proportion of recently activated cells released from lymphoid tissues.

We believe that our observations cannot be explained by contamination of the lymphocyte or EC preparations with other blood cells. First, although activated platelets represent a potential source of IL-1 [41 ], we established by flow cytometry using an anti-integrin {alpha}IIb mAb that the T-cell preparations were effectively depleted of contaminating platelets. Secondly, the T cells underwent rigorous depletion of NK cells [42 ] and monocytes [43 ], such that the final preparations contained no CD14 or CD56 positive cells detectable by FACS and did not respond to PHA in the absence of added accessory cells. Lastly, HUVEC were used at third passage and DMEC, at passages 4–6, at which there are no detectable mononuclear cells that might have derived from the umbilical veins or skin used for EC isolation.

Although we used mismatched combinations of T cells and EC, we believe that an allogeneic response is unlikely to explain the observations. First, contact-dependent EC activation could also be elicited with purified CD4+ T cells, which are unable to respond to HLA class II, negative, resting, allogeneic EC in the absence of accessory cells [28 , 31 , 32 ]. Secondly, it is very unlikely that any T-cell activation in the cocultures is required, because, although fixation of T cells prevented HLA class II upregulation, this procedure had no effect on the capacity of T cells to stimulate VCAM-1 expression. Furthermore, we have also found that preincubating T cells with the protein synthesis inhibitor emetine had no inhibitory effect on their capacity to activate EC but prevented the upregulation of CD40L in response to PMA and ionomycin (unpublished results). It should, however, be noted that CD8+ T cells are able to mount an allogeneic response against resting, unactivated EC, leading to the induction of EC class II MHC expression, largely mediated by T-cell secretion of IFN-{gamma} [31 , 32 , 44 ]. In this regard, although fixed, resting CD4+ T cells were similar to unfixed, resting CD4+ T cells, we observed that fixed, unfractionated, resting T cells consistently showed a slightly reduced capacity to upregulate VCAM-1 compared with unfixed, unfractionated, resting T cells. It remains possible, therefore, that this could be related to the inhibition by fixation of an allogeneic component to the EC-VCAM-1 expression mediated by CD8+ T cells.

The transwell experiments demonstrate that direct cell-cell contact is required to achieve EC adhesion-molecule expression. This is not a nonspecific effect of contact, because coculture with neutrophils failed to stimulate E-selectin or VCAM-1 expression. It is very unlikely that T-EC contact induces the release of a T-cell or EC-derived soluble factor capable of EC activation, because fixed T cells, but not conditioned media from fixed T cells or T-EC cocultures, are able to induce VCAM-1. The fact that fixed T cells were able to activate adhesion-molecule expression also argues against the effect being mediated by transfer of a cytoplasmic component(s) between the two cell types, as has been shown to be possible [45 ].

Taken together, our results indicate that the direct activating effect of T cells is mediated by a factor(s) expressed on the T-cell surface membrane. We think it is unlikely that the effect is a result of expression of IL-1{alpha} or TNF-{alpha} on the T-cell surface for several reasons. (1) Resting T cells express negligible amounts of these cell-surface cytokines [46 47 48 ]. (2) Recombinant IL-1RA, together with a cocktail of mAb against IL-1{alpha}, IL-1ß, TNF-{alpha}, TNF-ß, and TNF-R p55 mAb, had no inhibitory effect on the induction of VCAM-1 by resting T cells. The inclusion in these experiments of reagents that block cytokine receptors is particularly important, because this ensures that even cytokines secreted into the intercellular contact points between the two cell types should be inhibited. Furthermore, this cocktail of neutralizing reagents only partially inhibited the induction of EC-VCAM-1 by phorbol ester and ionomycin-activated T cells, indicating that a significant component of this response was mediated by an IL-1 and TNF-independent mechanism. (3) DMEC are largely unresponsive to recombinant IL-1{alpha} or IL-1ß in terms of VCAM-1 expression [49 ].

Given the contact dependence of the effects we have observed, we conducted experiments testing the role of a number of candidate surface molecules. Recent work has shown that ligation of CD40 on EC using CD40L fusion proteins or CD40L transfected cell lines can lead to upregulation of E-selectin and VCAM-1 expression [14 15 16 ]. Two lines of evidence indicate that this pathway is unlikely to be of primary importance to the capacity of freshly isolated, peripheral blood, resting T cells to activate EC. First, the resting T-cell populations added to the EC cultures were shown by flow cytometry to be essentially CD40L-negative. Secondly, an anti-CD40L mAb failed to inhibit VCAM-1 upregulation by resting or phorbol ester and ionomycin-activated T cells, despite clear inhibition of the stimulatory effect of P3 x 63.Ag8.653 cells transfected with human CD40L. A role for CD40 in T cell:EC interactions remains to be elucidated. In other experiments, we were unable to obtain any inhibitory effects with antibodies against PECAM-1 (CD31) and ICAM-1 (CD54) or LFA-3 (CD58), suggesting that a higher density of counter-receptors, ß2 integrins, and CD2, respectively, is not the explanation for the greater effects we observed of CD4+ CD45RO+ compared with CD4+ CD45RA+ T cells.

In conclusion, our data provide evidence for the existence of mechanism(s) whereby resting and activated T cells may stimulate EC adhesion-molecule expression through direct contact with EC. Because the effects we have observed are not readily explained by CD40-CD40L interactions, further work is now required to define the possible roles of other T-cell surface molecules, such as surface cytokines related to TNF- [50 ] and members of the recently described disintegrin and metalloprotease (ADAM) family [51 ]. Moreover, it will be important to determine the precise pathophysiological context in which T-cell contact-mediated activation of EC adhesion molecules occurs and at what point, in the cascade of adhesion events that occur during T-cell emigration through endothelium, the contact-dependent, activating event takes place.


   ACKNOWLEDGEMENTS
 
This study was funded in part by the Wellcome Trust and in part by a discretionary professorial award from the British Heart Foundation. We thank Professor R. Lechler, Dr. G. Lombardi, Dr. R. C. Landis, and Dr. F. Brennan for helpful discussions. We are grateful for the help of P. Kiely, P. Singh, Dr. L. Lovat, and M. McNamara for collection of foreskins and to the staff of the maternity unit of Hammersmith Hospital for the provision of umbilical cords.

Received October 11, 1999; revised March 12, 2000; accepted March 15, 2000.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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