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The Journal of Immunology, 1999, 162: 1677-1684.
Copyright © 1999 by The American Association of Immunologists

Activated T Cells Acquire Endothelial Cell Surface Determinants During Transendothelial Migration1

Ruth I. Brezinschek, Nancy Oppenheimer-Marks and Peter E. Lipsky2

Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75235


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Activated T cells acquire endothelial cell (EC) plasma membrane constituents during transendothelial migration. This was assessed using an in vitro model system in which human peripheral blood CD4+ T cells migrated through confluent monolayers of HUVEC. Flow cytometry of migrated CD4+ T cells demonstrated that activated, but not resting, T cells acquired a variety of endothelial surface determinants, including CD31, CD49d, CD54, CD61, and CD62E. The extracellular domains of these molecules were detected on migrated T cells with mAbs, including those directed to the ligand-binding regions. A number of approaches were employed to document that the acquisition of these molecules was uniquely accomplished by activated T cells and clearly involved transfer from both resting and TNF-{alpha}-activated EC. Acquisition of endothelial markers by activated T cells occurred as part of the transfer of membrane components, as migrating T cells acquired EC membranes prelabeled with the lipophilic dye, 3,3'-dihexadecyloxacarbocyanine perchlorate (DiOC-16), along with EC surface proteins. Thus, during transendothelial migration, activated T cells acquire endothelial membrane components, and as a result may deliver them to perivascular sites.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Extravasation of mononuclear cells, especially lymphocytes into perivascular tissue, is a critical step in the development and progression of immunologically driven inflammation. Based on in vitro findings, it is thought that the endothelium functions as an important regulator of inflammatory and immune responses by governing adherence and extravasation of T cells and other inflammatory cells at tissue sites of antigenic challenge (1). An initial event in the migration of T cells into tissue is their binding to vascular endothelial cells (EC)3 lining postcapillary venules. After this first step, T cells subsequently migrate through the EC layer and accumulate in the perivascular areas of the inflamed tissue. At such sites, a unique subset of memory CD4+ T cells defined by the bright expression of CD11a, CD26, and CD44 specifically exhibits the capacity to migrate through the endothelial barrier (2). Importantly, EC regulate the capacity of this intrinsically migratory T cell subset to access the tissue (2). Similar events appear to govern access of memory T cells to sites of inflammation in diverse locations, including the gut, the thyroid gland, the skin, the lung, and at sites of vascular inflammation (3, 4, 5, 6, 7, 8).

Cellular activation can alter the capacity of both the T cell and the EC to interact. Although T cells do not require stimulation to interact with EC, it has become apparent that activated CD4+ T cells migrate through endothelial barriers more effectively than resting CD4+ T cells (2, 9). Similarly, cytokine-stimulated EC express adhesion molecules not found on resting endothelium, such as VCAM-1 (CD106) and E-selectin (CD62E), that enhance their ability to bind T cells (10, 11). Thus, activation stimuli can alter the capacity of EC and T cells to interact, but whether the interaction between T cells and EC may alter the subsequent function of either or both cell types is less well delineated.

During T cell/EC interactions, cellular function could be altered in a variety of physiologic ways, including that mediated by the action of secreted cytokines, chemokines, and other effector molecules, as well as that resulting from a variety of ligand-receptor interactions with signaling potential. Additionally, cellular function could be altered by the transfer of cellular constituents released by ectocytosis (12) or as a result of apoptosis (13).

Over the course of studies on mechanisms of transendothelial migration, we observed that membrane blebs occurred on the surface of EC during their interaction with T cells. In the current study, we sought to determine whether this leads to the transfer of EC determinants to migrating T cells and the characteristics of this process. The results clearly indicate that activated CD4+ T cells acquire endothelial determinants during their transendothelial migration, regardless of the activation state of the endothelium.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures

EC were isolated from individual human umbilical cord veins by collagenase digestion, as previously described (9), and cultured in RPMI 1640, purchased from Whittaker Bioproducts (Walkersville, MD), which contained 10% heat-inactivated human serum (HS) from normal blood donors; 15% FCS, purchased from Sigma (St. Louis, MO); 24 µg/ml EC growth supplement, purchased from Collaborative Research (Bedford, MA); 12.5 U/ml heparin, purchased from Upjohn (Kalamazoo, MI); penicillin G (100 U/ml) and 2 mM L-glutamine, purchased from Whittaker Bioproducts; and gentamicin (50 mg/ml) and nystatin (100 U/ml). EC from the third through fifth passage were used for these studies. Following their removal from the culture flasks by typsinization in the presence of Puck’s EDTA, the EC were resuspended in RPMI 1640 culture medium supplemented with 10% HS, 24 µg/ml EC growth supplement, and 50 µg/ml ascorbic acid, purchased from Sigma, as previously described (9).

Preparation of CD4+ T cells

T cells were prepared by density-gradient centrifugation, adherent cell depletion, and passage over a nylon wool column, as previously described (9). CD4+ T cells were then isolated by a negative panning method, as previously described (2), using a mixture of mAb with specificities against CD8 (OKT8), HLA-DR (L243), CD14 (63D3), and CD11b (OKM1), all purchased from the American Type Culture Collection (ATCC, Manassas, VA); and CD16 (B73.1), kindly provided by Dr. G. Trinchieri (Fox Chase Cancer Center, Philadelphia, PA). The purity of the CD4+ T cells prepared by this method was always greater than 95%. The purified CD4+ T cells were washed and incubated overnight at 37°C in RPMI medium containing 10% FCS. In some cases, T cells were activated with phorbol dibutyrate (PDB) (10 ng/ml, 18 h, 37°C), purchased from Sigma, as previously described (2, 9), or by culture for 18 h (14) in tissue culture plates containing immobilized anti-CD3 mAb (64.1; 200 ng/ml).

Assay of transendothelial migration

The transendothelial migration assay was performed as previously described (2). Briefly, in the wells of 36-mm macrowell tissue culture plates, EC (4 x 106) were cultured to confluence on hydrated type I collagen gels (50% v/v) purchased from Vitrogen 100R, Celtrix (Palo Alto, CA). In certain experiments, EC were stimulated for 4 h (37°C) with TNF-{alpha} (400 U/ml) obtained from R&D Systems (Emeryville, CA). These conditions were sufficient to induce the expression of CD62E (E-selectin) and CD106 (VCAM-1), and to increase CD54 (ICAM-1) expression (2). Resting or PDB-activated CD4+ T cells (10 x 106), resuspended in 0.6% BSA in RPMI (RPMI/BSA), were incubated with the EC for 4 h at 37°C. Subsequently, nonadherent and migrated CD4+ T cells were recovered, as described previously (2).

In some experiments, EC on collagen gels were labeled according to manufacturer’s protocol with the lipophilic dye, DiOC-16, obtained from Molecular Probes (Eugene, OR), or with FITC-labeled anti-CD62E mAb (1.2B6) obtained from Southern Biotechnology (Birmingham, AL), or FITC-labeled isotype-matched control Ab (MOPC) purchased from Sigma, before assay. Subsequently, the cells were washed and then used in assays with CD4+ T cells. In other experiments, CD4+ T cells were depleted of CD4+/CD54+ T cells by a negative selection using the mAb R6.5 (anti-CD54), kindly provided by Dr. Robert Rothlein, Boehringer Ingelheim Pharmaceuticals (Ridgefield, CT). Subsequently, the T cells were preincubated (60 min, 37°C) in the absence or presence of the RNA synthesis inhibitor actinomycin D (10 µg/ml), purchased from Sigma, activated with PDB (50 ng/ml, 60 min), washed, and then added to EC monolayers.

Analysis of CD4+ T cell surface phenotype by flow cytometry

Cells in the initial and recovered populations were examined by direct staining, as previously described (2), with FITC- and/or PE-labeled mAb for the expression of various surface receptors, including CD69 (Leu23 PE), CD3 (Leu4 PE), and CD54 (Leu54 PE), all purchased from Becton Dickinson (Mountain View, CA); CD4 (Q4120 PE), purchased from Sigma; CD62E (1.2B6 FITC), purchased from Southern Biotechnology; and CD54 (R6.5 FITC), obtained from Dr. R. Rothlein of Boerhinger Ingelheim Pharmaceuticals, and coupled with FITC. FITC or PE directly labeled irrelevant mouse mAb used as negative controls were purchased from Becton Dickinson or Sigma to verify the staining specificity of the experimental mAb. In addition, indirect staining examined the expression of the surface determinant CD62E using several anti-CD62E mAb, including CL3 and CL37, gifts from Dr. C. Wayne Smith, Baylor College of Medicine (Houston, TX), and 1.2B6, kindly provided by Dr. D. Haskard, Hammersmith Hospital (London, U.K.). The expression of CD31 was examined using an anti-CD31 mAb (5.6E), obtained from Immunotech (Westbrook, ME). Isotype-matched control mAb, MOPC (IgG1), and P117 (IgG2a) were purchased from Sigma.

For direct staining, T cells were incubated with saturating concentrations of the indicated mAb for 30 min at 4°C, washed three times with PBS containing 2% HS and 0.1% sodium azide, fixed with 1% paraformaldehyde for 20 min, and washed again. When indirect staining was performed, incubation with the primary mAb was followed by counterstaining with FITC-conjugated goat anti-mouse Ig obtained from Cappel Research Products (Durham, NC), as described (14). After three washes, cells were fixed with 1% paraformaldehyde for 20 min and washed again. Analysis of cell surface marker expression was performed by flow cytometry using a Becton Dickinson FACScan. For all experiments, the FACScan was calibrated with calibration beads purchased from Becton Dickinson, to obtain comparable results. The lymphocytes were specifically analyzed by selective gating based on the parameters of forward and side scatter. The percentages of cells positive for green (FITC) and red (PE) fluorescence and the density of expression of specific determinants, as estimated by the mean fluorescence intensity, were calculated using the Becton Dickinson Lysis II program.

Quantification of apoptosis by flow cytometry

Cell cycle progression and apoptosis were monitored by quantitating cellular DNA content after staining with propidium iodide. After migration of CD4+ T cells and collagenase digestion of the collagen gels, EC were isolated from the digest by negative selection using an anti-CD3 mAb (OKT3), purchased from ATCC. Subsequently, the purified EC were fixed in ethanol:PBS (70% v:v, -20°C, 18 h), washed in cold PBS, and then resuspended in propidium iodide stain mixture containing 50 µg/ml propidium iodide, 0.05% Triton X-100, and 100 U/ml RNase, all purchased from Sigma. DNA content was then determined by flow cytometry (Becton Dickinson FACScan). At least 104 cells were analyzed in each sample. DNA content per cell was determined with CellQuest software.

Reverse-transcriptase PCR

Expression of CD62E mRNA in T cells and EC was determined as previously described (15). Briefly, RNA was isolated and purified from 2 x 106 TNF-{alpha}-activated or resting EC, as well as from 2 x 106 PDB activated or resting CD4+ T cells using the SNAP-O-SOL RNA/DNA isolation kit obtained from BIOTECX Laboratories (Houston, TX). First strand cDNA was synthesized using the Super Script Preamplification System for first strand cDNA synthesis, purchased from Life Technologies (Grand Island, NY). Aliquots (3 µl) of the cDNA were amplified in two separate reactions for CD62E and G6PD in 0.2-ml PCR tubes, as described previously (15). Briefly, a wax pellet was used to seal a 25-µl reaction mix containing MgCl2, obtained from Promega (Madison, MI), all four dNTPs purchased from Sigma, and specific primers for CD62E, 5'-AAG GTA CAC ACA CCT GGT TGC-3' and 5'-TTC TTC AGA GGA CAT ACA CTG-3', and G6PD, 5'-CTG GCG TAT TTT CAC CCC AC-3' and 5'-GGT TCA CCC ACT TGT AGG TG-3'. The primer pair for CD62E was identified using the Gene Works software release 2.3.1, purchased from Intelligenetics (Mountain View, CA). Thereafter, a second reaction mix containing Taq polymerase, Taq polymerase buffer, both obtained from Promega, and 3 µl from the first strand cDNA synthesis reaction in a total volume of 50 µl was added to the tube. The final concentration of the reagents was 50 mM KCl, 10 mM Tris Cl (pH 9), 0.1% Triton X-100, 1.5 mM MgCl2, 100 µM each dNTP, 50 pmol each primer, and 2 U Taq polymerase. The cycle program consisted of one cycle at 95°C for 5 min, 56–58°C for 30 s (depending on the melting temperature of the individual primer pair), 72°C for 1 min, followed by 30 cycles of 94°C for 1 min, 56–58°C for 30 s, 72°C for 1 min, followed by a 5-min primer extension time at 72°C. Aliquots of the PCR products were analyzed on a 1.5% agarose gel.

Southern blot analysis

PCR products were transferred by alkaline transfer onto a {zeta}-Probe GT membrane obtained from Bio-Rad (Hercules, CA), as previously described (15). Blots were incubated for at least 1 h at 52°C with Rapid-hyb buffer, purchased from Amersham Life Science (Amersham, U.K.). Amplified target cDNA was detected by incubation of the blots at 52°C for at least 2 h in Rapid-hyb buffer containing target DNA-specific {gamma}-32P-labeled probes. Probes were labeled for oligo end labeling, as previously described (15), using the T4 polynucleotide kinase obtained from Promega. Specific probes for CD62E (5'-GTA CCA ATA CAT CCT GCA GTG G-3') and G6PD (5'-ATT GAC CTC AGC TGC ACA TTC C-3') were prepared using the Gene Works software (release 2.3.1).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Migrating CD4+ T cells are enriched in CD54+ T cells

Compared with nonmigrated T cells, migrated, resting T cells (Fig. 1GoA), and resting T cells that had migrated through a TNF-{alpha}-stimulated EC monolayer (Fig. 1GoB), CD4+ T cells that had been activated by PDB were greatly enriched in CD54+ cells after migrating through resting (Fig. 1GoC) or TNF-{alpha}-stimulated EC monolayers (Fig. 1GoD). Moreover, greater numbers of CD54+ T cells were recovered than were contained in the initial CD4+ T cell population (3.9 ± 1.3 x 106 initial CD54+ T cells, 5.8 ± 1.6 x 106 recovered CD54+ T cells), indicating that CD54 had been induced or acquired from the EC. The next experiments addressed these possibilities.



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  		FIGURE 1. Expression of CD54 by initial, nonadherent, and migrated CD4+ T cells. Control (resting) (A, B) or PDB-activated (C, D) CD4+ T cells were incubated (4 h, 37°C) with resting (A, C) or TNF-{alpha}-activated (B, D) EC. Subsequently, nonadherent and migrated T cells were isolated and analyzed by flow cytometry using indirect immunofluorescence to detect CD54. Results are expressed as the percentage of T cells in the indicated populations that expressed CD54 (n = 6).

 
Highly purified CD4+ T cells were depleted of CD54-expressing cells and then activated with PDB, in the absence or presence of the RNA synthesis inhibitor, actinomycin D. As shown in Fig. 2GoA, 11% of the control CD4+CD54- T cells expressed CD54, and 98% of the CD4+CD54- T cells expressed CD69 when activated in the absence of actinomycin D. In contrast, when actinomycin D-treated CD4+CD54- T cells were activated, no CD54 expression was induced. In contrast, CD69, which can be up-regulated without de novo protein synthesis (16), was induced, but only by 35% of the activated T cells. Of importance after migration through EC, 32% of the CD4+CD54- T cells activated with PDB in the presence of actinomycin D became CD54+ (Fig. 2GoB). These findings indicate that migration did not induce CD54 expression by CD4+ T cells, but rather that the migrating CD4+ T cells acquired CD54 from the EC during the migration process (Fig. 2Go).



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  		FIGURE 2. A, Acquisition of CD54 by migrating PDB-activated CD4+ T cells is not caused by de novo synthesis of CD54 by T cells. CD4+CD54- T cells isolated by negative selection were treated with control medium (upper panel) or actinomycin D (10 ng/ml) (lower panel), after which time they were stimulated with PDB (50 ng/ml, 1 h, 37°C). Subsequently, the cells were analyzed for CD3 (OKT3-FITC, x-axis) and CD54 (R6.5-PE, y-axis) expression (left panels) or CD3 (x-axis) and CD69 (Leu23-PE, y-axis) (right panels). B, Actinomycin D-treated, PDB-activated CD4+CD54- T cells were incubated with TNF-{alpha}-activated EC (4 h, 37°C), after which time nonadherent and migrated T cells were recovered and analyzed by flow cytometry using direct immunofluorescence to detect CD3 (OKT3-FITC, x-axis) and CD54 (R6.5-PE, y-axis).

 
PDB-activated CD4+ T cells acquire CD31 and CD49b as a result of migration

As shown in Fig. 3Go, A and B, migrating PDB-activated CD4+ T cells contained an increased percentage of CD31+ and CD49b+ cells, compared with that found in migrating resting CD4+ T cells. Similar results were found when PDB-activated CD4+ T cells migrated through TNF-{alpha}-activated EC, whereas resting CD4+ T cells remained largely CD31- and CD49b- after migrating through activated EC. It should be noted that although the activation period was relatively short (18 h), it resulted in a small increase in the percentage of CD49b+ cells in the initial population, whereas the expression of CD31 remained unchanged by PDB activation.



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  		FIGURE 3. Expression of CD31 (A) and CD49b (B) by initial, nonadherent, and migrating CD4+ T cells. Control (resting) or PDB-activated CD4+ T cells were incubated (4 h, 37°C) with resting or TNF-{alpha}-activated EC. Subsequently, nonadherent and migrated T cells were recovered and analyzed by flow cytometry using indirect immunofluorescence to detect CD31 and CD49b. Staining with an isotype-matched control mAb also is shown (thin line). Results representative of five (CD49b) and six (CD31) experiments are shown.

 
PDB-activated CD4+ T cells express CD62E and CD61 after transendothelial migration

After transendothelial migration through either activated or resting EC, PDB-activated CD4+ T cells also acquired CD62E and CD61 (Fig. 4Go and data not shown). In contrast, minimal expression of either CD62E or CD61 was noted by migrating resting CD4+ T cells. Only when resting CD4+ T cells migrated through TNF-{alpha}-activated EC was some CD62E acquired (Fig. 4Go, B compared with A), but to a lesser extent than when PDB-activated T cells migrated through either resting or TNF-{alpha}-activated EC (Fig. 4Go, C and D, respectively). Whereas 14.1 ± 1.4% and 28.6 ± 3.7% of resting T cells that had migrated through resting and TNF-{alpha}-activated EC monolayers, respectively, expressed CD62E, 35.1 ± 9.1% and 64 ± 5.1% of PDB-stimulated T cells that had migrated through resting and TNF-{alpha}-activated EC, respectively, were CD62E positive. Similar results were obtained regardless of whether anti-CD62E mAb CL3, CL37, or 1.2B6 was used to detect CD62E on the T cells (data not shown).



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  		FIGURE 4. Migrating PDB-activated CD4+ T cells acquire CD62E. Control (resting) (A, B) or PDB-activated (C, D) CD4+ T cells were incubated (4 h, 37°C) with resting (A, C) or TNF-{alpha}-activated (B, D) EC. Subsequently, nonadherent and migrated T cells were recovered and analyzed by flow cytometry using indirect immunofluorescence with mAb to CD62E (1.2B6). Results are expressed as the percentage of T cells in the indicated populations that expressed CD62 (n = 8).

 
The initial CD4+ T cell population also did not express CD61, regardless of the activation status of the T cells. However, PDB-activated CD4+ T cells became greatly enriched in CD61+ cells after migrating through TNF-{alpha}-activated EC monolayers (data not shown). Similar results were found when PDB-activated T cells migrated through resting EC (data not shown). This is in contrast to experiments with activated EC and resting T cells in which CD61+ T cells were not found in the migrating population.

To ensure that EC markers were being detected on migrating CD4+ T cells, they were double stained for CD62E and CD3. As shown in Fig. 5Go, no initial CD3+CD62E+ T cells were found. The migrating T cell population, however, was greatly enriched in CD3, CD62E double-positive T cells.



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  		FIGURE 5. CD62E+ cells in the migrating population are CD3+ T cells. PDB-activated CD4+ T cells were incubated with TNF-{alpha}-activated EC (4 h, 37°C), after which time nonadherent and migrated T cells were recovered and analyzed by flow cytometry using a FITC-conjugated mAb to detect CD62E (x-axis) and a PE-conjugated mAb to detect CD3 (y-axis).

 
Migrating anti-CD3-activated CD4+ T cells acquire EC surface determinants

As shown in Fig. 6Go, the acquisition of EC membrane determinants was not unique to PDB-activated CD4+ T cells, inasmuch as anti-CD3-activated T cells migrating through TNF-{alpha}-activated EC were also greatly enriched in CD62E+ and CD61+ T cells compared with resting T cells that migrated through TNF-{alpha}-activated EC.



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  		FIGURE 6. Acquisition of CD62E and CD61 by migrated anti-CD3-activated CD4+ T cells. Following incubation in the presence or absence of immobilized anti-CD3 mAb (18 h, 37°C), anti-CD3-activated or control (resting) T cells were incubated with TNF-{alpha}-activated EC (4 h, 37°C), after which time nonadherent and migrated T cells were recovered and analyzed for the expression of CD62E and CD61.

 
CD4+ T cells do not express CD62E mRNA

To rule out the possibility that T cells are able to express CD62E, reverse-transcriptase PCR of highly purified resting and activated CD4+ T cells was performed. As shown in the upper panel of Fig. 7Go, neither resting (lane 2) nor PDB-activated (lane 3) CD4+ T cells express CD62E mRNA compared with resting EC (lane 4) and TNF-{alpha}-activated EC (lane 5). The lower panel of Fig. 7Go demonstrates that equal amounts of cDNA were loaded on the gel, as judged by the amplification of G6PD cDNA.



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  		FIGURE 7. Activated CD4+ T cells do not express CD62E mRNA. mRNA was isolated from resting and TNF-{alpha}-activated EC, as well as control (resting) and PDB-activated T cells (TC), after which time CD62E cDNA was amplified by reverse-transcriptase PCR and analyzed by Southern hybridization.

 
The association of CD62E with migrating PDB-activated CD4+ T cells occurs independently of the major active adhesion domain of CD62E

Additional experiments were conducted in which EC monolayers were labeled with FITC-conjugated anti-CD62E mAb directed to the ligand binding site of the molecule, to investigate whether transfer of mAb-labeled CD62E from EC to T cells would occur. As shown in Fig. 8Go, CD4+ T cells migrating through anti-CD62E FITC-labeled EC became greatly enriched in green fluorescent cells, indicating that transfer of labeled anti-CD62E mAb from EC to migrating T cells had occurred. In contrast, initial T cells and nonadherent T cells contained no cells expressing the anti-CD62E mAb. Since the anti-CD62E mAb was directed to the major ligand binding site (17), the results imply that transfer did not involve binding of CD62E to a known ligand on the activated T cell.



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  		FIGURE 8. The association of CD62E with migrated PDB-activated CD4+ T cells occurs independently of the ligand binding domain of CD62E. TNF-{alpha}-activated EC were incubated with FITC-conjugated anti-CD62E mAb (1.2B6) that has adhesion-blocking activity or an isotype-matched FITC-conjugated control mAb (30 min, 37°C), after which time the monolayers were washed and incubated with PDB-activated CD4+ T cells. Initial CD4+ T cells (thin lined histogram) and the recovered nonadherent and migrated T cell populations were analyzed by flow cytometry for FITC fluorescence.

 
The acquisition of EC surface determinants by migrating CD4+ T cells is a property of activated, and not resting T cells

To determine whether the ability to acquire EC-specific surface determinants is a characteristic of activated rather than resting CD4+ T cells, PDB-activated T cells (CD69+) and resting T cells (CD69-) were allowed to migrate simultaneously through TNF-{alpha}-activated EC. As shown in Fig. 9Go, only activated, CD69+ T cells acquired CD62E surface expression. Even the simultaneous presence of activated T cells did not induce CD62E acquisition by resting CD69- T cells.



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  		FIGURE 9. Acquisition of CD62E by migrated CD4+ T cells is a property of activated, but not resting T cells. PDB-activated CD69+, or resting CD69-CD4+ T cells, or a mixture of both resting and activated CD4+ T cells were incubated with resting or TNF-{alpha}-activated EC (4 h, 37°C), after which time migrated T cells were recovered and analyzed by flow cytometry using a FITC-conjugated mAb to detect CD62E (x-axis) and a PE-conjugated mAb to detect CD69 (y-axis).

 
The appearance of EC-specific determinants on the surface of PDB-activated T cells is associated with the transfer of EC cell membranes

To delineate whether EC surface marker acquisition by PDB-activated T cells was associated with membrane transfer, migration was examined using EC that were prelabeled with the lipophilic dye, DiOC-16. Once this dye is bound to membrane lipids, it does not reenter aqueous solution (18). Initial, nonadherent, and migrating T cells were stained with PE-conjugated anti-CD3 mAb and analyzed for the appearance of DiOC-16 fluorescence. As shown in Fig. 10Go, migrating PDB-activated T cells were greatly enriched in DiOC-16 fluorescence compared with migrating resting T cells. A much smaller number of nonmigrating PDB-activated T cells also were found to have acquired DiOC-16 fluorescence. In contrast, nonmigrating resting T cells were devoid of DiOC-16 fluorescence. Additional experiments were conducted to determine whether EC-specific transmembrane proteins acquired by migrating T cells were transferred in association with EC membranes. As shown in Fig. 11Go, most (70%) activated migrating T cells that acquired CD62E from EC also became positive for DiOC-16 fluorescence. In contrast, migrating resting T cells showed only minimal acquisition of CD62E and a modest increase in DiOC-16 fluorescent cells.



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  		FIGURE 10. Migrated PDB-activated CD4+ T cells acquire endothelial membranes prelabeled with the lipophilic dye, DiOC-16. Resting or TNF-{alpha}-activated EC were incubated with DiOC-16 (5 mg/ml, 30 min, 37°C), washed extensively, and than incubated with control (resting) or PDB-activated CD4+ T cells. Subsequently, the T cells in the initial, nonadherent, and migrated populations were recovered and stained with a PE-conjugated anti-CD3 mAb and analyzed by flow cytometry for the expression of DiOC-16 (green fluorescence, x-axis) and CD3 (red fluorescence, y-axis).

 


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  		FIGURE 11. Migrated PDB-activated CD4+ T cells simultaneously acquire CD62E and endothelial membranes prelabeled with DiOC-16. Resting and TNF-{alpha}-activated EC were incubated with DiOC-16 (5 mg/ml, 30 min, 37°C), washed extensively, and than incubated with control (resting) or PDB-activated CD4+ T cells. Subsequently, T cells in the initial nonadherent and migrated populations were recovered and stained with a PE-conjugated anti-CD62E mAb and analyzed by flow cytometry for the expression of DiOC-16 (green fluorescence, x-axis) and CD62E (red fluorescence, y-axis).

 
Activated CD4+ T cells induce programmed cell death in human EC

Since apoptosis is accompanied by plasma membrane remodeling, including redistribution of phospholipids and membrane blebbing (13), EC were analyzed to determine whether they were induced to undergo apoptosis after the migration of activated CD4+ T cells. As shown in Fig. 12Go, the low level of apoptosis occurring in resting EC was negligibly affected by the transendothelial migration of resting T cells (11 and 15% apoptosis of resting EC in the absence and presence of resting T cells, respectively). TNF-{alpha} slightly increased endothelial apoptosis that was not affected by the transendothelial migration of resting T cells (19 and 22% apoptosis of TNF-{alpha}-activated EC in the absence and presence of resting T cells, respectively). In contrast, apoptosis of EC was markedly increased by the transmigration of activated CD4+ T cells. This was observed regardless of the activation state of the endothelium (34 and 42% apoptosis of resting and TNF-{alpha}-activated EC, respectively, in the presence of activated T cells) (Fig. 12Go).



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  		FIGURE 12. Activated CD4+ T cells induce programmed cell death in human EC. Control (resting) or PDB-activated CD4+ T cells were incubated (3 h, 37°C) with resting (open bar) or TNF-{alpha}-activated (hatched bar) EC, after which time the EC were isolated and analyzed for apoptosis by flow cytometry using propidium iodide.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of the current studies provide a number of new insights into the nature of the interactions of activated CD4+ T cells with EC, and imply that both the phenotype of the T cell and the physiology of the EC are modified as a result of these interactions. We conclude that during the transendothelial migration of activated CD4+ T cells, there is transfer to the T cells of endothelial membranes and surface proteins. Migrating resting CD4+ T cells do not acquire membrane determinants, even if activated CD4+ T cells are also present. Moreover, activated or resting T cells that do not bind to or migrate through endothelium acquire markedly less EC membrane determinants. Finally, acquisition of endothelial constituents by migrating activated CD4+ T cells appears to be independent of the activation state of the EC, although additional molecules expressed by TNF-{alpha}-activated EC appear to also be transferred when activated CD4+ T cells migrated through activated EC.

A number of different observations are consistent with the conclusion that activated migrating CD4+ T cells acquired endothelial surface molecules, including CD31, CD49b, CD54, CD61, and CD62E. Thus, greater numbers of CD4+CD54+ T cells were recovered at the end of the experiments than were present in the initial T cell population. In addition, the appearance of CD54 on activated migrating CD54-CD4+ T cells supports the conclusion that endothelial-specific determinants were acquired by transfer from the EC, as this occurred when T cell RNA synthesis was inhibited. Moreover, molecules such as CD61 and CD62E are not expressed by T cells, but double staining demonstrated that migrating CD3+ T cells expressed both CD61 and CD62E. The data, therefore, indicate that during the process of transendothelial migration, activated CD4+ T cells are able to acquire a variety of surface determinants from EC. That transfer apparently occurs without specificity is suggested by the variety of endothelial molecules acquired by the migrating activated T cells. It should be pointed out that CD106 (VCAM-1) expression could not be examined because staining for this molecule was ineffective following collagenase digestion used to recover migrated CD4+ T cells.

Inasmuch as EC are able to generate soluble forms of CD54 and CD62E (19), it was conceivable that activated CD4+ T cells bound soluble endothelial molecules by means of counter-receptors, such as CD11a/CD18 (LFA-1) and cutaneous lymphocyte Ag. There are, however, several observations that indicate that this possibility is unlikely. First, CD54 was detected on migrated T cells with a mAb, R6.5, that blocks binding of both CD11a/CD18 and CD11b/CD18 (Mac-1) (20). Therefore, if CD54 were bound to either of these counter-receptors, it would not have been detected by R6.5. In addition, blocking mAb against CD11a and CD11b were not able to inhibit the acquisition of CD54 by migrating T cells (data not shown). CD62E acquisition was also detected with a mAb to the lectin domain, the major ligand binding site of this molecule (21). Moreover, prelabeling EC with mAb to the binding site on CD62E did not affect the transfer of EC-derived CD62E to migrating activated CD4+ T cells. In addition, Western blot analysis of membrane preparations of migrating activated CD4+ T cells indicated that the T cells had acquired the 115-kDa full-length transmembrane form of CD62E, and not the truncated soluble form of the molecule (data not shown) (19). Finally, migrating T cells acquired molecules, such as CD49b, for which they have no known receptors. These results are all consistent with the conclusion that counter-receptor-mediated acquisition of cleaved endothelial surface molecules by activated T cells could not explain the current results. Rather, the data are more consistent with the conclusion that acquisition of endothelial markers by migrating activated CD4+ T cells results from the transfer of membrane fragments or vesicles containing intact transmembrane surface molecules oriented in a right-side-out manner that permitted detection with mAb to extracellular domains, including those directed to known ligand binding sites.

These conclusions are supported by experiments with DiOC-16-labeled EC monolayers, the results of which showed an association between the transfer of EC plasma membrane lipids and the appearance of endothelial-specific membrane proteins on the surface of PDB-activated T cells. A number of reports have described the release of plasma membrane constituents by a variety of cell types, including EC, in response to injury or stress or as part of normal metabolic processes (22). Ectocytosis, as this has been designated (12), involves the shedding of plasma membrane fragments or right-side-out vesicles in response to such perturbations. This response can have a positive effect on cells by permitting them to overcome injury by modification of their plasma membranes. It also may be an indication of injury and impending programmed cell death, however, since vesiculation is an early feature of the cellular response to stimuli that induce apoptosis (13). Whether occurring in the context of ectocytosis or apoptosis, vesiculation by EC appears to play a role in the transfer of endothelial membranes and proteins to migrating activated T cells.

The nature of the endothelial response to transmigrating CD4+ T cells may be varied. Thus, at the early time points measured by the current studies, apoptosis clearly was increased following the transendothelial migration of activated T cells. Inasmuch as membrane blebbing and release are very early features of apoptosis and also may occur as a normal cell repair mechanism, it is uncertain whether EC stimulated to undergo programmed cell death by interaction with activated CD4+ T cells were the sole source of transferred membrane constituents. It is possible that EC membrane vesicles elaborated constitutively, or by EC undergoing reparative membrane reorganization in response to transmigrating activated CD4+ T cells, were the source of some or all of the membrane material transferred to migrating activated CD4+ T cells.

In a subset of EC, it seems likely that apoptosis resulted from their interaction with transmigrating activated CD4+ T cells. The precise mechanism by which programmed cell death is induced by EC that support transendothelial migration of CD4+ T cells is uncertain at the present time. Activated CD4+ T cells can elaborate TNF-{alpha}, and TNF-{alpha} is known to induce apoptosis of EC (23, 24). However, TNF-{alpha}-induced apoptosis of EC was not evident until at least 10 h of exposure to TNF-{alpha} (24). Since apoptosis was found to be induced within 4 h of contact with activated CD4+ T cells, it is not likely that T cell-derived TNF-{alpha} alone induced EC apoptosis. Indeed, in the current studies, large concentrations of TNF-{alpha} induced only modest apoptosis of EC, and that was markedly enhanced by activated CD4+ T cells. Thus, it is likely that other cytokines and/or membrane receptor interactions might be involved in the induction of EC apoptosis. Although the nature of these molecules is unknown at this time, EC are known to express CD95 (Fas), and apoptosis of EC was noted following the ligation of receptors for thrombospondin and oxidized low density lipoprotein (25, 26, 27). It is unknown whether any of these is involved in apoptosis of EC induced by activated CD4+ T cells. Fas-Fas ligand interactions are an unlikely candidate, however, since fresh T cells activated with phorbol ester alone express minimal Fas ligand, and ligation of CD95 alone on EC is insufficient to induce apoptosis. Signaling through other EC surface molecules is likely to be necessary for activated CD4+ T cells to induce this programmed cell death of EC.

It is noteworthy that following T cell migration through resting endothelium, a subset of EC became CD62E+, presumably as a result of interactions with activated CD4+ T cells (data not shown). Whether this was mediated by ligation of specific receptors and/or soluble mediators is uncertain at this time. IL-3, TNF-{alpha}, and IFN-{gamma}, which are all produced by activated T cells, have been shown to stimulate CD62E synthesis by EC (10, 28, 29). Moreover, reports have indicated that monocyte interactions with EC induce endothelial expression of CD62E (30). In addition, agonists of protein kinase C as well as neuropeptides, bacterial endotoxin, and Plasmodium falciparum similarly induce endothelial CD62E expression (31, 32, 33, 34). Thus, the results of the current studies imply that activated CD4+ T cells can induce EC to express CD62E, as has been noted previously (35, 36), that can be transferred during transendothelial migration.

That only activated and not resting T cells acquired endothelial determinants suggests that activated CD4+ T cells have a unique capacity to bind endothelial membranes. It is unlikely that acquisition was merely the result of the ability of the T cells to induce endothelial plasma membrane shedding, as transendothelial migration of a combination of resting and activated CD4+ T cells resulted in acquisition of endothelial determinants only by the migrating activated T cells. Previous studies have identified the phenotype of the major CD4+ T cell subset that exhibits a transendothelial migratory capacity as being CD45RO+, CD45RA-, CD26bright, CD11abright, and CD44bright (2). This is the major subset of migratory CD4+ T cells, regardless of activation status. Since the major migratory CD4+ T cell population is similar regardless of the activation state, and since migrating resting CD4+ T cells do not acquire endothelial determinants, the data suggest that activation events induce this capacity in CD4+ T cells. Thus, an additional property of activated CD4+ T cells that exhibit a capacity for transendothelial migration appears to be their ability to bind endothelial plasma membrane fragments or vesicles. Taken together, the results suggest that the acquisition of endothelial membranes requires intimate contact with the plasma membrane fragments or vesicles, as is likely to occur during transendothelial migration. Whether a unique set of receptors expressed by activated, but not resting, CD4+ T cells is involved in the acquisition of EC membrane receptors is unknown.

At this time, the initiating events in the development of inflammatory disease are not known. Some studies have suggested that an early event may involve disruption of the vascular endothelium, although the precise nature of the insult has not been identified (37, 38). It is intriguing to speculate that transendothelial migration of activated CD4+ T cells may play a role in the initial insult. Thus, changes in the endothelium resulting from the induction of programmed cell death may cause disruption of the vasculature. This could contribute to the admission of both inflammatory cells and also proinflammatory serum components into the tissue.

During transendothelial migration, activated CD4+ T cells might also acquire EC membrane constituents and surface proteins. This may provide a mechanism to deliver endothelial determinants to perivascular tissues. Thus, by arming extravasating T cells with molecules previously shown to be involved in B cell and macrophage costimulation (CD54) (39), endothelial angiogenesis (CD62E) (40), and extracellular matrix organization (CD61 and CD49b) (41), T cells at the expense of the endothelium may influence the organization, activity, and cellular content of chronically inflamed tissues.


   Acknowledgments
 
We thank Drs. Dorian Haskard, C. Wayne Smith, and Robert Rothlein for generously providing some of the mAbs used in these studies.


   Footnotes
 
1 This work was supported by Grants AR39169 and AR/OD420 from the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Peter E. Lipsky, Department of Internal Medicine, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75235-8884. E-mail address: Back

3 Abbreviations used in this paper: EC, endothelial cell; DiOC-16, 3,3'-dihexadecyloxacarbocyanine perchlorate; HS, human serum; PDB, phorbol dibutyrate; PE, phycoerythrin. Back

Received for publication July 8, 1998. Accepted for publication September 28, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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