Download Transendothelial Migration Surface Determinants During Activated T

Activated T Cells Acquire Endothelial Cell
Surface Determinants During
Transendothelial Migration
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of June 18, 2017.
Ruth I. Brezinschek, Nancy Oppenheimer-Marks and Peter E.
Lipsky
J Immunol 1999; 162:1677-1684; ;
http://www.jimmunol.org/content/162/3/1677
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References
Activated T Cells Acquire Endothelial Cell Surface
Determinants During Transendothelial Migration1
Ruth I. Brezinschek, Nancy Oppenheimer-Marks, and Peter E. Lipsky2
E
xtravasation 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 CD41 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– 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 CD41 T
cells migrate through endothelial barriers more effectively than
resting CD41 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 CD41 T cells
acquire endothelial determinants during their transendothelial migration, regardless of the activation state of the endothelium.
Materials and Methods
Department of Internal Medicine, University of Texas Southwestern Medical Center,
Dallas, TX 75235
Received for publication July 8, 1998. Accepted for publication September 28, 1998.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by Grants AR39169 and AR/OD420 from the National
Institutes of Health.
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: peter.lipsky@
email.swmed.edu
3
Abbreviations used in this paper: EC, endothelial cell; DiOC-16, 3,39-dihexadecyloxacarbocyanine perchlorate; HS, human serum; PDB, phorbol dibutyrate; PE,
phycoerythrin.
Copyright © 1999 by The American Association of Immunologists
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 mg/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 mg/ml EC growth supplement, and 50 mg/ml ascorbic acid, purchased from Sigma, as previously described (9).
0022-1767/99/$02.00
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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 CD41 T cells migrated through confluent monolayers of
HUVEC. Flow cytometry of migrated CD41 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-a-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. The Journal
of Immunology, 1999, 162: 1677–1684.
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MIGRATING ACTIVATED T CELLS ACQUIRE ENDOTHELIAL CELL DETERMINANTS
Preparation of CD41 T cells
T cells were prepared by density-gradient centrifugation, adherent cell depletion, and passage over a nylon wool column, as previously described
(9). CD41 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 CD41 T cells prepared
by this method was always greater than 95%. The purified CD41 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).
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
CD41 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, 220°C, 18 h), washed in cold PBS, and then
resuspended in propidium iodide stain mixture containing 50 mg/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.
Assay of transendothelial migration
Analysis of CD41 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
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 3
106 TNF-a-activated or resting EC, as well as from 2 3 106 PDB activated
or resting CD41 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 ml) 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-ml reaction mix containing
MgCl2, obtained from Promega (Madison, MI), all four dNTPs purchased
from Sigma, and specific primers for CD62E, 59-AAG GTA CAC ACA
CCT GGT TGC-39 and 59-TTC TTC AGA GGA CAT ACA CTG-39, and
G6PD, 59-CTG GCG TAT TTT CAC CCC AC-39 and 59-GGT TCA CCC
ACT TGT AGG TG-39. 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 ml
from the first strand cDNA synthesis reaction in a total volume of 50 ml
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 mM
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 z-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 g-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 (59-GTA CCA ATA CAT CCT GCA GTG G-39) and
G6PD (59-ATT GAC CTC AGC TGC ACA TTC C-39) were prepared
using the Gene Works software (release 2.3.1).
Results
Migrating CD41 T cells are enriched in CD541 T cells
Compared with nonmigrated T cells, migrated, resting T cells (Fig.
1A), and resting T cells that had migrated through a TNF-a-stimulated EC monolayer (Fig. 1B), CD41 T cells that had been activated by PDB were greatly enriched in CD541 cells after migrating through resting (Fig. 1C) or TNF-a-stimulated EC monolayers
(Fig. 1D). Moreover, greater numbers of CD541 T cells were recovered than were contained in the initial CD41 T cell population
(3.9 6 1.3 3 106 initial CD541 T cells, 5.8 6 1.6 3 106 recovered
CD541 T cells), indicating that CD54 had been induced or acquired from the EC. The next experiments addressed these
possibilities.
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The transendothelial migration assay was performed as previously described (2). Briefly, in the wells of 36-mm macrowell tissue culture plates,
EC (4 3 106) 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-a (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 CD41 T cells (10 3 106), resuspended in 0.6% BSA in
RPMI (RPMI/BSA), were incubated with the EC for 4 h at 37°C. Subsequently, nonadherent and migrated CD41 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 CD41 T cells. In other experiments, CD41 T cells were depleted of
CD41/CD541 T cells by a negative selection using the mAb R6.5 (antiCD54), 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 mg/ml), purchased from Sigma, activated with
PDB (50 ng/ml, 60 min), washed, and then added to EC monolayers.
The Journal of Immunology
1679
Highly purified CD41 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.
2A, 11% of the control CD41CD542 T cells expressed CD54, and
98% of the CD41CD542 T cells expressed CD69 when activated
in the absence of actinomycin D. In contrast, when actinomycin
D-treated CD41CD542 T cells were activated, no CD54 expression was induced. In contrast, CD69, which can be up-regulated
FIGURE 3. Expression of CD31 (A) and CD49b (B) by initial, nonadherent, and migrating CD41 T cells. Control (resting) or PDB-activated
CD41 T cells were incubated (4 h, 37°C) with resting or TNF-a-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.
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 CD41CD542 T cells activated with PDB
in the presence of actinomycin D became CD541 (Fig. 2B). These
findings indicate that migration did not induce CD54 expression by
CD41 T cells, but rather that the migrating CD41 T cells acquired
CD54 from the EC during the migration process (Fig. 2).
FIGURE 2. A, Acquisition of CD54 by migrating PDB-activated CD41
T cells is not caused by de novo synthesis of CD54 by T cells.
CD41CD542 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 CD41CD542 T cells were incubated with TNF-a-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 CD41 T cells acquire CD31 and CD49b as a
result of migration
As shown in Fig. 3, A and B, migrating PDB-activated CD41 T
cells contained an increased percentage of CD311 and CD49b1
cells, compared with that found in migrating resting CD41 T cells.
Similar results were found when PDB-activated CD41 T cells migrated through TNF-a-activated EC, whereas resting CD41 T cells
remained largely CD312 and CD49b2 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
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FIGURE 1. Expression of CD54 by initial, nonadherent, and migrated
CD41 T cells. Control (resting) (A, B) or PDB-activated (C, D) CD41 T
cells were incubated (4 h, 37°C) with resting (A, C) or TNF-a-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 5 6).
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MIGRATING ACTIVATED T CELLS ACQUIRE ENDOTHELIAL CELL DETERMINANTS
FIGURE 5. CD62E1 cells in the migrating population are CD31 T
cells. PDB-activated CD41 T cells were incubated with TNF-a-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).
percentage of CD49b1 cells in the initial population, whereas the
expression of CD31 remained unchanged by PDB activation.
PDB-activated CD41 T cells express CD62E and CD61 after
transendothelial migration
After transendothelial migration through either activated or resting
EC, PDB-activated CD41 T cells also acquired CD62E and CD61
(Fig. 4 and data not shown). In contrast, minimal expression of
either CD62E or CD61 was noted by migrating resting CD41 T
cells. Only when resting CD41 T cells migrated through TNF-aactivated EC was some CD62E acquired (Fig. 4, B compared with
A), but to a lesser extent than when PDB-activated T cells migrated
through either resting or TNF-a-activated EC (Fig. 4, C and D,
respectively). Whereas 14.1 6 1.4% and 28.6 6 3.7% of resting T
cells that had migrated through resting and TNF-a-activated EC
monolayers, respectively, expressed CD62E, 35.1 6 9.1% and
64 6 5.1% of PDB-stimulated T cells that had migrated through
resting and TNF-a-activated EC, respectively, were CD62E positive. Similar results were obtained regardless of whether antiCD62E mAb CL3, CL37, or 1.2B6 was used to detect CD62E on
the T cells (data not shown).
FIGURE 6. Acquisition of CD62E and CD61 by
migrated anti-CD3-activated CD41 T cells. Following incubation in the presence or absence of immobilized anti-CD3 mAb (18 h, 37°C), antiCD3-activated or control (resting) T cells were
incubated with TNF-a-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.
Migrating anti-CD3-activated CD41 T cells acquire EC surface
determinants
As shown in Fig. 6, the acquisition of EC membrane determinants was not unique to PDB-activated CD41 T cells, inasmuch
as anti-CD3-activated T cells migrating through TNF-a-activated EC were also greatly enriched in CD62E1 and CD611 T
cells compared with resting T cells that migrated through TNFa-activated EC.
CD41 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
CD41 T cells was performed. As shown in the upper panel of Fig.
7, neither resting (lane 2) nor PDB-activated (lane 3) CD41 T
cells express CD62E mRNA compared with resting EC (lane 4)
and TNF-a-activated EC (lane 5). The lower panel of Fig. 7 demonstrates that equal amounts of cDNA were loaded on the gel, as
judged by the amplification of G6PD cDNA.
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FIGURE 4. Migrating PDB-activated CD41 T cells acquire CD62E.
Control (resting) (A, B) or PDB-activated (C, D) CD41 T cells were incubated (4 h, 37°C) with resting (A, C) or TNF-a-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 5 8).
The initial CD41 T cell population also did not express CD61,
regardless of the activation status of the T cells. However, PDBactivated CD41 T cells became greatly enriched in CD611 cells
after migrating through TNF-a-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
CD611 T cells were not found in the migrating population.
To ensure that EC markers were being detected on migrating
CD41 T cells, they were double stained for CD62E and CD3. As
shown in Fig. 5, no initial CD31CD62E1 T cells were found. The
migrating T cell population, however, was greatly enriched in
CD3, CD62E double-positive T cells.
The Journal of Immunology
FIGURE 7. Activated CD41 T cells do not express CD62E mRNA.
mRNA was isolated from resting and TNF-a-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.
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. 8, CD41 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.
The acquisition of EC surface determinants by migrating CD41
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
CD41 T cells, PDB-activated T cells (CD691) and resting T cells
(CD692) were allowed to migrate simultaneously through TNF-
FIGURE 9. Acquisition of CD62E by migrated CD41 T cells is a property of activated, but not resting T cells. PDB-activated CD691, or resting
CD692CD41 T cells, or a mixture of both resting and activated CD41 T
cells were incubated with resting or TNF-a-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 PEconjugated mAb to detect CD69 (y-axis).
a-activated EC. As shown in Fig. 9, only activated, CD691 T cells
acquired CD62E surface expression. Even the simultaneous presence of activated T cells did not induce CD62E acquisition by
resting CD692 T cells.
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. 10, 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. 11, 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.
Activated CD41 T cells induce programmed cell death in
human EC
FIGURE 8. The association of CD62E with migrated PDB-activated
CD41 T cells occurs independently of the ligand binding domain of
CD62E. TNF-a-activated EC were incubated with FITC-conjugated antiCD62E mAb (1.2B6) that has adhesion-blocking activity or an isotypematched FITC-conjugated control mAb (30 min, 37°C), after which time
the monolayers were washed and incubated with PDB-activated CD41 T
cells. Initial CD41 T cells (thin lined histogram) and the recovered nonadherent and migrated T cell populations were analyzed by flow cytometry
for FITC fluorescence.
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 CD41 T cells.
As shown in Fig. 12, 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-a slightly increased endothelial apoptosis that was not affected by the transendothelial migration of resting T cells (19 and 22% apoptosis of
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The association of CD62E with migrating PDB-activated CD41
T cells occurs independently of the major active adhesion
domain of CD62E
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MIGRATING ACTIVATED T CELLS ACQUIRE ENDOTHELIAL CELL DETERMINANTS
TNF-a-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 CD41 T cells. This was observed regardless of the activation state of the endothelium (34 and
42% apoptosis of resting and TNF-a-activated EC, respectively, in
the presence of activated T cells) (Fig. 12).
Discussion
The results of the current studies provide a number of new insights
into the nature of the interactions of activated CD41 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
CD41 T cells, there is transfer to the T cells of endothelial membranes and surface proteins. Migrating resting CD41 T cells do not
acquire membrane determinants, even if activated CD41 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 CD41 T cells appears to be independent of the activation state of the EC, although additional molecules expressed by TNF-a-activated EC appear to also be
transferred when activated CD41 T cells migrated through
activated EC.
A number of different observations are consistent with the conclusion that activated migrating CD41 T cells acquired endothelial
surface molecules, including CD31, CD49b, CD54, CD61, and
CD62E. Thus, greater numbers of CD41CD541 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 CD542CD41 T cells supports the conclusion
that endothelial-specific determinants were acquired by transfer
FIGURE 11. Migrated PDB-activated CD41 T cells simultaneously acquire CD62E and endothelial membranes prelabeled with DiOC-16. Resting and TNF-a-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 CD41 T cells. Subsequently, T cells in the initial nonadherent and migrated populations were recovered and stained with a PEconjugated anti-CD62E mAb and analyzed by flow cytometry for the expression of DiOC-16 (green fluorescence, x-axis) and CD62E (red
fluorescence, y-axis).
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 CD31 T cells expressed both CD61 and CD62E. The data,
therefore, indicate that during the process of transendothelial migration, activated CD41 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 CD41 T cells.
FIGURE 12. Activated CD41 T cells induce programmed cell death in
human EC. Control (resting) or PDB-activated CD41 T cells were incubated (3 h, 37°C) with resting (open bar) or TNF-a-activated (hatched bar)
EC, after which time the EC were isolated and analyzed for apoptosis by
flow cytometry using propidium iodide.
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FIGURE 10. Migrated PDB-activated CD41 T cells acquire endothelial
membranes prelabeled with the lipophilic dye, DiOC-16. Resting or TNFa-activated EC were incubated with DiOC-16 (5 mg/ml, 30 min, 37°C),
washed extensively, and than incubated with control (resting) or PDBactivated CD41 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).
The Journal of Immunology
In a subset of EC, it seems likely that apoptosis resulted from
their interaction with transmigrating activated CD41 T cells. The
precise mechanism by which programmed cell death is induced by
EC that support transendothelial migration of CD41 T cells is
uncertain at the present time. Activated CD41 T cells can elaborate TNF-a, and TNF-a is known to induce apoptosis of EC (23,
24). However, TNF-a-induced apoptosis of EC was not evident
until at least 10 h of exposure to TNF-a (24). Since apoptosis was
found to be induced within 4 h of contact with activated CD41 T
cells, it is not likely that T cell-derived TNF-a alone induced EC
apoptosis. Indeed, in the current studies, large concentrations of
TNF-a induced only modest apoptosis of EC, and that was markedly enhanced by activated CD41 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 (25–27). It is unknown whether any of these is involved in
apoptosis of EC induced by activated CD41 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 CD41 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 CD62E1, presumably as a
result of interactions with activated CD41 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-a, and IFN-g, 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 (31–34). Thus, the results of the current studies
imply that activated CD41 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 CD41 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 CD41 T cells resulted in acquisition of endothelial determinants only by the migrating activated T cells. Previous studies have identified the
phenotype of the major CD41 T cell subset that exhibits a transendothelial migratory capacity as being CD45RO1, CD45RA2,
CD26bright, CD11abright, and CD44bright (2). This is the major subset of migratory CD41 T cells, regardless of activation status.
Since the major migratory CD41 T cell population is similar regardless of the activation state, and since migrating resting CD41
T cells do not acquire endothelial determinants, the data suggest
that activation events induce this capacity in CD41 T cells. Thus,
an additional property of activated CD41 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
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Inasmuch as EC are able to generate soluble forms of CD54 and
CD62E (19), it was conceivable that activated CD41 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 CD41 T cells. In addition, Western blot analysis
of membrane preparations of migrating activated CD41 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 CD41 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 DiOC16-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 CD41
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 CD41 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 CD41 T cells, were the source of some or all of the
membrane material transferred to migrating activated CD41 T
cells.
1683
1684
MIGRATING ACTIVATED T CELLS ACQUIRE ENDOTHELIAL CELL DETERMINANTS
17.
18.
19.
20.
21.
22.
23.
24.
25.
Acknowledgments
26.
We thank Drs. Dorian Haskard, C. Wayne Smith, and Robert Rothlein for
generously providing some of the mAbs used in these studies.
27.
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migration. Whether a unique set of receptors expressed by activated, but not resting, CD41 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 CD41 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 CD41 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.