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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 3 (February 1), 1999:
pp. 886-896
Activation of Peripheral Blood T Cells by Interaction and Migration
Through Endothelium: Role of Lymphocyte Function
Antigen-1/Intercellular Adhesion Molecule-1 and Interleukin-15
By
David Sancho,
María Yáñez-Mó,
Reyes Tejedor, and
Francisco Sánchez-Madrid
From the Servicio de Inmulogía, Hospital de la Princesa,
Universidad Autónoma de Madrid, Madrid, Spain.
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ABSTRACT |
Cell adhesion molecules have a key role in the migration of T cells
to inflammatory foci. However, the effect of the endothelial-lymphocyte interaction on the activation of the latter cells remains unresolved. We have studied the effect of resting and stimulated endothelial cells
(ECs) on the activation of peripheral blood T cells (PBTLs), as
assessed by the expression of CD69 and CD25 activation antigens. The
incubation of PBTLs with tumor necrosis factor- -activated EC
monolayers, either alive or fixed, induced the expression of CD69 but
not CD25, preferentially in the CD8+
CD45RO+ cell subset. Furthermore, it induced the
production of cytokines such as IFN- , but not that of interleukin-2
(IL-2) and IL-4. EC treated with other stimuli such as IL-1 ,
IFN-, or lipopolysaccharide also showed the same proactivatory
effect on T cells. Lymphocyte activation was almost completely
inhibited by blocking anti-CD18 and anti-intercellular adhesion
molecule-1 (anti-ICAM-1) monoclonal antibodies (MoAbs), but only
slightly affected by MoAbs against CD49d, vascular cell adhesion
molecule-1, and anti-IL-15. In addition, the interaction of PBTL with
immobilized ICAM-1 induced CD69 expression in the same memory T-cell
subset. IL-15 induced T-cell activation with expression of CD69 and
CD25, and production of IFN-, and its effect was additive with that
triggered by cell adhesion to either EC or immobilized ICAM-1. The
transmigration of PBTLs through either confluent EC monolayers or
ICAM-1-coated membranes also induced efficiently the expression of
CD69. When IL-15 was used as chemoattractant in these assays, a further
enhancement in CD69 expression was observed in migrated cells. Together
these results indicate that stimulated endothelium may have an
important role in T-cell activation, through the lymphocyte function
antigen-1/ICAM-1 pathway, and that IL-15 efficiently cooperates in this
phenomenon. These observations could account for the abundance of
CD69+ cells in the lymphocytic infiltrates of several
chronic inflammatory diseases.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE DEVELOPMENT AND progression of
chronic, immune-mediated inflammatory diseases is dependent on the
persistent infiltration of mononuclear cells into the affected
tissues.1 Although different cell types have been
identified in inflammatory infiltrates, T cells seem to play
a central role in the initiation and perpetuation of chronic
inflammation.2 Adhesion receptors, including the lymphocyte
function antigen-1 (LFA-1; CD11a/CD18) and very late antigen-4
(CD49d/CD29) integrins, mediate the firm adhesion and transmigration of
T cells through activated endothelial cells (ECs) by binding to their
counter-receptors, intercellular adhesion molecule-1 (ICAM-1)/ICAM-2
and vascular cell adhesion molecule-1 (VCAM-1).3,4 In
addition, adhesion molecules play an important role in signal
transduction and in the costimulation of T cells during
their activation through the TCR/CD3 complex.5,6
Interleukin-15 (IL-15) is synthesized by several cell types and is
involved in the proliferation and activation of T cells.7,8 This cytokine also induces inflammatory cell recruitment in vivo and
T-cell migration in chemotactic assays in vitro.9-11 In
addition, IL-15 has a noticeable effect on lymphocyte
polarization and redistribution of adhesion molecules and chemokine
receptors.12,13 The widespread tissue expression of IL-15
mRNA is not consistent with IL-15 protein synthesis because this
cytokine seems to be subjected to a significant posttranscriptional
regulation.14,15 These multiple levels of regulation
prevent undesirable IL-15 expression in tissues. IL-15 has been
detected at sites of chronic inflammation, such as the rheumatoid
arthritis (RA) synovium,16 where it is involved in the
recruitment and activation of T cells, thus facilitating macrophage
activation and tumor necrosis factor- (TNF-) synthesis, which is crucial for joint destruction.17 In this
regard, it has been proposed that T-cell-macrophage interactions are
mediated by the LFA-1/ICAM-1 adhesion pathway and the binding of CD69, which is expressed at high levels in T cells from chronic
inflammatory lesions, with its putative ligand.17
The phenotype of T cells that have migrated to chronic
inflammatory sites is uncommon and somehow paradoxical; these cells show an increased expression of CD69 and CD45RO with a weak expression of the intermediate activation antigen CD25.1,18,19 The
phenotype of these infiltrating T cells could be explained by their
selective migration from the peripheral blood during the recirculation
of these cells. Conversely, the phenotype of these cells may be
acquired during their recruitment through the activated endothelium at inflamed tissues. In this regard, it has been reported that the majority of T cells in the rheumatoid synovium are nonspecifically recruited bystander T lymphocytes.2 However, the possible
role of the endothelium in the activation of T cells that migrate to inflammatory foci is still under discussion.20-23
In this study, we used an in vitro model to analyze the effect of
endothelium on the activation of peripheral blood T cells (PBTLs). We
found that TNF--activated endothelium induces the expression of
CD69 and synthesis of IFN- on PBTLs and that this effect is mediated
through the LFA-1/ICAM-1 interaction. The role of IL-15 in the
induction of T-cell activation in the presence or in the absence of ECs
or ICAM-1 was also analyzed. Our results indicate that the
transmigration through endothelium in response to IL-15 may account for
the antigen-independent activation of T cells observed in chronic inflammation.
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MATERIALS AND METHODS |
Reagents, cytokines, monoclonal antibodies (MoAbs), and protein
substrata.
Recombinant human (rh)IL-15, and rhMIP-1 (purity >98%, endotoxin
level <0.1 ng/mg cytokine) were obtained from PeproTech EC Ltd
(London, UK); rhTNF- (specific activity 5 × 107
U/mg, purity >95%) was provided by Genetech (San Francisco, CA); rhIFN- (specific activity 4.75 × 107 U/mg) was
purchased from Genzyme Diagnostics (Cambridge, MA); rhIL-1 (specific
activity 4 × 108 U/mg) was obtained from Promega
(Madison, WI); and lipopolysaccharide (LPS) (Escherichia coli
055:B5) and phorbol myristate acetate (PMA) were purchased from Sigma
Chemical Co (St Louis, MO). Brefeldin A and the calcium ionophore
A23187 were obtained from Calbiochem (La Jolla, CA).
The following murine MoAbs to human antigens were used: the anti-CD3
(Leu 4, phycoerythrin [PE] or peridinin chlorophyll protein [PerCP]
conjugated), anti-CD4 (Leu3a, PE conjugated), anti-CD8 (Leu2a, PE
conjugated), anti-CD14 (LeuM3, PE conjugated), anti-CD25 (anti-IL-2R,
fluorescein isothiocyanate [FITC] conjugated), anti-CD45RO (Leu45RO,
PE conjugated), anti-HLA-DR (HLA-DR, FITC conjugated). All were purchased from Becton Dickinson (Mountain View, CA). The
anti-CD69 (TP1/55, biotin conjugated), anti-CD14 (MO2), anti-CD16 (KD1), anti-CD19 (Bu12), anti-ICAM-1 (RR1/1, kindly provided by Dr R. Rothlein, Boehringer Ingelheim, Ridgefield, CT), anti-VCAM-1 (4B9, a
generous gift of Dr J. Harlan, University of Washington, Seattle, WA),
anti-CD18 (Lia3/2), anti-CD49d (HP2/1), and the anti-MHC I (W6/32) have
been previously described.24-28 The anti-IL-15 MoAb was
provided by Immunex (Seattle, WA). The anti-hIL-2 PE (MQ1-17H12), the
anti-hIL-4 PE (8D48), the anti-hTNF- PE (Mab 11), and the
anti-hIFN- PE (4S.B3) were purchased from Pharmingen (San Diego,
CA). P3X63 myeloma protein (IgG1, kappa) was used as negative control
in immunofluorescence studies.
Recombinant chimeric ICAM-1-Fc and VCAM-1-4D-Fc, consisting of the
total extracellular domains fused to IgG1 Fc fragment, were obtained as
described.29 Briefly, COS-7 cells were transiently transfected with pICAM-1-Fc and pVCAM-1-4D-Fc (ICAM-1 and VCAM-1-4D cDNAs cloned in pCD8IgG1). After 4 days, culture supernatants were
precipitated with ammonium sulphate, and thereafter chimeric proteins
were isolated by using protein A coupled to Sepharose (Pharmacia Fine
Chemicals, Uppsala, Sweden). Fibronectin (FN) and Poly-L-Lysine (PLL)
were purchased from Sigma, and bovine serum albumin (BSA) from
Boehringer Mannheim GmbH (Mannheim, Germany).
Cells and cell lines.
Peripheral blood lymphocytes (PBLs) were isolated from fresh human
blood by Ficoll-Hypaque density gradient centrifugation (Pharmacia),
followed by two steps of cell adherence on plastic flasks (Costar Corp,
Cambridge, MA) at 37°C for 1 hour. Peripheral blood T lymphocytes
(PBTLs) for chemotactic assays were purified from PBLs by
incubation with a mixture of anti-CD19 (Bu12), anti-CD16 (KD1), and
anti-CD14 (MO2) MoAbs for 30 minutes at 4°C. Then, cells were
washed twice and incubated with magnetic beads (Dynabeads; Dynal, Oslo,
Norway) conjugated with goat anti-mouse IgG, at a 1:20 target/bead
ratio for 1 hour on a rotating wheel at 4°C. The negative selected
cell population was always >97% CD3+ as assessed by flow
cytometry analysis. Isolated cells were washed and resuspended in RPMI
1640 (Flow Lab, Irvine, Scotland) containing 10% fetal calf serum
(FCS; Flow Lab) for the assays.
The human dermal microvascular EC line HMEC-1 has
previously been described30 and was kindly provided by Drs
Ades and Lawley (Centers for Disease Control and Prevention and Emory
University School of Medicine, Atlanta, GA). These cells were grown in
MCDB 131 medium (GIBCO-BRL, Paisley, Scotland) supplemented with 20% FCS, 10 ng/mL epithelial growth factor (Promega), 1 µg/mL
hydrocortisone (Sigma), 20 mmol/L Hepes, and 10 mmol/L L-glutamine.
Human umbilical vein endothelial cells (HUVECs) were isolated and
cultured as previously described.31 Cells were seeded on
tissue culture flasks or dishes coated with gelatin 0.5% and grown in
199 medium (Bio Whitaker, Verviers, Belgium) supplemented with 20%
FCS, 50 IU/mL penicillin, 50 µg/mL streptomycin (ICN
Biomedicals, Costa Mesa, CA), 250 µg/mL fungizone (Squibb Industria
Farmacéutica, Barcelona, Spain), 50 µg/mL EC growth supplement
(prepared from bovine brain), and 100 µg/mL heparin (Sigma), and used
up to the third passage.
Lymphocyte transmigration assays.
The migration of T cells through a confluent monolayer of HMEC-1 was
assayed in chemotaxis Transwell cell culture chambers (Costar), as
described.29 The chemotaxis chambers are separated by a
polycarbonate membrane of 6.5-mm diameter, 10-µm thickness, and
8-µm diameter pore size. Filters were coated on their
upper surface with fibronectin and layered with HMEC-1, and the lower wells of the chambers were then filled with culture medium. The integrity of the EC layer was assessed by diffusion of a trypan blue-albumin complex across the endothelial monolayer, as
described.32 Only cell monolayers with permeability  0.5%
were used for transendothelial migration assays. Each transendothelial
migration assay was performed using lymphocytes from a single donor and
HMEC-1 cells from the same batch. Additional transmigration assays were
performed in Transwell chambers of 3-µm pore size coated with either
purified fibronectin (20 µg/mL) or ICAM-1 (20 µg/mL).
Transmigration assays were performed in MCDB-131 + RPMI-1640 (1:1)
culture media supplemented with 0.5% human serum albumin. A total of
106 T lymphocytes (100 µL) were poured into the upper
chamber and 600 µL of medium with different stimuli were deposited in
the lower chamber. As control, T cells were incubated with these
cytokines in the absence of ECs. PBTLs from the upper and lower
chambers as well as from control plates were obtained after 16 hours of incubation at 37°C and 5% CO2, and counted and
analyzed for their expression of activation antigens.
Culture of T cells with EC and protein substrata, detection of
intracellular cytokines, and immunofluorescence flow cytometry
analysis.
Confluent EC cultures plated in 24-well plates (Costar) precoated with
0.5% gelatin were stimulated with TNF- (20 ng/mL), IL-1 (1 ng/mL), IFN- (250 U/mL), or LPS (50 ng/mL) for 20 hours at 37°C
to induce a maximal expression of the adhesion molecules ICAM-1 and
VCAM-1. In most of the assays, the EC monolayers were then fixed with
1% formaldehyde for 10 minutes and then extensively washed with Tris
buffer saline. PBLs were then added at 5 × 105
cells/well and incubated for 18 hours. Afterwards, cells were collected, and when indicated, adherent and nonadherent PBLs were carefully separated as previously described.23 In the rest
of the assays a population enriched in adherent cells (70% to 80%) was analyzed. To study the effect of the interaction of T cells with
different protein substrata on the expression of activation antigens,
24-well plates were coated with different concentrations of purified
proteins in 0.1 mol/L Tris pH 8.2 and incubated at 4°C for 16 hours. Plates were washed and used in the assay, as stated above. For
the detection of the production of cytokines, T cells were incubated
for 48 hours with different stimuli and brefeldin A (1 µg/mL) was
added during the last 10 hours of culture to inhibit the secretion of
cytokines. PBLs were stimulated with PMA (50 ng/mL) and the calcium
ionophore A23187 (1 µmol/L) in the presence of brefeldin A for 10 hours and used as positive control for the detection of IL-2, TNF-,
and IFN-. PBLs were activated with anti-CD3 and cultured with IL-2
and IL-4 for 48 hours before stimulating them with PMA and ionophore in
the presence of brefeldin A as stated above for obtaining the positive
control for the production of IL-4.
Lymphocytes were collected after the assays and incubated for 30 minutes at 4°C in phosphate-buffered saline (PBS) with the primary
MoAb, washed twice in PBS, 2% normal human serum, 0.1% sodium azide,
and then incubated for 30 minutes at 4°C with the appropriate
secondary antibody: FITC-conjugated rabbit anti-mouse F(ab')2 (Dako, Glostrup, Denmark), PE-conjugated
avidin (Pierce, Rockford, IL), or FITC-conjugated avidin D (Vector,
Burlingame, CA). Then, cells were additionally labeled with PE-, FITC-,
and PerCP-conjugated MoAbs. For intracellular cytokine staining, the collected cells were first stained for the cell surface markers (biotinylated CD69 plus avidin-FITC and CD3 PerCP) and then fixed in
4% paraformaldehyde and permeabilized with 0.1% saponin. Then the
anti-cytokine PE MoAbs were incubated for 30 minutes at 4°C in the
presence of 0.1% saponin, washed, and analyzed. A minimum of 5,000 cells was analyzed by two- or three-color immunofluorescence on a
FACScan flow cytometer (Becton Dickinson) using the Lysis II software.
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RESULTS |
Effect of ECs on the expression of activation markers by PBTLs.
We examined the influence of endothelium on the activation state of
PBTLs, monitorized by the expression of CD69, CD25, and HLA-DR. There
was a significant increase in CD69 expression on PBTLs after their
coculture with TNF--activated HUVEC monolayers for 18 hours,
whereas nonstimulated EC did have a weak effect (Fig 1A and
Table 1). To determine whether the effect of
TNF--activated endothelium on T lymphocytes was mediated through
adhesion molecules or soluble factors secreted by ECs, we studied the
induction of activation markers in both adherent and nonadherent PBTLs
(Table 1). We found that the T cells that were unable to adhere to
activated ECs only showed a slight increase in the expression of CD69,
whereas the adhered lymphocytes showed a noticeable increase in the
expression of CD69 as well as a modest but consistent enhancement in
HLA-DR+ cells (Table 1). The expression of CD97, another
early activation antigen, was upregulated in the same manner as CD69
(data not shown). Additional experiments showed that culture
supernatants from either resting or TNF--activated EC failed to
induce CD69 expression on PBTLs (not shown). The induction of CD69
expression by TNF--activated ECs in PBTLs was independent of the
concentration of monocytes present in the cell cocultures (data not
shown). The expression of CD25 was not induced in either adherent or
nonadherent cells (Fig 1A and Table 1). We therefore focused our
studies in a population enriched in adherent cells (70% to 80%) in
the rest of the assays.
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| Fig 1.
ECs induce the expression of CD69 on PBTLs. (A)
ECs induce the expression of CD69 but not CD25 on PBTLs. PBLs were
incubated alone (control) or on fixed TNF--activated EC monolayers
(TNF-EC). After 18 hours of culture, CD69 and CD25 expression on
CD3+ cells was analyzed. Dotted lines represent negative
control corresponding to P3X63. Numbers correspond to the percent of
positive cells. A representative experiment out of three is shown. (B)
Induction of CD69 T-cell expression by ECs activated with different
stimuli. ECs were incubated with medium alone (untreated) or with
IFN- (250 U/mL), TNF- (20 ng/mL), LPS (50 ng/mL), and IL-1 (1 ng/mL) for 20 hours at 37°C, fixed, and extensively washed. Then,
PBLs pretreated or not with the blocking anti-ICAM-1 RR1/1 MoAb were
added to wells containing medium or fixed HUVEC. After 18 hours, CD69
expression in CD3+ T cells was measured. The percentage
of induction over the control level is shown as arithmetic mean ± 1 standard deviation (SD). The results correspond to three independent
experiments. ( ), EC; ( ), EC + anti-ICAM-1. (C) Effect of
adhesion receptor blockade on EC-mediated activation of T cells. PBLs
preincubated 30 minutes at 4°C with blocking anti-CD18 Lia3/2 and
anti-CD49d HP2/1 MoAbs were added to confluent monolayers of resting
and TNF--activated ECs and incubated for 18 hours. Then, the
expression of CD69 on CD3+ cells was analyzed by
two-color flow cytometry. Control cells corresponded to PBLs incubated
in the absence of ECs. The percentage of induction over the control
level is shown as arithmetic mean ± 1 SD, and the results correspond
to three independent experiments. ( ), no MoAb; (), anti-CD18;
( ), anti-CD49d; (), anti-CD18 + anti-CD49d.
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Table 1.
Expression of CD69, CD25, and HLA-DR by PBTLs Either
Adhered or Not Adhered to Resting (EC) and TNF--Activated (TNF-EC)
Human Umbilical Vein ECs
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Next, we assayed whether ECs activated by stimuli other than TNF-
were also able to induce CD69 expression in PBTLs. ECs were activated
for 20 hours with the indicated stimuli and then fixed, before the
incubation with PBLs. As shown in Fig 1B, the stimulation of HUVEC by
IFN-, LPS, and IL-1 also enabled them to induce the expression of
CD69 on T cells. We next characterized by three-color flow cytometry
analysis the subset of PBTLs that was preferentially activated by ECs.
We found that a larger fraction of CD8+ cells expressed
CD69 as compared with CD4+ lymphocytes
(Table 2). However, because
CD4+ cells are more abundant, both cell subsets comparably
contributed to the pool of CD3+ CD69+
lymphocytes. On the other hand, CD45RO+ cells were more
responsive to ECs compared with the reciprocal CD45RO subset (Table 2).
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Table 2.
Induction of CD69 Expression in Different
Subpopulations of CD3+ Cells in Response to Activated
ECs or ICAM-1
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Involvement of the LFA-1/ICAM-1 adhesion pathway on the activation of
T cells.
The above data prompted us to explore the role of 2 and 1
integrins in CD69 induction by using blocking MoAbs. The blockade of
the LFA-1/ICAM-1 adhesion pathway by the anti-CD18 Lia3/2 MoAb or the
anti-ICAM-1 RR1/1 MoAb abrogated the activation of PBTLs by ECs
activated with different stimuli. In contrast, the anti-CD49d HP2/1 and
the anti-VCAM-1 4B9 MoAb only slightly affected the expression of CD69
(Fig 1B and C and Table 3). The inhibitory effect of anti-ICAM-1 MoAb was partial in the case of
IFN--activated endothelium, and was increased by the addition of
MoAb anti-LFA-3 and anti-CD2 (data not shown). These effects were
observed in cell cocultures with both intact and fixed ECs. A blocking
anti-major histocompatibility class I (anti-MHC class I) MoAb did not
display any significant effect (Table 3).
To further confirm the involvement of LFA-1/ICAM-1 adhesion pathway on
the activation of PBTLs on interaction with endothelium, we studied the
effect of purified EC adhesion molecules on the expression of CD69 by T
cells. As shown in Fig 2A,
the binding of T cells to immobilized ICAM-1 also induced a significant
expression of CD69, an effect that was abolished by a blocking
anti-CD18 MoAb (data not shown). In contrast, other adhesion receptor
ligands such as VCAM-1 and FN as well as other substrata such as BSA or PLL did not affect CD69 expression (Fig 2A and B). The expression of
CD69 induced by interaction with ICAM-1 was dose dependent with an
evident effect at 5 µg/mL of ICAM-1 and a maximal induction at 20 µg/mL (Fig 2B). In contrast, the induction of CD25 expression was not
observed under these experimental conditions, at any of the doses of
ICAM-1 tested (data not shown). Kinetics studies on CD69 expression by
PBTLs in response to ICAM-1 showed a slight increment after 4 hours of
incubation, reaching a maximum at 24 hours, and remaining significantly
elevated for several days (Fig 2C). The phenotypic characterization of
CD69+ T lymphocytes activated by ICAM-1 also showed a
predominance of CD8+ and CD45RO+ cells (Table
2).
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| Fig 2.
Induction of CD69 expression by T-cell
interaction with immobilized ICAM-1. (A) PBLs were added to wells
precoated with BSA (1%), rsICAM-1 (20 µg/mL), rsVCAM-1 (20 µg/mL),
FN (20 µg/mL), and PLL (40 µg/mL). After 18 hours of incubation at
37°C, CD69 expression was measured in CD3+ cells by
two-color flow cytometry. The results of three independent experiments
are depicted as arithmetic mean ± 1 SD. *, P < .01 compared
with BSA (Mann-Whitney U test). (B) ICAM-1 dose-dependent
T-cell activation. PBLs were incubated on wells precoated with
different concentrations of rsICAM-1 () or rsVCAM-1 ( ). After 18 hours, CD69 expression was analyzed in CD3+ cells. The
results of four independent experiments are represented as arithmetic
mean ± 1 SD. (C) Kinetics analysis of CD69 T-cell expression induced
by rsICAM-1 and IL-15. PBLs were incubated on BSA (1%) or rsICAM-1 (20 µg/mL) precoated wells, and then IL-15 (5 ng/mL) was added where
indicated. After 4, 16, 24, 40, 62, and 120 hours, cells were collected
and CD69 expression was measured in CD3+ cells. The
results of three independent experiments are shown as arithmetic mean ± 1 SD. ( ), BSA; (), ICAM-1; (), BSA + IL-15; (),
ICAM-1 + IL-15.
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T-cell activation by adhesion molecules and IL-15.
It has been described that IL-15 is a T-cell stimulatory cytokine with
chemotactic properties that plays a relevant role in chronic
inflammatory processes.17 Chemotactic cytokines seem to act
in concert with adhesion molecules to direct extravasation to sites of
inflammation.33 We therefore analyzed the effect of IL-15,
either alone or in combination with EC or ICAM-1, on the induction of
CD69 expression in T cells. We found that this cytokine was able to
upregulate the expression of CD69 on T cells in a dose- and
time-dependent manner (Figs 2C and 3A),
similar to what occurred with the interaction of PBTLs with immobilized ICAM-1. Unlike the former activation, IL-15 was also able to upregulate the expression of CD25. As expected, the effect of IL-15 on PBTLs was
prevented by a blocking anti-IL-15 MoAb (Fig 3A). Because it has been
reported that IFN- and TNF- stimulate IL-15 production by EC and
this IL-15 can be cell-surface associated,11 we studied the
contribution of endothelial IL-15 to the induction of CD69 expression.
The blocking anti-IL-15 MoAb produced a slight but consistent
inhibition in the induction of CD69 by nonfixed TNF--activated EC,
but no significant inhibitory effect on the activation promoted by
fixed TNF--activated EC (Fig 3B).
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| Fig 3.
Effect of IL-15 in the activation of PBTLs. (A)
Dose-dependent effect of IL-15 on the expression of CD25 () and CD69
() by PBTLs. PBLs were incubated with IL-15 (5, 20, 45, 70, 100, or
150 ng/mL) by 18 hours in the absence (closed symbols) or presence
(open symbols) of a blocking anti-IL-15 MoAb (5 µg/mL). Then, the
expression of CD25 and CD69 was analyzed in CD3+ cells by
two-color flow cytometry. The results of three independent experiments
are shown as arithmetic mean ± 1 SD. (B) Contribution of endothelial
IL-15 to the induction of CD69 in T cells. PBLs were cocultured with
nonfixed (left) or fixed (right) TNF--activated endothelium for 18 hours in the presence or absence of a blocking anti-IL-15 MoAb (5 µg/mL) and/or a blocking anti-CD18 MoAb. Then, the expression
of CD69 was analyzed in CD3+ cells by two-color flow
cytometry. The results of three independent experiments are shown as
arithmetic mean ± 1 SD. *, P < .05 compared with untreated
(Mann-Whitney U test).
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IL-15 and EC had an apparent additive effect on the induction of CD69,
but not CD25 (Table 3 and Fig
4A). This cooperative induction was preferentially exerted on the
CD45RO+ subset of T cells (Fig 4B). This cytokine also
collaborated with immobilized ICAM-1 to induce CD69 in T cells (Fig
4C). The expression of CD69 obtained by the cooperative effect of EC
and IL-15 was reverted to the levels induced by IL-15 alone by
anti-CD18 MoAb, weakly affected by anti-CD49d MoAb, and not inhibited
by the anti-VCAM-1 and anti-MHC class I MoAbs (Table 3).
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| Fig 4.
IL-15 cooperates with TNF--activated ECs
and ICAM-1 in the induction of CD69 T-cell expression. (A) Activated
ECs do not enhance the induction of CD25 promoted by IL-15 in T cells.
PBLs incubated in the presence or absence of fixed TNF--activated
ECs (TNF-EC and control, respectively) were stimulated for 18 hours in
the presence or absence of IL-15 (15 ng/mL). Cells were collected and
CD69/CD25 expression of CD3+ cells was analyzed by
three-color flow cytometry. Numbers correspond to the percent of
positive cells. A representative experiment out of three is shown. (B)
Additive effect of IL-15 and activated ECs on the induction of CD69 in
CD45RO memory T cells. PBLs were incubated alone (control) or on fixed
TNF--activated EC monolayers in the presence or absence of IL-15.
After 18 hours of culture, CD69/CD45RO expression of CD3+
cells was analyzed by three-color flow cytometry. Numbers correspond to
the percent of positive cells. A representative experiment out of three
is shown. (C) Additive effect of IL-15 and ICAM-1 on the induction of
expression of CD69 by T cells. PBLs were plated on BSA (1%) or ICAM-1
(20 µg/mL) precoated wells in the presence or absence of IL-15 (25 ng/mL). After 18 hours, cells were collected and CD69 expression in
CD3+ T cells was measured and is shown as percentage of
positive T cells. A representative experiment out of five is shown.
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Induction of T-cell cytokine production by activated endothelium and
IL-15.
The effect of the interaction between fixed TNF--activated
endothelium and T cells was also studied by intracellular detection of
cytokines with specific MoAbs. As shown in
Table 4, neither TNF--activated EC nor
IL-15 were able to induce the production of IL-2 or IL-4. IL-15 alone
induced a mild TNF- production and a higher synthesis of IFN- in
T cells. The interaction of T cells with activated EC increased the
levels of IFN- predominantly in the CD69+ subset of T
cells and this effect was additive with that of IL-15 (Table 4 and
Fig 5). In addition, a blocking anti-ICAM-1
MoAb was able to inhibit the induction of IFN- production by
activated EC, further indicating the involvement of the LFA-1/ICAM-1
pathway in the functional activation of these cells (Table 4 and Fig 5). Cell cycle analysis in T cells after different periods of incubation with activated EC did not show any increment of cells in M
or G2 phases, which is in accordance with the failure to induce T-cell
production of IL-2 and IL-4 cytokines (data not shown, and Table
4). Hence, the unusual activation state reached by these
cells during the interaction with endothelium is not accompanied by
cell proliferation.
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| Fig 5.
Production of IFN- by T cells in the presence of
TNF--activated ECs and IL-15. PBLs were cultured alone (BSA) or on
TNF--activated ECs (TNF-EC) in the presence or absence of IL-15 (25 ng/mL) and/or anti-ICAM-1. After 48 hours of culture the
expression of CD69 and intracellular IFN- was measured in T cells. A
representative experiment out of four is shown.
|
|
Induction of T-cell activation during transmigration.
We then studied the effect of T-cell transmigration through HMEC-1
microvascular ECs on the induction of the expression of activation
antigens by PBTLs. In static assays, T cells were induced to express
CD69 by their interaction with the microvascular ECs: 17.6% ± 4.8% and 27.5% ± 7.3% by nonactivated and TNF--activated HMEC-1, respectively. In transmigration assays using IL-15 and MIP-1
as chemotactic agents,10,11,34 the migrated T lymphocytes showed a significantly higher expression of CD69 compared with nonmigrated T cells (Fig 6A). As expected,
the transmigration through TNF--stimulated HMEC-1 induced the
highest expression of CD69. In these assays, the activatory effect of
IL-15 on PBTLs was also additive with that of HMEC-1 cells (Fig 6A, and
data not shown). In contrast, MIP-1 did not increase the level of CD69 expression by itself and did not show any cooperative effect with
HMEC-1 monolayers (Fig 6A, and data not shown).
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| Fig 6.
Induction of T-cell activation during transmigration. (A)
Additive effect of transendothelial migration and IL-15 on the
expression of CD69 by T cells. PBTLs (>97% CD3+) were
added to the upper compartment of Transwell chemotaxis chambers
precoated with resting (HMEC-1) or TNF--activated HMEC-1 cells
(TNF-HMEC-1). In the lower chamber MIP1 (20 ng/mL) or IL-15 (15 ng/mL) were added as chemoattractants. As control, T cells were
incubated with these cytokines in the absence of ECs. After 16 hours,
control, upper chamber, and lower chamber T cells were separately
collected and the expression of CD69 in CD3+ cells was
measured by two-color flow cytometry. The thin lines and percentages
correspond to the upper chamber T cells (NM, nonmigrated) and the thick
lines and percentages to T cells migrated to the lower chamber (M,
migrated).The proportion of migrated T cells for MIP1 is 25.1%
(HMEC-1) and 30.6% (TNF-HMEC-1), and for IL-15 is 33.8% (HMEC-1) and
37.3% (TNF-HMEC-1). A representative experiment out of three is
shown. (B) Additive effect of transmigration across ICAM-1-coated
membranes and IL-15 on T-cell activation. PBTLs were plated in the
upper compartment of FN (20 µg/mL) or rsICAM-1 (20 µg/mL) precoated
Transwell chemotaxis chambers and MIP1 (20 ng/mL) or IL-15 (15 ng/mL) were added as chemoattractants in the lower chamber. Control
cells were incubated as in (A). After 16 hours, T cells from both
chambers were collected separately and the expression of CD69 in
CD3+ cells was measured. The thin lines and percentages
correspond to nonmigrated (NM) T cells from the upper chamber and the
thick lines and percentages are referred to migrated (M) cells from the
lower chamber. The proportion of migrated cells for MIP1 is 32.4%
(FN) and 37.1% (ICAM-1), and for IL-15 is 42.8% (FN) and 47.6%
(ICAM-1). A representative experiment out of three is shown.
|
|
To further examine the role of ICAM-1 in the activation of purified
PBTLs, we performed transmigration assays in chemotaxis Transwell
chambers coating the polycarbonate membrane (3-µm diameter pore size)
with either ICAM-1 or FN. ICAM-1 exerted its proactivatory effect on T
cells that migrated in response to IL-15 or MIP-1 (Fig 6B). As in
other assays, IL-15 cooperated with the activation induced by the
transmigration across ICAM-1 and the migrated subpopulation showed the
highest level of CD69 expression (Fig 6B). FN did not show any
proactivatory effect (data not shown); however, differences in CD69
expression between the migrated and nonmigrated cells were observed
when the chemotaxis was induced by IL-15, likely because of a higher
migratory capacity of IL-15-activated T cells (Fig 6B). As it was
found in other experiments, the phenotype of the cells activated in the
transmigration assays mainly corresponded to CD8+ and
CD45RO+ cells (data not shown).
| |
DISCUSSION |
The activation of T cells in vivo is usually induced through the
TCR/CD3 complex by immunogenic peptides bound to MHC molecules. However, different evidences indicate that most T cells that are recruited and activated at sites of chronic inflammation do not respond
to the triggering antigens.2,35 Little is known about the
mechanisms involved in the nonspecific recruitment and activation of
these cells. Different cytokines are able to activate T cells in vitro
in an antigen-independent manner. However, the doses and combinations
of cytokines used in such studies do not fully explain the pattern of
T-cell activation at sites of chronic inflammation.8,36 In
addition, at these sites (eg, RA synovium), there is a relative absence
of T-cell-derived cytokines,37 and proactivatory
cytokines, such as IL-15, are detected at a lower concentration than
that required for the activation of T lymphocytes.8,17
Therefore, it is important to explore the possible alternative
mechanisms of T-cell activation and recruitment that occur at sites of
chronic inflammation.
It has previously been proposed that preactivated cells preferentially
migrate toward sites of chronic inflammation. In this regard, it has
been described, by using short-time transendothelial migration assays,
that the migrated population is slightly enriched in CD69+
cells.21,22,38 Herein, we have explored the possible
involvement of activated endothelium in the activation and recruitment
of resting lymphocytes as an alternative mechanism that accounts for
the abundance of activated bystander T cells in chronic inflammatory cell infiltrates. A previous report described that a long-time coculture of peripheral blood mononuclear cells with
IL-1-stimulated ECs resulted in the induction of CD69 in T
cells.23 We have observed in a similar model using PBLs
cultured in the presence of both resting and TNF--stimulated ECs
that this proactivatory effect is independent of the presence of
monocytes, thus ruling out the possibility of an indirect activation of
T cells by monocyte-derived cytokines. In addition, we report the key
role of the LFA-1/ICAM-1 adhesion pathway and the additive effect of
IL-15 on the activation of PBTLs during this interaction. The
interaction with activated ECs through the LFA-1/ICAM-1 pathway is able
to induce the production of IFN- on CD69+ T cells, which
is additive to the response induced by IL-15. In contrast, ECs were
unable to stimulate the secretion of IL-4, IL-2, or TNF-. T cells
did not proliferate in response to activated ECs in agreement with the
absence of IL-2 and the low expression of CD25. Although we did not
perform coculture experiments using autologous T cells and ECs, the
possibility of an allogeneic stimulatory effect can be ruled out both
by the lack of induction of CD25 expression and by the absence of
effect of the blocking anti-MHC MoAb on CD69 upregulation.
The involvement of cell adhesion molecules in the activation of PBTLs
induced by their interaction with ECs was initially suggested by (1)
the failure of EC culture supernatants to activate T cells; (2) the
ability of fixed ECs to reproduce the activation effect on PBTLs
observed with alive cells; and (3) resting ECs, which express low
levels of ICAM-1, only slightly induced CD69 expression on T cells,
whereas activated ECs, which strongly upregulate ICAM-1 and VCAM-1,
induced a much higher expression of CD69. Our data showing both the
inhibition of CD69 expression with anti-LFA-1/ICAM-1 MoAb as well as
the induction of CD69 on interaction of PBTLs with immobilized ICAM-1
underscore the importance of the LFA-1/ICAM-1 adhesion pathway in
T-cell activation by ECs. The activation of PBTLs by ECs was
preferentially exerted on the CD45RO+ T-cell subset. This
is in accordance with the fact that these memory T cells express a
higher density of LFA-1 than naive T cells, preferentially bind to
endothelium, and show an intrinsic migratory capacity, thus leading to
their accumulation in chronic inflammatory
infiltrates.38-40 The persistence of CD69 T-cell expression observed on the interaction of PBTLs with ICAM-1 is clearly distinct to
that described after stimulation with anti-CD3 MoAbs or phorbol esters,
which induce a transient increment in CD69 and CD25 expression and a
return to basal levels after 48 hours.24
The relevant role played by the cytokine IL-15 in chronic inflammatory
lesions has been reported.9,16 The IL-15 produced in the
rheumatoid synovium can recruit and activate T cells, which in turn
amplify and perpetuate the inflammatory phenomenon through the
induction of monocyte-derived TNF- via a cell-to-cell
contact-dependent mechanism. Both CD69 and LFA-1 have been postulated
as the cell surface molecules that mediate the interaction of T cells
with monocytes, leading to the secretion of TNF- and the subsequent induction of a cascade of proinflammatory cytokines that are
responsible for joint destruction.17 Nevertheless, at least
in the case of RA, the induction of CD69 expression is not completely
explained by the action of IL-15 alone, because the levels of IL-15
detected in RA synovial fluid are lower than those required to trigger a high expression of CD69.8,17 Our data show that IL-15 is able to induce in vitro the expression of CD69 and CD25 on T cells. Therefore, the phenotype induced by IL-15 does not completely correspond with that observed in chronic inflammatory infiltrates, where T cells display a high expression of CD69 with only intermediate levels of CD25.1,18,19,41 However, this phenotype
corresponds to that induced by the coordinate effect of activated
endothelium plus IL-15, with a high expression of CD69 and a mild
induction of CD25. The recently reported activation of the binding
capacity of LFA-1 to ICAM-1 by IL-15 could explain this cooperative
effect of IL-15 in EC-induced T-cell activation.11
In the transendothelial migration process, T cells interact with EC
mainly via the LFA-1/ICAM-1 adhesion pathway.3 Our studies
on the phenotypic characterization of T cells that transmigrate across
confluent monolayers of the human microvascular EC line HMEC-1 showed
the highest expression of CD69 in T cells that were able to
transmigrate. This induction of CD69 expression was much higher when T
cells transmigrated across TNF--activated HMEC-1 than through
nonactivated HMEC-1. Furthermore, by using an ICAM-1-coated Transwell
migration system, we could reproduce the high induction of CD69 in the
migratory subpopulation and this proactivatory effect was additive with
IL-15. All these results suggest that the induction of CD69, but not
CD25, during transmigration is mediated by the LFA-1/ICAM-1
interaction, as occurs during interaction with EC monolayers. On the
other hand, the higher CD69 expression found in the migratory
subpopulation of T cells in the FN-coated Transwell chambers when IL-15
is used as chemoattractant is likely because of the higher migratory
capacity of the IL-15-activated T cells.
In summary, our data suggest that the activated endothelium at sites of
chronic inflammation, along with IL-15, is able to induce, via the
LFA-1/ICAM-1 adhesion pathway, the activation and recruitment of PBTLs,
preferentially the CD8+ CD45RO+ cell subset.
This phenomenon could be responsible for the phenotype (CD69high, CD25dull) and the pattern of
cytokines (Th1 response, absence of IL-2, and proliferation) observed
in chronic inflammatory T-cell infiltrates. The relevance of LFA-1 in
these conditions could be further emphasized by the existence of an
LFA-1-activating environment that has been found in the rheumatoid
synovial fluid.42 Therefore, the maintenance of T-cell
activation in chronic inflammatory sites could result from the
interaction of these LFA-1 high-avidity state T cells with resident
cells expressing ICAM-1.43,44 In addition, cytokines might
potentiate this T-cell activation. This model provides a mechanism for
the recruitment and activation of CD45RO+ T cells at sites
of chronic inflammation, where there is a relative absence of
T-cell-derived cytokines, such as IL-2.
| |
ACKNOWLEDGMENT |
We are especially grateful to Drs R. González-Amaro, M. Nieto,
and M.A. del Pozo for critical reading of the manuscript; to E. Moreno
for excellent editorial assistance; and to M.Vitón for technical support.
| |
FOOTNOTES |
Submitted April 6, 1998; accepted September 25, 1998.
Supported by grant SAF96/0039 from Plan Nacional de Investigación
y Desarrollo and by Grant No. 08.3/0011/1997 from Comunidad Autónoma de Madrid.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Francisco Sánchez-Madrid, Servicio de
Inmunología, Hospital de la Princesa, Universidad
Autónoma de Madrid, Diego de León, 62, 28006-Madrid, Spain;
e-mail: fsmadrid/princesa{at}hup.es).
| |
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Top
Abstract
Introduction