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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 7 (October 1), 1998:
pp. 2389-2398
Salicylates Inhibit Adhesion and Transmigration of T Lymphocytes by
Preventing Integrin Activation Induced by Contact With Endothelial
Cells
By
Roberto Gerli,
Cristina Paolucci,
Paolo Gresele,
Onelia Bistoni,
Stefano Fiorucci,
Christopher Muscat,
Silvia Belia,
Alberto Bertotto, and
Vincenzo Costantini
From the Institute of Internal Medicine and Oncological Sciences,
Center for the Study of Rheumatic Diseases; Institute of Internal and
Vascular Medicine; Department of Clinical and Experimental Medicine,
Gastroenterology Section; Department of Cellular and Molecular Biology
and Institute of Pediatrics, University of Perugia, Perugia, Italy.
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ABSTRACT |
The inhibition of cyclooxygenase does not fully account for the
spectrum of activities of nonsteroidal antiinflammatory drugs. It is
evident, indeed, that regulation of inflammatory cell function may
contribute in explaining some of the effects of these drugs. Tissue
recruitment of T cells plays a key role in the development of chronic
inflammation. Therefore, the effects of salicylates on T-cell adhesion
to and migration through endothelial cell monolayers on collagen were
analyzed in an in vitro static system. Aspirin and sodium salicylate
reduced the ability of unstimulated T cells to adhere to and
transmigrate through cytokine-activated endothelium. Although
salicylates did not modify the expression of integrins on T cells, they
blunted the increased adherence induced by the anti- 2
monoclonal antibody (MoAb) KIM127 and prevented the appearance of an
activation-dependent epitope of the CD11/CD18 complex, recognized by
the MoAb 24, induced by contact with endothelial cells. Salicylates also induced an increase of intracellular calcium
([Ca2+]i) and activation of protein kinase
C (PKC) in T cells, but not cell proliferation and interleukin (IL)-2
synthesis. The reduction of T-cell adhesiveness appears to be dependent
on the increase in[Ca2+]i levels, as it
could be reversed by blocking Ca2+ influx, but not by
inhibiting PKC. Moreover, ionomycin at concentrations giving an
increase in [Ca2+]i similar to that
triggered by aspirin, strictly reproduced the T-cell phenotypic and
functional changes induced by salicylates. Aspirin reduced T-cell
adhesion and migration also ex vivo after infusion to healthy
volunteers. These data suggest that the antiinflammatory activity of
salicylates may be due, at least in part, to an interference with the
integrin-mediated binding of resting T lymphocytes to activated
endothelium with consequent reduction of a specific T-cell recruitment
into inflammatory sites.
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INTRODUCTION |
NONSTEROIDAL ANTIINFLAMMATORY drugs
(NSAIDs) are very effective in the treatment of inflammatory
disorders, although patient compliance is often compromised by
NSAID-induced gastric mucosal injury.1 It is thought that
the pharmacologic effect of these drugs is mainly due to the inhibition
of prostaglandin (PG) synthesis.2 However, the precise
mechanism of action of NSAIDs has not been completely clarified, as the
inhibition of PG synthesis does not account for the entire spectrum of
NSAID antiinflammatory activities.3 Doses of
acetylsalicylic acid (aspirin, ASA) necessary to treat chronic
inflammatory diseases, indeed, are much higher than those required to
inhibit PG synthesis.3 Moreover, there is an increasing body of evidence to suggest that NSAIDs derive significant
antiinflammatory effects from non-PG-dependent mechanisms, including
perturbation of the cell membrane lipid bilayer, modification of
cytokine production, and inhibition of a number of intracellular events
including transmembrane anion transport, oxidative phosphorylation in
mitochondria, and activation of transcription factor nuclear
factor-kB.2-6 None of these, however, fully explains the
antiinflammatory action, as well as the side effects, of these drugs.
In recent years, there have been several lines of evidence to support
the hypothesis that polymorphonuclear cells (PMN) play a key role in
the pathogenesis of NSAID-induced gastric mucosal injury and that PMN
adherence to vascular endothelium, mediated by an interaction between
integrins and their ligands expressed on activated endothelial cells,
is one of the early and pivotal events in the process leading to
NSAID-induced gastric damage.7-14
The enhanced leukocyte adherence to endothelial cells induced by
NSAIDs, however, appears to represent a paradoxical effect in view of
the antiinflammatory action of these drugs. Indeed, leukocyte adhesion
to and migration through endothelium represent the initial and
fundamental steps in mounting an inflammatory response.15-20 Several adhesion molecules, which bind
specific counterreceptors on endothelial cells, are involved in these
processes. Among them, integrins, in particular the
2-integrins and the 1-integrin very late
antigen (VLA)-4, mediates the firm leukocyte attachment to
endothelium, a prerequisite for transendothelial migration and cell
extravasation to the inflamed tissues.18-20
These mechanisms regulate adhesion and migration also of T lymphocytes,
which play a fundamental role in the development of chronic
inflammation, such as synovitis in rheumatoid
arthritis.16,21-24 However, data regarding the effects of
NSAIDs on T-cell functions and, in particular, on T-cell/endothelium
interaction are scanty and controversial.25-29 In the
present study, we showed that ASA and sodium salicylate (NaS) can
reduce the ability of T lymphocytes to adhere to endothelial cell
monolayers and to transmigrate into collagen in an in vitro static
model. This inhibitory effect appears to be mediated, at least in part,
by a Ca2+-dependent interference with the activation of
-integrins, which mediate T-cell adhesion.
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MATERIALS AND METHODS |
Cell purification.
Peripheral blood mononuclear cells (PBMC), monocytes, and T cells were
isolated as already reported,30 and resuspended in RPMI-1640 supplemented with 10% fetal calf serum (FCS), 4 mmol/L L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin
(complete medium) (GIBCO-BRL, Gaithersburg, MD). Platelets and monocyte contamination in each purified T-cell suspension, as assessed by
microscope examination and nonspecific esterase staining, was less than
1%.
Monoclonal antibodies (MoAbs) and reagents.
Anti-CD3 was purified from supernatant of hybridoma cells obtained from
the American Type Cell Culture Collection (Rockville, MD). The
anti-L-selectin MoAb DREG-200 was kindly supplied by Dr Dorian O. Haskard (Rheumatology Unit, Hammersmith Hospital, London, UK).
Anti-CD29 (4B4) was purchased from Coulter Immunology (Hialeah, FL),
anti-CD11a (IOT16) and anti-CD18 (IOT18) from Immunotech (Marseille,
France), anti-CD11b (OKM1) from Ortho Diagnostic Systems Inc (Raritan,
NJ), anti-CD11c (Leu-M5) from Becton Dickinson (San Jose, CA), and
anti-CD49d from Dakopatts (Copenhagen, Denmark). The KIM127 MoAb,
stimulating CD18-dependent cell adhesion,31 was kindly
provided by Dr Martyn Robinson (Celltech Therapeutics Ltd, Berkshire,
UK). The anti-1 integrin MoAb TS 2/16 was kindly donated
by Dr Francisco Sanchez-Madrid (Hospital da la Princesa, Madrid,
Spain).32 The MoAb 24, recognizing an activation-dependent epitope of all subunits of CD11a/CD18 complex,33 was a kind gift of Dr Nancy Hogg (Imperial Cancer Research Fund, London, UK).
Human recombinant (r) tumor necrosis factor- (TNF-) (specific activity, 6.8 × 107 U/mg) was
graciuosly provided by Dr E. Allievi (Knoll Farmaceutici S.p.A., Milan,
Italy). Human r interleukin-1 (IL-1) (specific activity, 1.3 × 107 U/mg), phorbol myristate acetate (PMA), and
ionomycin were purchased from Sigma Chemical Co (St Louis, MO). The
kinase inhibitors staurosporin, H-7 and calphostin C, were obtained
from Biomol (Plymouth Meeting, PA). Type I collagen solution extracts
from porcine skin (Cellmatrix I-A) were purchased from Nitta Gelatin,
Inc (Osaka, Japan). Endothelial cell growth factor was purified from
bovine hypothalamus extract, as described.34 ASA and NaS
were purchased from Sigma. Because it is thought that a total serum
salicylate concentration of 250 to 350 µg/mL is associated with
optimal antiinflammatory effects,35 a final concentration
of 300 µg/mL for both drugs was chosen for the experiments. In some
of these, 30 and 600 µg/mL final concentrations were also used to
evaluate the dose-dependency of the observed results.
Cell phenotype.
T-cell surface phenotyping was performed with a two-color
immunofluorescence staining technique using isotype-specific goat antimouse antibodies (Southern Biotechnology Associates, Birmingham, AL) conjugated with either fluorescein or phycoerythrin as developing reagents for each MoAb. This procedure has been described in detail elsewhere.30 For the evaluation of salicylates' activity
on the expression of adhesion molecules on the T-cell surface, parallel experiments using PBMC simultaneously stained with anti-CD3 and the
specific antiadhesion molecule MoAb were performed to rule out
nonwanted effects on membrane molecules induced by T-cell purification
procedures. Because no phenotypic differences between T-cell and PBMC
suspensions were found, only the results obtained with purified T-cell
samples were given. Negative controls and tests to prove the
specificity of the isotype-specific antisera were performed for each
experiment as described.30
Culture of human umbilical vein endothelial cells (HUVEC).
Endothelial cells were harvested from HUVEC treated with 0.1%
collagenase (Type I; Boehringer Mannheim, Mannheim,
Germany), by a previously described method.36 Cells were
grown on 5% gelatin-coated 25-cm2 culture flask (Falcon;
Becton Dickinson) in M-199 (Sigma) supplemented with 20% FCS,
antibiotics, 90 µg/mL porcine intestinal heparin (Sigma), and 100 µg/mL endothelial cell growth factor. Cells were passaged using
trypsin-EDTA (GIBCO). Identification of cultured endothelial cells was
confirmed by their cobblestone structure and by immunohistochemistry,
using a rabbit antihuman von-Willebrand factor antibody (Dakopatts) and
fluorescein isothiocyanate (FITC)-conjugated goat antirabbit IgG
antibody. Nearly 100% of the cells showed a positive staining. These
experiments used cells in passages 2 to 3 only. To prepare the
collagen-coated wells, the concentration of collagen in the aqueous
stock solution (3 mg/mL) was adjusted to 1 mg/mL with pH 3.0 HCl/distilled water. Collagen gels were prepared in a 15-mL polystyrene
tube (Falcon) by rapidly mixing 8 vol of the diluted collagen solution,
1 vol of 10× M199, and 1 vol of 0.2 mol/L HEPES plus 0.08 N
NaOH/distilled water. A total of 100 µL of the solution was seeded
into each well of a 24-well plate (Falcon) and incubated at 37°C
until the solution became gel. A total of 500 µL of HUVEC suspensions
containing 2 × 105 cells were added to the
collagen-coated 24-well plate. Culture media was changed every 3 days.
T-cell adhesion to and migration through HUVEC monolayers cultured
on collagen gels in an in vitro static experimental system.
HUVEC monolayers were precultured for at least 3 days on collagen gels
in the plastic 24-well plates before treatment with rTNF- (10 ng/mL)
or rIL-1 (10 IU/mL), as already described.37 After
washing, the HUVEC monolayers were incubated with T-cell suspensions
(2.0 × 106/well) at 37°C. At the selected times,
unbound T cells were removed from the surface of the HUVEC monolayers
by gently washing with warmed M-199 + 0.1% bovine serum albumin (BSA).
The HUVEC were then incubated for 20 minutes at 37°C with 0.4%
EDTA in phosphate-buffered saline (PBS) to remove adherent T cells.
Almost all adherent T cells could be detached from the surface of HUVEC
with this treatment. Monolayers were then treated for another 30 minutes with 0.4% EDTA to remove HUVEC from the surface of collagen
gels, this process being monitored with phase microscopy to confirm
removal of HUVEC. After monolayers were washed out with PBS, the
collagen gels containing migrating T cells were treated with 0.05%
collagenase/Hanks' balanced salt solution (HBSS) at
37°C for 3 minutes two to three times with continuous pipetting to
release the T cells. After each pipetting with collagenase, FCS (10%
at final concentration) was added to the solution containing cells to
diminish the enzyme activity. The number of adherent and migrating T
cells was counted by hemocytometer and analyzed by flow cytometry. No
changes in the expression of CD3 and other adhesion molecules were
found on T cells after collagenase treatment.
Cell proliferation assay.
In vitro T-cell proliferation (5 × 104/well) was
performed in triplicate in complete medium in 96-well round-bottomed
microwell plates (Nunc, Roskilde, Denmark), as described.30
For positive controls, anti-CD3 was placed in a 96-well flat bottom
microtiter plate (No. 3596; Costar, Data Packaging Corp, Cambridge, MA)
and incubated at room temperature for 1 hour. The wells were then washed twice in medium to remove nonadherent MoAb before T cells were
added. A total of 20 IU/mL of human rIL-2 (Janssen, Beerse, Belgium;
specific activity, 9.0 × 106 U/mg) was added to
plastic immobilized anti-CD3 MoAb to obtain full proliferation in
highly purified T-cell cultures. This was measured by
(3H)-TdR incorporation (0.5 Ci, specific activity, 25 Ci/mmol; Amersham Int, Amersham, UK) during the last 6 hours of culture
and counting in a liquid scintillation counter. In some experiments, a
minimal amount of autologous monocytes (1% to 2% in the final cell
suspension), instead of rIL-2, was added to purified T-cell cultures.
Assay for IL-2 secretion.
A commercially available radioimmunoassay (RIA) kit was
used to detect IL-2 levels in filtered cell culture supernatants after 48-hour cultures (Medgenix Diagnostics SA, Fleurus, Belgium), according
to manufacturer's instructions.
Intracellular [Ca2+]i measurement.
Concentration of [Ca2+]i was measured in
T-cell suspensions using fura-2/AM (Sigma), a Ca2+
fluorescent ester chelator and indicator.38 T cells (1 × 106/mL) were suspended in standard incubation
solution, 5 mmol/L fura-2/AM was added, and incubated at 37°C for
30 minutes. Cells were washed twice in iced buffer solution,
resuspended in either standard medium or in a Ca2+-free
medium containing 1 mmol/L EGTA (Sigma) and transferred in a quartz
cuvette under continuous stirring with the temperature thermostatically
maintained at 37°C. Fluorescence was measured by a Hitachi 2000 spectrophotometer (Pabisch, Milan, Italy) by adjusting the excitation
wavelengths sequentially at 340- and 380-nm and the emission
wavelengths at 510 nm. Levels of [Ca2+]i were
calculated according to Tsien et al.39 The
fura-2/AM-Ca2+ signal was calibrated at the end of each
experiment by adding 125 µmol/L digitonin followed by 2.5 mmol/L EGTA
in Tris base, pH 8.3. The [Ca2+]i levels were
measured in resting T cells (basal) or in T cells stimulated with
different concentrations of ionomycin, ASA, or NaS. To ascertain
whether salicylates increased Ca2+ influx, manganese
(Mn2+) was used as a Ca2+
surrogate.40 Fura-2-loaded cells were resuspended in a
Ca2+-free buffer and stimulated with ASA or NaS.
Fluorescence was excited at 360 nm, ie, the isosbestic wavelenght at
which Ca2+ does not affect fura-2 fluorescence and changes
in fluorescence intensity are only caused by Mn2+
quenching. Emission was recorded at 505 nm. Maximal Mn2+
quenching was calibrated in each preparation at the end of recording with digitonin.
Evaluation of protein kinase C (PKC) activity.
PKC activity was evaluated by a previously described method using the
binding of [20(n)-3H]-phorbol-12,13-dibutirrate
([3H]PdBu) (Amersham Int; specific activity, 16.2 Ci/mmol) to intact cells and based on the principle that some of the
phorbol esters can interact with the site for diacylglycerol on
PKC.41 T cells (2 × 106/mL) were washed
with Locke's solution and incubated for 30 minutes with the same
solution containing 10 nmol/L of [3H]PdBu. PKC activity
was evaluated using adequate negative and positive controls represented
respectively by the nonactivated enzyme and by the enzyme maximally
activated with 100 µmol/L of norepinephrine. Nonspecific binding was
determined in the presence of 1 µmol/L unlabeled PMA. After
incubation, T cells were washed with cold Locke's solution, dried, and
resuspended in 0.5% Triton. Aliquots of each suspension were used for
liquid scintillation counting. Staurosporin (1 nmol/L), H-7 (100 µmol/L), or calphostin C (1 µmol/L) were used in the assay as PKC
inhibitors, when indicated.
In vivo experimental infusion of aspirin.
After giving informed consent, three healthy volunteers had a sample of
venous blood taken. Then, 500 mg of aspirin (lysine acetylsalicylate,
Aspegic; Synthelabo, Milan, Italy) was injected intravenously over 1 to
2 minutes and other blood samples were taken 30 and 120 minutes after
the injection to analyze T-cell adhesion and migration ability, as
described above.
Statistical analysis.
Owing to the nonnormal distribution of samples, the Kruskal-Wallis
analysis of the variance was adopted for the statistical evaluation of
the results. Values of P < .05 were chosen for rejection of
the null hypothesis.
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RESULTS |
Effect of salicylates on T-cell capacity to adhere to HUVEC and migrate
into collagen.
Unstimulated T lymphocytes, when exposed for 30 minutes to unstimulated
HUVEC monolayers, had a low adhesive (mean ± standard error of mean
[SEM], 10% ± 5%) and migratory ability (0.2% ± 0.2%). HUVEC activation by rTNF- led to a significant increase in
adhesiveness and migration of T cells, which was detectable 1 hour
after triggering (16% ± 3% and 1.3% ± 0.7%; P < .01) and which was maximal 4 hours after addition of the cytokine (26% ± 8% and 4.7% ± 3%; P < .001). Similar results
were obtained when rIL-1 was used as HUVEC activating stimulus (data
not shown). The binding was mainly mediated by both 2-
and 1-integrins, as T-cell/HUVEC interaction was
markedly reduced by saturating concentrations of anti-CD18
( 73.9%) or anti-CD29 MoAbs (60.7%). An almost complete
inhibition of T-cell adhesion to endothelium was obtained when
anti-CD18 and anti-CD29 were used together in the in vitro assay
(93.8%). L-selectin instead had a minor role in the attachment
of T cells to HUVEC in this experimental condition, as the
anti-L-selectin MoAb DREG-200 only marginally inhibited T-cell binding
to HUVEC (12.6%).
Experiments were then designed to determine the effects of ASA or NaS
on adhesion and migration of resting T cells. As our preliminary data
showed that negligible numbers of T cells adhered to and migrated
through unstimulated HUVEC, we used only activated HUVEC. Preincubation
of resting T lymphocytes with either ASA or NaS for 30 minutes resulted
in a dose-dependent decrease in adherence to HUVEC monolayers with an
inhibitory effect plateauing at 300 µg/mL
(Table 1). A more prolonged incubation with
ASA or NaS (1 to 2.5 hours) had similar inhibitory effects, while a
shorter incubation (15 minutes) led to a lesser inhibition of T-cell
adherence to HUVEC (data not shown). ASA or NaS treatment produced also
a dose-dependent reduction of T-cell migratory capacity (Table 1). No
effects were obtained by pretreatment of HUVEC with various
concentrations of the two drugs for different incubation times (data
not shown).
Effect of salicylates on T-cell integrin expression.
In an attempt to investigate the molecular basis of the inhibitory
activity of ASA and NaS, we analyzed the expression of integrins, which
play a key role in endothelial/T-cell interaction in an in vitro static
system,42,43 on unstimulated T cells. Flow cytometry
analysis showed that the basal expression of CD11b (mean fluorescence
intensity [MFI], 91 ± 18) was slightly, but significantly,
increased after treatment for 30 minutes with 300 µg/mL or 600 µg/mL of ASA (+12.8% ± 4% and +13.8% ± 3%, respectively; P < .05) or NaS (+10.1% ± 4% and +12.5% ± 4%; P < .05), whereas CD18, CD11a, CD11c, CD29,
and CD49d molecule expression was unchanged (data not shown).
Effect of salicylates on integrin activation.
Our phenotypic data do not explain the negative interference by ASA or
NaS on the firm T-cell/endothelium adhesion obtained in our in vitro
model. The lack of quantitative changes in surface expression of
integrins, however, does not rule out a possible interference by
salicylates with qualitative changes of the molecules, as it is known
that the conformational state of integrins plays a critical role in the
adhesion of T cells to endothelium.15,16 Thus, to verify
the effect of salicylates on integrin function, we used the
anti-2 MoAb KIM127 and the anti-1 TS2/16,
able to directly induce the active conformation of the respective
molecules.31,32 A strong enhancement of T-cell adhesion and
migration was induced by the anti-2, while a minor, but
significant, increase in adhesion was obtained with the
anti-1 MoAb (Table 2).
Pretreatment with ASA (Table 2) or NaS (data not shown) of T cells
prevented the enhancement in adhesion triggered by these stimulating
MoAbs. It is worth noting that salicylates were also able to
significantly reduce T-cell migration induced by the KIM127 MoAb,
thereby suggesting an effect of the drug on 2-mediated
binding.
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Table 2.
Effect of ASA Preincubation (300 µg) on T-Cell
Adherence and Migratory Ability Triggered by Anti-1-
and Anti-2-Integrin Stimulating MoAb
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On the basis of these results, as well as previous observation showing
an induction of integrin activation on the surface of PMN by simple
contact with activated endothelium and/or
platelets,44 we wondered whether the attachment of resting
T lymphocytes to activated endothelial cells is able to activate
integrins and whether salicylates can block this activation. To test
this hypothesis, we analyzed the expression of an activation-dependent
epitope of the CD11/CD18 complex, recognized by the MoAb
24,33,45,46 on the surface of T cells before and after
adhesion to and transmigration through HUVEC. The results reported in
Table 3 show that the 24 epitope was
undetectable on unstimulated T cells, but it significantly increased on
T cells adherent to activated HUVEC. Interestingly, maximal antigen
expression was displayed by the migrated T-cell fraction. Neither ASA
nor NaS induced the 24 epitope on the surface of unstimulated T cells.
However, T-cell preincubation with 300 µg/mL ASA for 30 minutes
strongly reduced the expression of 24 epitope triggered by the contact
with HUVEC on both adherent and migrated cell fractions (Table 3). NaS
preincubation gave similar results (data not shown). Thus, taken
together, our data suggest that salicylates are able to decrease the
adhesion of resting T cells to cytokine-activated HUVEC by preventing
T-cell integrin activation induced by contact with endothelium.
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Table 3.
Expression Induced by Activated-HUVEC of the 2
Integrin Activation Reporter Epitope Recognized by the MoAb 24: Effect
of T-Cell Preincubation With 300 µg/mL ASA for 30 Minutes
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Effect of salicylates on [Ca2+]i
levels and PKC activation.
Several studies have shown that changes in the integrin conformational
state on T cells are modulated by intracellular signals triggered by
activating stimuli.47,48 Therefore, we were interested in
evaluating whether salicylates are able to elicit intracellular events,
which usually follow T-cell activation.49 As shown in Fig 1A and B, both ASA and NaS were able to
induce a dose-dependent increase of [Ca2+]i,
which reached a maximal increase of fivefold to sixfold over basal with
600 µg/mL ASA or NaS.
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| Fig 1.
Time-course of Ca2+-mobilization induced by
ASA (A), NaS (B), or ionomycin (C) in T cells loaded with Fura 2/AM.
Cells were incubated in complete medium and stimulated after 60 seconds
(arrow) with different concentrations of salicylates or ionomycin.
Results of experiments in which T cells were incubated in
Ca2+-free medium in the presence of 1 mmol/L EGTA are
also shown (A and B). Data are expressed as the mean SEM of 20 determinations.
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Ionomycin induced a concentration-dependent increase of
[Ca2+]i level, the effect being detectable at
1 nmol/L and maximal at 100 nmol/L (data not shown). Fig 1C shows that
the magnitude of [Ca2+]i increase obtained
with optimal ASA or NaS concentrations was reproduced by the effect of
suboptimal concentration of ionomycin (50 nmol/L).
Salicylate-induced [Ca2+]i mobilization was
largely dependent on a Ca2+ inflow, as demonstrated by the
rapid quenching of fura-2 signals induced by adding Mn2+ to
the extracellular medium (Fig 2) and by the
fact that the [Ca2+]i increase was completely
abolished by preincubating the cells with 1 mmol/L of EGTA (Fig 1A and
B).
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| Fig 2.
Effect of Mn2+ addition to Fura 2/AM-loaded
T cells incubated with ASA. Mn2+ was used as substitute
for Ca2+ and the more pronounced the
quenching of the Fura 2/AM signal, the higher the
Mn2+ influx. Cells were incubated in a
Ca2+-free medium and stimulated (arrow) with ASA or no
agent (medium). After incubation for 60 seconds, 25 µmol/L
Mn2+ was added to the cell suspension. Fluorescence
intensity was normalized to 100% just before Mn2+
addition. Data are expressed as the mean SEM of 10 determinations. (*)
P < .01
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As Fig 3 shows, the incubation of T cells
with 300 µg/mL of ASA or NaS led to a significant activation of PKC,
close to the maximum obtained with norepinephrine, which was abolished
by the PKC inhibitor staurosporin. Two other PKC inhibitors, H7 and
calphostin C, were also able to inhibit the PKC stimulation produced by
ASA and NaS to a similar extent (data not shown). Significant PKC activation was also obtained with 30 or 600 µg/mL of both drugs (data
not shown).
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| Fig 3.
Effect of ASA and NaS on PKC activity in T lymphocytes.
Positive controls were represented by maximal PKC activation obtained
with 100 µmol/L norepinephrine (NE). Data are expressed as the mean
SEM of four separate experiments. P < .001 versus basal
values (*) or versus the respective values obtained with medium alone
(**).
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Effect of EGTA and PKC inhibitors on adhesive and migratory ability
of salicylate-treated T cells.
To check whether [Ca2+]i increase
and/or PKC activation were relevant for the observed inhibition
by salicylates of T-cell adhesion and migration, experiments using the
Ca2+ chelator EGTA, as well as different PKC inhibitors,
were performed (Table 4). The ASA-induced
decrease of T-cell adhesiveness and migration was almost completely
reversed by the addition of EGTA, whereas staurosporin, calphostin C,
or H7 did not significantly affect the functional T-cell changes
induced by ASA. Overlapping results were obtained in similar
experiments using NaS instead of ASA (data not shown).
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Table 4.
Effect of EGTA and Three Different PKC Inhibitors on
Adhesive and Migratory Ability of Aspirin (ASA)-Treated T Cells
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Modulatory effect of [Ca2+]i increases
on 2-integrin induced T-cell function.
Having shown a role of [Ca2+]i rather than
PKC in the observed effect of salicylates on T-lymphocyte adhesion and
migration, we first verified whether specific stimulation of
2-integrin is able to increase the [Ca2+]i
levels of resting T lymphocytes and subsequently analyzed the possible
influence of salicylates on this intracellular event. The data shown in
Fig 4 indicate that the binding of KIM127
MoAb to 2-integrin triggers a significant enhancement of
[Ca2+]i levels and that preincubation of T
cells with ASA for 30 minutes blunts this increase. Moreover, it is of
interest to note that a similar inhibitory effect on the KIM127-induced
[Ca2+]i increase was observed when T cells
were preincubated for 30 minutes with the suboptimal concentration of
ionomycin able to mimic the [Ca2+]i increase
induced by ASA (Fig 4).
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| Fig 4.
Effect of ASA or ionomycin pretreatment of T cells on
[Ca2+]i mobilization induced by the
anti-2-integrin MoAb KIM127. Cells were incubated with
300 µg ASA, 50 nmol/L ionomycin, or medium for 30 minutes at
37°C, washed, and then tested for mobilization of
[Ca2+]i. Arrow indicates addition of KIM127
MoAb (medium [ ], ASA [ ], and ionomycin pretreatment [ ])
or its solvent (control [ ]). Data are expressed as the mean SEM of
five determinations.
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Unstimulated T lymphocytes were then pretreated for 30 minutes with a
suboptimal concentration of ionomycin, washed, and tested in the
adhesion system, as well as analyzed by flow cytometry for the MoAb 24 staining. The simple incubation with this concentration of ionomycin
did not induce the activation conformational change of 2-integrin on
T-cell surface (Table 5). However,
ionomycin pretreatment induced a decrease not only of T-cell adhesion
and migration, but also of the binding of MoAb 24 induced by contact with HUVEC on adherent and migrated cell fractions.
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Table 5.
Effects of Suboptimal Concentration of Ionomycin on
T-Cell Adhesive and Migratory Ability and Surface Expression of the
Integrin Activation Reporter Epitope Recognized by the 24 MoAb
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In vitro effect of salicylates on T-cell (3H)-TdR
incorporation and IL-2 synthesis.
Increase in [Ca2+]i and activation of PKC are
usually followed by complete T-cell activation.49
Therefore, we analyzed the possibility that salicylates could induce
T-cell proliferation and IL-2 production. The basal T-cell
(3H)-TdR incorporation with medium alone (620 ± 250 cpm, n = 3) was enhanced by immobilized anti-CD3 MoAb plus monocytes to
1,850 ± 430 (P .01), 7,860 ± 1,290 (P < .001), and 18,380 ± 4,230 cpm (P < .001) after 24, 48, or
72 hours of culture, respectively. Similar results were obtained when
rIL-2 was added to immobilized anti-CD3. On the contrary, T-cell
incubation with 300 µg/mL of ASA for a period ranging from 6 to 72 hours did not change the basal (3H)-TdR incorporation (715 ± 330 cpm at 24 hours, 760 ± 350 cpm at 48 hours, and 610 ± 290 cpm at 72 hours, not significant [NS]). In addition,
no effects were obtained with 30 and 600 µg/mL of ASA. NaS incubation
of T cells gave similar results (data not shown). After 48 hours of
culture, the levels of IL-2 in the supernatant of T-cell cultures
stimulated with immobilized anti-CD3 plus 1% monocytes were increased
(14.4 ± 3.9 IU/mL, n = 3) with respect to those detected in the
supernatants of resting T cells (<1 IU/mL, P < .001). The
incubation with 30 to 600 µg/mL of either ASA or NaS did not induce
any detectable IL-2 activity after 6, 12, 24, and 48 hours of culture
(<1 IU/mL).
In vivo effect of salicylates on adhesive and migratory ability of T
cells.
Finally, we evaluated whether the observed in vitro effects of
salicylates on T-cell adhesion and migration were relevant to their
antiinflammatory effects in vivo. Adhesion and migration ability of T
cells isolated from the peripheral blood of three healthy volunteers
before and after intravenous injection of 500 mg of aspirin was
examined. T-cell adhesion and migration ability in the in vitro system
was significantly decreased 30 minutes after aspirin injection and the
inhibitory effect persisted 2 hours after
(Fig 5).
View larger version (15K):
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| Fig 5.
Effect of in vivo injection of ASA on adhesive and
migratory in vitro ability of T lymphocytes. Data are expressed as the
mean SEM of three separate experiments. P < .05 ( ) and
P < .01 (*) versus basal values (time 0).
|
|
| |
DISCUSSION |
The present study shows that salicylates, such as ASA or NaS, decrease
the ability of unstimulated T cells to adhere to cytokine-activated HUVEC monolayers and transmigrate through them into collagen in an in
vitro static model, which simulates vessel wall and extracellular matrix.37 In these experimental conditions, the adhesion of T cells to HUVEC is mainly mediated by surface molecules of the integrin family.42,43 However, our flow cytometry studies
showed that salicylates fail to significantly modify the constitutive expression of integrins on the membrane of resting T cells. Moreover, the slight increase in the expression of the CD11b molecule, already demonstrated for PMN,50 cannot account for the observed
decrease in T-cell adhesion induced by salicylates.
On the other hand, integrins also exist in either active or inactive
state. Activated T lymphocytes exhibit a greater capacity to adhere to
and transmigrate through endothelium than resting T cells and this
functional characteristic can be largely explained by upregulation of
the binding affinity of integrins induced by activation, without
changes in the level of surface expression.15,16 We have
confirmed that the selective activation of 1- or
2-integrins, induced by specific MoAbs,31,32
leads to a significant enhancement of T-cell adhesion to activated
HUVEC. As already shown in previous studies, 2-integrins
play a prominent role in the firm attachment to HUVEC and
transendothelial migration of T cells, as their specific activation
leads to a striking increase in adhesion of T cells and is associated
with a significant increase in the number of migrated
cells.42 The present demonstration that preincubation of T
cells with ASA prevents the stimulating effects of
anti-2 MoAb on adhesion and migration suggests that
salicylates block the integrin conformational changes induced by T-cell
activation.
The in vivo activation of T cells can be triggered by different
mechanisms at the site of inflammation.51,52 Among these, the simple contact with endothelium seems to play a major role in
inducing activation antigens on leukocyte surface.44,53 We
have shown that the attachment to HUVEC induces the expression on the
T-cell surface of a 2 integrin activation epitope, which is correlated with optimal adhesive function.33,45,46 This novel observation adds further knowledge on the effect of
endothelium/T-lymphocyte interaction. In this setting, it is tempting
to speculate that activation of 2-integrins induced by
HUVEC enhances not only adhesive, but also migratory capacity of T
lymphocytes, as our experiments have shown that the expression of the
neo-epitope recognized by the 24 MoAb was maximal in the migrated cell
fraction. Thus, our results, showing that salicylates block the
HUVEC-induced appearance of the 24 epitope on T cells, strongly suggest
that these drugs can interfere with the recruitment of T lymphocytes toward inflammatory sites by preventing the conformational change of
2-integrins induced by contact with endothelial wall. We
cannot rule out that the inhibitory effects of salicylates could be
also exerted on other surface molecules involved in emigration. Among these, for example, CD31/platelet-endothelial cell adhesion molecule-1 (PECAM-1) seems to play a key role in the process of
diapedesis between endothelial cells.15 However, PECAM-1
seems to be preferentially involved in the emigration of PMN,
monocytes, and only a subset of activated T cells,15,54
thereby suggesting a minor role of this molecule in the migratory
ability of the whole T-cell population.
An expanding literature indicates that integrin activation is dependent
on signals coming from the extracellular environment, but is also
regulated by intracellular events in an "outside-in" and
inside-out" signaling system able to modulate cell adhesion and
behavior.15,17,47,48,52 It is of interest that some NSAIDs
exert an inhibitory activity on the activation of PMN produced by
proinflammatory mediators by interfering with early events of signal
transduction.55,56 Moreover, it has been proven that aspirin-like drugs, including sodium diclofenac, indomethacin, or
piroxicam, induce an increase in [Ca2+]i, as
well as a transient activation of PKC in human T
lymphocytes.28,57 The latter observations have been
confirmed by the present study using salicylates.
Because it is known that both iCa2+ and PKC are implicated
in the regulation of cell adhesion,47,48,52,58 we attempted to address the question of whether the salicylate-induced activation of
these twin intracellular signals has a biologic relevance in reducing
integrin-mediated adhesiveness of T cells. EGTA, blocking Ca2+ influx, completely reversed the inhibitory effects of
salicylates on T-cell adhesion and migration. On the contrary,
different PKC inhibitors, with various degrees of selectivity, were
unable to restore normal adhesion and migration of T cells treated with ASA or NaS. This suggests that the salicylate-triggered decrease in
adhesion and migration of T lymphocytes is a Ca2+, rather
than PKC-dependent, phenomenon.
There is now evidence that Ca2+ is a significant player in
a complex, feedback coordinated, signaling repertoire, which can modulate leukocyte adhesion. The 2-integrin binding, for
example, triggers rapid mobilization of
[Ca2+]i,59,60 and, on the other
hand, cell adhesion via 2-integrins is directly
modulated by changes in [Ca2+]i
levels.61 Some of our experimental observations support the link between the increase of [Ca2+]i and the
decrease of integrin-mediated T-cell adhesion induced by salicylates.
ASA or NaS inhibit integrin-mediated adhesion of T cells to HUVEC, as
well as [Ca2+]i increase induced by MoAb
stimulation of 2-integrin. In addition, a concentration
of ionomycin, which gives an increase in
[Ca2+]i similar to that triggered by
salicylates, is able to reduce the expression of 24 epitope elicited by
HUVEC, as well as the adhesion and migration of T cells. Interestingly,
this concentration of ionomycin, similar to salicylates, does not
induce the 2-integrin activation conformational epitope.
In contrast, higher ionomycin concentrations ( 100 nmol/L) induce
significant binding of the MoAb 24 on T-cell surface (unpublished
personal data, November 1997) and enhance
-integrin-mediated T-cell adhesion to endothelium.61,62
Taken together, our data indicate that the inhibitory effect exerted by
salicylates on the adhesion of resting T cells to HUVEC may be mediated
by triggering a Ca2+ flux, which makes the cell
unresponsive to the subsequent Ca2+-mediated integrin
activation induced by endothelial contact. In this context, the
demonstration that [Ca2+]i depletion in T
cells prevents an increase in cell adhesion triggered by anti-CD3 or
anti-CD2 MoAbs would agree with this hypothesis.62
PKC and [Ca2+]i play a crucial role in T-cell
activation, as they are the two main second messengers generated by the
activation of the phosphatidylinositol pathway, which takes place after
T-cell receptor stimulation.49,63 However, our data
demonstrate that neither ASA nor NaS promote significant T-cell
proliferation or IL-2 synthesis. It is conceivable that, depending on
the nature of the stimulating ligand, rise in
[Ca2+]i and activation of PKC not necessarily
lead to the entire range of the T-cell functional repertoire, but may
selectively activate a discrete response of T lymphocytes. This is
consistent with recent observations showing that the degree of T-cell
activation is dependent on the kinetics and the extent of
[Ca2+]i elevation and on the type of PKC
isoform involved.64,65
The pharmacologic and clinical relevance of these data may be
considerable. The present results, showing that ASA or NaS are able to
reduce the capacity of circulating resting T lymphocytes to adhere to
and transmigrate through endothelial cells activated by proinflammatory
cytokines, indicate that salicylates may exert an inhibitory effect on
the aspecific recruitment of T cells, which have the potential to
contribute to the augmentation and persistence of the inflammatory
process. This activity might also be of some relevance to the known
protective effect of ASA against ischemic cardiovascular diseases in
view of the involvement of extravasated T cells in the processes
leading to atheroma formation and plaque disruption.66 In
this setting, it is worth noting that our in vitro results were
strictly reproduced by the in vivo administration of
antiinflammatory doses of ASA to healthy volunteers, where a very
strong inhibitory effect on the capacity of circulating T cells to
attach to and migrate through endothelium was seen. These findings are
in apparent contrast with previous studies showing an increase of PMN
adhesion to gut endothelium induced by ASA.8-12 However,
the effects of ASA on leukocytes may be tissue-specific or vary
according to the activation state of endothelium. In addition, the
enhanced PMN adhesion is usually not associated with increased migration (Gerli R., et al, manuscript in
preparation).12 It is interesting to note that
NaS, which does not share either the cyclooxygenase inhibition or the
ulcerogenic actions of ASA,67-69 does not enhance leukocyte
adherence to gut endothelium,12 but, like ASA, reduces
adherence and migration of T cells. This suggests that the observed
effects on T-cell adhesion and migration are probably not dependent on
the inhibition of cycloxygenase and PG metabolism. This concept seems
to be also supported by the present observation that the ASA or NaS
concentrations exerting maximal T-cell inhibition were equivalent to
the higher concentrations leading to optimal antiinflammatory effects
in vivo, rather than to those sufficient to inhibit PG
production.35
In conclusion, we have shown that salicylates can interrupt the
activation of 2-integrins on T lymphocytes with
consequent reduction of their capacity to adhere to and migrate through
endothelium. This effect appears to be mediated by a drug-induced
increase in [Ca2+]i. Although NSAIDs are able
to enhance [Ca2+]i in several cell
types,38 the mechanism of [Ca2+]i
mobilization is unclear. In human lymphocytes, ASA induces alterations
in membrane fluidity, suggesting that partitioning of salicylates into
plasma membrane lipids might increase cell-membrane permeability.28,57,70 Compatible with these findings is our observation showing that exposure to salicylates stimulates mainly Ca2+ inflow.
We are aware that our system, exploring only intermediate steps in
T-cell/endothelium interaction, does not allow to rule out an
interference of salicylates with other phases of the adhesion cascade.
In this context, results from our laboratories appear to confirm that
these drugs exert inhibitory effects on T-cell rolling along the vessel
wall through an interference with L-selectin. Further definition of the
influence of different NSAIDs on the ability of T lymphocytes to adhere
to endothelial cells will provide new insights into the mechanisms of
action of aspirin and its derivatives.
| |
FOOTNOTES |
Submitted February 6, 1998;
accepted May 20, 1998.
Address reprint requests to Roberto Gerli, MD, Institute of Internal
Medicine and Oncological Sciences, Center for the Study of Rheumatic
Diseases, Policlinico di Monteluce, I-06122 Perugia, Italy.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Dr Dorian O. Haskard (Rheumatology Unit, Hammersmith Hospital,
London, UK) for providing the DREG-200 (anti-L-selectin) MoAb, Dr
Martyn Robinson (Celltech Therapeutics Ltd, Berkshire, UK) for the
anti-2 integrin MoAb KIM127, Dr Francisco Sanchez-Madrid (Hospital da la Princesa, Madrid, Spain) for the anti-2
integrin MoAb TS 2/16, and Dr Nancy Hogg (Leukocyte Adhesion
Laboratory, Imperial Cancer Research Fund, London, UK) for the MoAb 24.
| |
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Abstract
Introduction
Abstract