Download CD4 CD25 Foxp3 Regulatory T Cells Protect the Proinflammatory

CD4ⴙCD25ⴙFoxp3ⴙ Regulatory T Cells Protect the
Proinflammatory Activation of Human Umbilical Vein
Endothelial Cells
Shaolin He, Ming Li, Xuming Ma, Jing Lin, Dazhu Li
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Objective—To investigate the role of CD4⫹CD25⫹forkhead box P 3 (Foxp3)⫹ T-regulatory cells (Tregs) in protecting the
activation and function of human umbilical vein endothelial cells (HUVECs) induced by proinflammatory stimulus and
the mechanisms of it.
Methods and Results—ECs play a major role in atherogenic initiation, changing their quiescence into activated phenotypes
to support every phase of the inflammatory process. HUVECs were incubated alone, with Tregs or CD4⫹CD25⫺ T cells
in the presence of anti–CD3 monoclonal antibodies for 48 hours, and then were stimulated with or without oxidized
low-density lipoprotein/lipopolysaccharide for an additional 24 hours. Tregs are able to induce alternative expression of
immune phenotypic markers of activated HUVECs by down modulating CD86 and to inhibit the adhesion molecule,
such as vascular cell adhesion molecule-1 (VCAM-1) and proinflammatory cytokine (eg, monocyte chemoattractant
protein-1 and interleukin 6), response of HUVECs to oxidized low-density lipoprotein/lipopolysaccharide.
Moreover, Tregs downregulate proinflammatory factor nuclear factor-␬B activation and induce resistance to
suppression of anti-inflammatory factor Kruppellike factor 2 in HUVECs induced by a proinflammatory stimulus.
Mechanism studies reveal that Treg-mediated suppression of HUVEC proinflammatory cytokines and adhesion
molecule expression impaired by oxidized low-density lipoprotein/lipopolysaccharide require cell contact by
cytotoxic T-lymphocyte antigen-4 and CD86 and by soluble factors (mainly interleukin 10 and transforming growth
factor [TGF]-␤).
Conclusion—Tregs may exert their protective effects against atherogenesis in part through inducing an immune-inhibitory
phenotype of ECs involving cytotoxic T-lymphocyte antigen-4 – dependent cell-to-cell contact and also requiring soluble
factors (mainly interleukin 10 and TGF-␤). (Arterioscler Thromb Vasc Biol. 2010;30:2621-2630.)
Key Words: atherosclerosis 䡲 regulatory T cells 䡲 endothelial cells adhesion molecules 䡲 endothelial function
䡲 immune system 䡲 oxidized lipids
A
therosclerosis is a chronic inflammatory disease of the
arterial wall, in which both innate and adaptive immune
responses contribute to disease initiation and progression.1,2
The role of the immune system in atherosclerosis has received considerable interest in recent years. However, sufficient knowledge to justify the immune modulatory mechanisms has not yet been obtained.
Recent studies from several groups suggest that subtypes
of T cells, called T-regulatory cells (Tregs), previously shown
to maintain immunologic tolerance, inhibit the development
and progression of atherosclerosis.3– 6 Interestingly, the suppressive effects of these cells are not restricted to the adaptive
immune system, including CD4⫹ T, CD8⫹ T, natural killer,
or B cells. These cells can also affect the activation and
function of innate immune cells (eg, monocytes, macro-
phages, and dendritic cells) and neutrophils7; however, their
effects on endothelial cells (ECs) are less well-known.
Previous studies have shown that alloantigen-specific
CD8⫹CD28⫺ T-suppressor cells can induce interleukin
immunoglobulin-like transcript 3⫹ (ILT3⫹) ILT4⫹ tolerogenic ECs, inhibiting alloreactivity.8,9 Because ECs are an
important target of antigen-specific T-effector cells (in transplantation and in numerous autoimmune diseases), Tregs that
inhibit EC activation and/or render ECs tolerogenic are
likely to have a beneficial effect in the treatment of
rejection, autoimmunity, and possibly atherosclerosis.
Thus, the identification and characterization of Tregs
regulating EC activation, inflammation, and dysfunction
could be crucial in deciphering the effects and mechanisms
of Tregs in atherosclerosis. Herein, we assessed the role of
Received on: February 25, 2010; final version accepted on: September 23, 2010.
From the Department of Cardiology, Institute of Cardiovascular Diseases, Union Hospital, Tongji Medical College, Huazhong University of Science
and Technology, Wuhan, China.
Drs He and Li contributed equally to this work.
Correspondence to Dazhu Li, MD, Department of Cardiology, Institute of Cardiovascular Diseases, Union Hospital, Tongji Medical College, Huazhong
University of Science and Technology, Wuhan, China. E-mail [email protected]
© 2010 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
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DOI: 10.1161/ATVBAHA.110.210492
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Figure 1. Identification of CD4⫹CD25⫹
Foxp3⫹ T cells. CD25⫹CD4⫹ T cells
were sorted from peripheral blood
mononuclear cells (PBMCs) of healthy
volunteers. A and B, Fluorescence-activated cell sorter analysis shows the sort
purity of CD25⫹CD4⫹ T cells (A) and
Foxp3 expression in sorted subsets (B).
The numbers display the frequency of
cells within indicated populations. The
bars in B indicate the marker gate for
Foxp3⫹ cells. Different ratios of magnetic
bead–sorted CD4⫹CD25⫹ T cells (Tregs)
from PBMCs of healthy volunteers were
incubated with effector CD4⫹CD25⫺ T
cells (Tcon) in the presence of irradiated
antigen presenting cells. C, Thymidine
uptake was used for assessment of
effector cell proliferative indexes. D, The
comparative effect of CD4⫹CD25⫹ T
cells (Tregs) expressed as percentage
inhibition of CD4⫹CD25⫺ T cells (Tcon).
cpm indicates counts per minute.
CD4⫹CD25⫹Foxp3⫹ regulatory T cells in protecting the
proinflammatory activation of human umbilical vein ECs
(HUVECs) and the mechanisms of it.
rated, HUVECs were harvested, and supernatants were collected for
further experiments.
Methods
The supplemental information provides detailed descriptions of the
following procedures: (1) flow cytometry for the detection of human
leukocyte antigen (HLA) DR, CD86, CD80, and VCAM-1; (2)
ELISA for the detection of monocyte chemoattractant protein
(MCP)-1, IL-6, IL-10, and TGF-␤; (3) real-time RT-PCR for the
detection of VCAM-1, MCP-1, IL-6, and Kruppellike factor (KLF)
2; and (4) electrophoretic mobility shift assay for the detection of
nuclear factor (NF)-␬B.
HUVEC Culture and Preparation of
Low-Density Lipoprotein and Copper-Oxidized
Low-Density Lipoprotein
HUVEC culture was performed as previously described.10 Lowdensity lipoprotein (LDL) and copper-oxidized LDL were prepared
as previously described.11,12 The supplemental information (available online at http//:atvb.ahajournals.org) provides descriptions of
the extended protocols.
Isolation and Purification of Tregs and Coculture
of T-Effector and Tregs
Isolation and purification of Tregs were performed using the method
of Dieckmann et al.13 Assays were performed using a previously
published method.14 The supplemental information provides descriptions of the extended protocols.
Coculture of HUVECs and Tregs
HUVECs that have grown to 90% confluence on 6-well plates
(1⫻106 cells per well) were washed 3 times with PBS and cultured
in serum-free medium for 24 hours for synchronization. Then,
nonadherent cells were removed and culture medium was changed.
For coculture experiment, HUVECs were cultured without T cells,
with Tregs (CD25⫹), or with CD4⫹CD25⫺ T cells (CD25⫺) for 48
hours in the presence of anti–CD3 monoclonal antibodies (mAbs)
(50 ng/mL) and then with oxidized LDL (ox-LDL) (40 ␮g/mL)/
lipopolysaccharide (LPS) (1 ␮g/mL) (Escherichia coli 0111:B4;
Sigma, St. Louis, Mo) for an additional 24 hours; as a control group
(control), HUVECs were cultured without anything but serum-free
Ml99 medium for 24 hours. The concentrations of ox-LDL and LPS
are those commonly used to investigate the inflammatory response in
vitro.15–18 After the incubation period, floating T cells were aspi-
Detailed Procedures
Transwell Experiments and Fixation of Tregs
Details of Transwell (TW) experiments are given in the supplemental
information. The detailed methods for fixation of Tregs are performed as previously described,19 which may be seen in the
supplemental data.
Data Analysis
All data are expressed as means of duplicate samples from 3 to 5
independently performed experiments. Data are presented as
mean⫾SEM. The significance of differences was estimated by
ANOVA, followed by Student-Newmann-Keuls multiple comparison tests. P⬍0.05 was considered significant. All statistical analyses
were performed with computer software (SPSS, version 11.0; SPSS
Inc, Chicago, Ill).
Results
ⴙ
Identification of CD4 CD25ⴙFoxp3ⴙ T Cells
We sorted CD4⫹CD25⫹ T cells from peripheral blood
mononuclear cells of healthy volunteers. As expected, the
purity of CD4⫹CD25⫹ T cells was ⬎93% (Figure 1A) and
most of the sorted CD4⫹CD25⫹ T cells were Foxp3⫹ (Figure
1B). To assess whether cells with a CD4⫹CD25⫹Foxp3⫹
T-cell (Treg) phenotype also displayed functional Treg char-
He et al
Tregs May Modulate the Activation and Function of Huvecs
acteristics, we tested their capacity to suppress the proliferation of autologous cocultured CD4⫹CD25⫺ T cells (Tcon)
after activation with anti–CD3 mAbs; we found that Tregs
can efficiently suppress Tcon cell proliferation in a dosedependent manner (Figure 1C and 1D).
Effects of Tregs on the Expression of Immune
Phenotypic Markers of HUVECs
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HUVECs were cocultured without T cells, with Tregs (CD25⫹),
or with CD4⫹CD25⫺ T cells (CD25⫺) in the presence of
anti–CD3 mAbs for 48 hours and then with or without (control)
ox-LDL for an additional 24 hours. The expression of HLA DR,
CD86, and CD80 in HUVECs was determined by flow cytometry. Ox-LDL impaired HUVECs (without T cells) displayed an
activating phenotype by high expression of HLA DR
(15.3⫾3.0%, P⬍0.001) and CD86 (39.4⫾5.1%, P⬍0.001)
compared with a resting phenotype of HUVECs (control)
(3.5⫾1.3% and 6.2⫾4.9%, respectively). Treg-treated HUVECs
(CD25⫹) significantly decreased CD86 (20.8⫾6.4%) expression
compared with that in a system without T cells (39.4⫾5.1%,
P⬍0.01) or a CD25⫺ system (33.2⫾4.0%, P⬍0.05). The
expression of HLA DR was significantly increased in a CD25⫹
(27.4⫾4.4%, P⬍0.01) and CD25⫺ (36.8⫾3.5%, P⬍0.001)
system compared with that in a system without T cells
(15.3⫾3.0%), whereas the levels of CD80 in the parallel
experiments have no significant difference among the 4 groups
(control, 0.8⫾0.8%; without T cells, 1.7⫾0.7%; CD25⫹,
1.3⫾1.1%; CD25⫺, 1.6⫾1.2%; all P⬎0.05) (Figure 2). We also
found that the effects of Tregs on the expression of immune
phenotypic markers were seen with other proinflammatory
agents, such as LPS (Figure 2).
Tregs Downregulate VCAM-1, MCP-1, and IL-6
Responses of HUVECs to ox-LDL/LPS
HUVECs were cocultured without T cells, with Tregs
(CD25⫹), or with CD4⫹CD25⫺ T cells (CD25⫺) in the
presence of anti–CD3 mAbs for 48 hours and then with or
without (control) ox-LDL/LPS for an additional 24 hours.
The protein levels of VCAM-1, MCP-1, and IL-6 of the
HUVECs were determined by flow cytometry and ELISA.
HUVECs exposed to ox-LDL have a significant increased
expression of VCAM-1 (58.0⫾5.2%, P⬍0.001), MCP-1
(47.4⫾5.8 ng/mL, P⬍0.001), and IL-6 (10.8⫾1.4 ng/mL,
P⬍0.001) compared with that from a control system
(0.6⫾0.3%, 4.9⫾2.7 ng/mL, and 0.5⫾0.2 ng/mL, respectively). Treg-treated HUVECs significantly decreased
VCAM-1 (13.6⫾4.0%), MCP-1 (20.6⫾2.0 ng/mL), and IL-6
(3.9⫾0.8 ng/mL) expression compared with that from the
system without T cells (VCAM-1, 58.0⫾5.2%, P⬍0.001;
MCP-1, 47.4⫾5.8 ng/mL, P⬍0.01; and IL-6, 10.8⫾1.4
ng/mL, P⬍0.01) or the CD25 ⫺ system (VCAM-1,
58.3⫾5.1%, P⬍0.001; MCP-1, 47.7⫾4.0 ng/mL, P⬍0.001;
and IL-6, 9.3⫾1.7 ng/mL, P⬍0.01) (Figure 3A and 3B).
Similar results occurred in Treg-treated HUVECs exposed to
LPS (Figure 3A and 3B). To exclude the possible contributions of Treg-derived proinflammatory cytokines in these
assays, HUVECs were precultured without T cells, with
Tregs (CD25⫹), or with CD4⫹CD25⫺ T cells (CD25⫺) in the
presence of anti–CD3 mAbs for 48 hours; T cells were
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depleted by using anti–CD2 beads, and the HUVECs were
stimulated with or without (control) ox-LDL/LPS for an
additional 24 hours. A similar result was obtained (Figure
3C). More important, these latter experiments also demonstrate that the suppressive effects persist even once Tregs are
removed from the assay.
The mRNA levels of VCAM-1, MCP-1, and IL-6 were
also determined by real-time RT-PCR analysis. As shown in
supplemental Figure, IA, GAPDH was used as endogenous
control (C), and the amount of target was normalized to
endogenous control gene. Coincident with the protein activity
data previously given, VCAM-1, MCP-1, and IL-6 mRNA
expression levels were reduced in CD25⫹ cultures relative to
cultures without T cells or CD25⫺.
To explore whether there was a threshold effect for
Treg-mediated suppressive expression of VCAM-1, MCP-1,
and IL-6 in mRNA levels in HUVECs, HUVECs (1⫻106
cells per well) were cultured with or without various concentrations of anti–CD3 mAb–pretreated Tregs (1⫻105, 2.5⫻
105, and 5⫻105 cells per well, respectively) in the presence of
ox-LDL for 24 hours; then, HUVEC proinflammatory cytokine expression levels were measured. A dose-dependent
effect on HUVEC proinflammatory cytokine expression was
noted in HUVECs (supplemental Figure, IB).
Tregs Downregulate NF-␬B Activation in
HUVECs, Induced by a Proinflammatory Stimulus
NF-␬B is a ubiquitous transcription factor that regulates
expression of proinflammatory and antiapoptotic genes; it is
thought to play an important role in driving the inflammatory
response.20 Therefore, we examined NF-␬B activity in
HUVECs cocultured with or without anti–CD3 mAb–activated Tregs after ox-LDL/LPS treatment. We performed an
electrophoretic mobility shift assay with specific oligonucleotide probes for the NF-␬B binding site regions. As shown in
Figure 4, the reduced proinflammatory cytokines were reflected at the transcriptional level by a clear decreased NF-␬B
upregulation on ox-LDL/LPS stimulation from Treg-treated
HUVECs (CD25⫹) (lane 4 or 7). In contrast, HUVECs
cultured only with ox-LDL/LPS (without T cells) or
HUVECs precultured with CD4⫹CD25⫺ T cells (CD25⫺)
displayed an increase in NF-␬B activation on ox-LDL/LPS
triggering (lane 3, 5, 6, or 8). The identity of the NF-␬B–
specific band was confirmed by competition analyses with
unlabeled oligonucleotide (competition) (lane 1).
Tregs Induce Resistance to Suppression of KLF2 in
HUVECs, Induced by a Proinflammatory Stimulus
Because the transcription factor KLF2 is considered an important mediator of the anti-inflammatory properties of the endothelium, we next investigated the influence of proinflammatory
stimuli, such as ox-LDL/LPS, on KLF2 mRNA levels in control
cells for various periods and after coculture with Tregs. As
depicted in Figure 5A, KLF2 mRNA expression was strongly
suppressed at the point of 6 hours after treatment of HUVECs
with ox-LDL/LPS, whereas the reduction observed in KLF2
mRNA levels induced by ox-LDL/LPS was significantly inverse
regulated in the presence of Tregs (Figure 5B).
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Figure 2. Tregs modulate
expression of HLA DR and
CD86 in HUVECs impaired by
ox-LDL/LPS. HUVECs were
cocultured without T cells (no
T), with Tregs (CD25⫹), or with
CD4⫹CD25⫺ T cells (CD25⫺)
in the presence of anti–CD3
mAbs for 48 hours and then
with or without ox-LDL/LPS for
an additional 24 hours; as a
control, HUVECs were cultured
without anything but serumfree Ml99 medium for 24
hours. A through C, The
expression of HLA DR, CD86,
and CD80 in HUVECs was
determined by flow cytometry
(A, B, and C, respectively). D,
Study data are representative
of at least 3 separate flow
cytometric analyses. ⫹ indicates CD25⫹ vs CD25⫺; §, no
T vs control; $, CD25⫹ vs no T
or CD25⫺; ***P⬍0.001;
##P⬍0.01; ###P⬍0.001;
⫹P⬍0.05; ⫹⫹P⬍0.01;
§P⬎0.05; $P⬎0.05.
Treg-Mediated Suppression of HUVEC Activation
and Function Requires Cell Contact and
Soluble Factors
To investigate whether suppression of HUVEC activation and
function depended on cell contact or soluble factors, we
cultured HUVECs without T cells, with Tregs in the presence
of anti–CD3 mAbs in either a coculture or a TW system.
After 48 hours of culture, the inserts were removed, and the
HUVECs in the lower well were stimulated with ox-LDL for
24 hours; as a control group (control), HUVECs were
cultured with medium for 24 hours. The expression of
VCAM-1 in HUVECs from the coculture system decreased
by 71.2% (P⬍0.001) compared with that in a system without
T cells (Figure 6A). By disrupting physical contact between
HUVECs and Tregs, the expression of VCAM-1 in HUVECs
from the TW system decreased by 46.5% (P⬍0.01) compared
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Tregs May Modulate the Activation and Function of Huvecs
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Figure 3. Tregs downregulate protein expression of VCAM-1, MCP-1, and IL-6 in HUVECs impaired by ox-LDL/LPS. Coculture was set
up as described in the legend to Figure 2. A, HUVECs were harvested, and expression of VCAM-1 on them was observed by flow
cytometry. B, Supernatants were collected, and MCP-1 and IL-6 were detected by ELISA. HUVECs were precultured without T cells (no
T), with Tregs (CD25⫹), or with CD4⫹CD25⫺ T cells (CD25⫺) in the presence of anti–CD3 mAbs for 48 hours; T cells were depleted by
using anti–CD2 beads; and the HUVECs were stimulated with ox-LDL/LPS for an additional 24 hours. C, Supernatants from different
groups were collected, and MCP-1and IL-6 were determined by ELISA assays. Data are expressed as mean⫾SEM of at least 3 independent experiments. ***P⬍0.001, ##P⬍0.01, ⫹⫹P⬍0.01, ⫹⫹⫹P⬍0.001.
with that in a system without T cells (Figure 6A). The MCP-1
and IL-6 in supernatants from all of the described groups
were also detected by ELISA assays; similar results were
obtained (Figure 6B). This partial reversal of suppression
could be because of the requirement of cell contact between
activated Tregs and ox-LDL–impaired HUVECs.
Previous studies have shown that cytotoxic T-lymphocyte
antigen-4 (CTLA-4) may be involved in the cell contact–
mediated negative regulation of Tregs21,22; thus, we hypothesized that activated Tregs are able to down modulate
proinflammatory cytokine expression on ECs impaired by
ox-LDL in a CTLA-4 – dependent manner. Although Tregs
are able to increase expression of CTLA-4 on stimulation,19
we need to confirm that this molecule was present in our
cultures. Flow cytometry assays showed that, after being
stimulated with anti–CD3 mAbs for 48 hours, viable Tregs
and activated fixed Tregs were strongly positive in high
expression of CTLA-4 (compared with resting Tregs or
resting fixed Tregs: all P⬍0.001); this indicated that activated
paraformaldehyde-fixed Tregs show a similar regulatory
capacity as viable cells (Figure 6C). This was a fundamental
study for further fixation and neutralization experiments. For
fixation and neutralization experiments, HUVECs were cultured without T cells, with activated fixed Tregs, or with
activated fixed Tregs added to anti–CTLA-4 mAbs (anti–
CTLA-4, eBioscience) or isotype in the presence of ox-LDL
for 24 hours; expression levels of VCAM-1, MCP-1, and IL-6
in HUVECs were then assayed. As shown in Figure 6D and
6E, VCAM-1, MCP-1, and IL-6 expression levels were
significantly inhibited in the activated fixed Tregs group,
which showed similar regulatory capacity as their norm
viable counterpart compared with that in the group without T
cells (P⬍0.01). However, the inhibitory effects of activated
fixed Tregs were significantly reversed when anti–CTLA-4
mAbs were added (P⬍0.01). CD86 and CD80 are the only 2
known ligands for CTLA-4. Therefore, anti–CD86 mAbs
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Figure 4.Tregs downregulate
NF-␬B activation in HUVECs
impaired by ox-LDL/LPS. The
coculture system was set up
as described in the legend to
Figure 2. An electrophoretic
mobility shift assay was performed with nuclear extracts
from HUVECs in different cultures to examine NF-␬B activity. A, lane 1, competition of
biotin-labeled probe with a
200-fold excess of unlabeled
probes; lane 2, HUVECs cultured alone (control); lane 3,
HUVECs cocultured with
ox-LDL (no T cells); lane 4,
HUVECs cocultured with Tregs
and ox-LDL (CD25⫹); lane 5,
HUVECs cocultured with
CD4⫹CD25⫺ T cells and
ox-LDL (CD25⫺); lane 6,
HUVECs cocultured with LPS
(no T); lane 7, HUVECs cocultured with Tregs and LPS (CD25⫹); and lane 8, HUVECs cocultured with CD4⫹CD25⫺ T cells and LPS
(CD25⫺). Nuclear protein extracted from the no T and CD25⫺ system (lanes 3, 5, 6, and 8) showed strong binding activity for the
NF-␬B oligonucleotide probe compared with that from the control or CD25⫹ system (lane 2, 4, 5, and 7). Binding was specifically inhibited by excess unlabeled NF-␬B oligonucleotide (lane 1). B, DNA binding activity of NF-␬B in different cultures was shown using the
relative measurement method. Data are expressed as mean⫾SEM of at least 3 independent experiments. ***P⬍0.001, ###P⬍0.001,
⫹⫹⫹P⬍0.001.
(anti-CD86) or anti–CD80 mAbs (anti-CD80) were also
added in HUVECs cultured with an activated fixed Tregs
system. We found that anti-CD86, but not anti-CD80, has a
similar reversion effect with the anti–CTLA-4 on the inhibitory effects of activated fixed Tregs. The isotype-matched
control mAbs had no effect (Figure 6D and 6E).
In the TW system, we found that inflammatory cytokine
levels were still markedly reduced compared with those in
HUVECs cultured alone (without T cells) (P⬍0.01), indicating that cell contact was only partly necessary and that
immunosuppressive cytokines may play a role in Tregmediated suppression on HUVEC proinflammatory cytokine
expression. To test the role of immunosuppressive cytokines
in the mechanism of regulation by Tregs, we incubated the
Tregs with anti–CD3 mAbs for 48 hours and then collected
the supernatants, added them to ox-LDL–treated HUVECs
supernatant (sup) for an additional 24 hours, and assayed the
expression of VCAM-1, MCP-1, and IL-6 in HUVECs. We
found that anti–CD3 mAb–treated Tregs can secrete immunosuppressive cytokines to inhibit VCAM-1 (37.1⫾4.3%,
P⬍0.01), MCP-1 (19.75⫾1.9 ng/mL, P⬍0.001), and IL-6
(4.92⫾0.9 ng/mL, P⬍0.01) expression in HUVECs compared with that in HUVECs cultured with ox-LDL alone
(62.3⫾4.9%, 44.11⫾3.0 ng/mL, and 9.72⫾1.3 ng/mL, respectively). These findings suggest that immune-suppressive
cytokines were required in Treg-mediated suppression of
Figure 5. Tregs downregulate
mRNA expression of KLF2 in
HUVECs impaired by ox-LDL/
LPS. HUVECs were cultured in
the continuous presence of
ox-LDL or LPS for 4, 6, 12, and
24 hours to allow identification of
early- and late-induced genes,
whereas nonstimulated controls
were taken at 0 hours. A, The
mRNA level of KLF2 was determined by real-time RT-PCR analysis. Expression levels of KLF2 in
HUVECs cultured alone or with
anti–CD3 mAb–activated Tregs,
followed by the addition of
ox-LDL (40 ␮g/mL) or LPS (1
␮g/mL) for 6 hours, are compared with expression levels in
control cultures. B, GAPDH was
used as an endogenous control.
Data are presented as
mean⫾SEM of triplicate wells
and are representative of at least
3 independent experiments.
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Tregs May Modulate the Activation and Function of Huvecs
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Figure 6. Treg-mediated suppression of HUVEC activation and function requires cell contact by CTLA-4 and CD86. HUVECs were cultured without T cells (no T) or with Tregs in the presence of anti–CD3 mAbs in either a coculture (CC) or a TW system. After 48 hours
of culture, the inserts were removed and HUVECs in the lower well were stimulated with ox-LDL; as a control, HUVECs were cultured
without anything but serum-free Ml99 medium. A and B, After cells were cocultured for 24 hours, HUVECs were harvested and the
expression of VCAM-1 on them was observed by flow cytometry (A); supernatants were collected, and MCP-1 and IL-6 were detected
by ELISA (B). Purified Tregs from peripheral blood mononuclear cells were divided into 4 fractions. One part was activated with anti–
CD3 mAbs for 48 hours and fixed with 2% paraformaldehyde (activated-fixed Tregs); the second part was fixed with 2% paraformaldehyde without activation (resting-fixed Tregs); the third part was stimulated with anti–CD3 mAbs for 48 hours (viable Tregs); and the last
part was left untreated (resting Tregs). C, Then, the expression of CTLA-4 in these 4 parts of Tregs was detected by flow cytometry.
HUVECs were cultured without T cells, with activated fixed Tregs, with activated fixed Tregs added to anti–CTLA-4 mAbs (anti–CTLA4), with activated fixed added anti–CD86 mAbs (anti-CD86), or with activated fixed added anti–CD80 mAbs (anti-CD80) or isotype
mAbs in the presence of anti–CD3 mAbs for 48 hours and then stimulated with ox-LDL. D and E, After cells were cocultured for 24
hours, HUVECs were harvested and the expression of VCAM-1 on them was observed by flow cytometry (D); supernatants were collected, and MCP-1 and IL-6 were detected by ELISA (E). Data are expressed as mean⫾SEM of at least 3 independent experiments.
*indicates V.S. no T; §, V.S. activated fixed Tregs; *P⬍0.05; **P⬍0.01; ##P⬍0.01; ⫹⫹P⬍0.01; §P⬎0.05.
HUVEC proinflammatory cytokine expression impaired by
ox-LDL (Figure 7B and 7C). Important recent advances in the
comprehension of the mechanisms of atherosclerosis provided evidence that the immune-inflammatory response in
atherosclerosis is modulated by regulatory pathways in which
2 anti-inflammatory cytokines (ie, IL-10 and TGF-␤) play a
critical role.2 Also, Tregs are able to produce immunesuppressive cytokines, such as IL-10 and TGF-␤, on stimulation.23 Therefore, we test whether IL-10 or TGF-␤ might be
involved in the supernatant-mediated suppression of proinflammatory cytokine expression in HUVECs impaired by
ox-LDL. First, we confirmed that these cytokines were
present in our cultures. ELISA assays showed that supernatants from the coculture system were strongly positive in the
expression of TGF-␤ (1.9⫾0.12 ng/mL, P⬍0.001) and IL-10
(678.1⫾27.8 pg/mL, P⬍0.001) compared with those in the
control (0.8⫾0.1 ng/mL and 143.3⫾25.0 pg/mL, respectively), without T cell (0.7⫾0.09 ng/mL and 132.9⫾24.2
pg/mL, respectively), or CD25⫺ system (0.6⫾0.14 ng/mL
and 128.5⫾26.6 pg/mL, respectively) (Figure 7A). Second,
neutralizing experiments were performed. Anti–IL-10, anti–
TGF-␤, or isotype mAbs were added to the sup system. After
treatment with anti–TGF-␤ or anti–IL-10 mAbs, the inhibitory effects of supernatants were significantly reduced; moreover, the suppression of VCAM-1, MCP-1, and IL-6 expression in HUVECs was markedly abrogated when both
neutralizing anti–IL-10 and neutralizing anti–TGF-␤ mAbs
were added. The isotype-matched control mAbs had no
effect. Furthermore, we cultured HUVECs with TGF-␤ mAbs
(final concentration, 1.9 ng/mL) or IL-10 mAbs (final concentration, 678.1 pg/mL) for 48 hours and then added
ox-LDL for another 24 hours. The expression of VCAM-1 in
HUVECs and MCP-1 and IL-6 from supernatants was measured and showed a significant reduction in HUVECs from the
TGF-␤ or IL-10 system compared with that from the no T-cell
system (Figure 7B and 7C).
Discussion
Atherosclerosis is driven by a chronic inflammatory process
within the arterial wall, initiated mainly in response to
2628
Arterioscler Thromb Vasc Biol
December 2010
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Figure 7. Role of immunosuppressive cytokines played in the mechanism of regulation by Tregs. We incubated Tregs in the presence
of anti–CD3 mAbs for 48 hours and collected the supernatants. HUVECs were cultured without T cells (no T), with anti–CD3 mAb–activated Tregs (coculture [CC]), with supernatants (sup), with sup added to anti–TGF-␤, with sup added to anti–IL-10, with sup added to
anti–TGF-␤ and anti–IL-10, with IL-10 mAbs, or with TGF-␤ or isotype mAbs in the presence of ox-LDL for 24 hours. The expression of
IL-10, TGF-␤ (A), VCAM-1 (B), MCP-1, and IL-6 (C) in HUVECs was assayed by flow cytometry or ELISA. As a control, HUVECs were
cultured without anything but serum-free Ml99 medium. Data are presented as the mean⫾SEM of at least 3 independent experiments.
* indicates V.S. no T; #, sup V.S. CC; ⫹, V.S. sup; §, isotype V.S. sup; *P⬍0.05; **P⬍0.01; ***P⬍0.001; #P⬍0.05; ##P⬍0.01; ⫹P⬍0.05;
⫹⫹P⬍0.01; ⫹⫹⫹P⬍0.001; §P⬎0.05.
endogenously modified structures, particularly oxidized lipoproteins or LPS that stimulates both innate and adaptive
immune responses, leading to further alteration of the vascular wall and promotion of disease progression and complications.2,24 The historical focus of immunologic studies on
regulation of atherogenesis has been on the functions of
infiltrating macrophages and T cells. However, recent reports
demonstrated that ECs play a major role in atherogenic
initiation, changing their quiescence into activated phenotypes to support every phase of the inflammatory process.25,26
ECs play an important role in many phases of immunologic
events. In inflammation, ECs exhibit increased adhesiveness
for host leukocytes and are involved in their recruitment to
the interstitium of the tissue.27 Furthermore, ECs could
effectively present alloantigens to lymphocytes, leading to
T-cell activation.28 Concerns in recent studies have been
focused on the mechanisms of costimulation for T-cell
activation delivered by ECs.29,30 In the present study, we have
examined the interactions of activated Tregs with ox-LDL/
LPS–impaired HUVECs and found a critical role of Tregs in
protecting the activation and function of HUVECs.
The important role of ECs in antigen presentation and
immunogenicity in vascular inflammation and autoimmune
responses has been recognized. ECs represent a highly
heterogeneous population of cells with the ability to modulate
the function of immune cells.31 The immunogenicity of ECs
is significantly upregulated when ECs are activated and then
can highly express HLA DR and costimulators, such as CD86
and CD80.24,32,33 Our studies showed that Tregs are able to
induce alternative expression of immune phenotypic markers
of activated HUVECs by down modulating CD86, which
extends previous reports that costimulatory molecule expression on ECs can provide important supplementary signals for
Tregs interacting with ECs. However, there is conflicting
evidence in the literature regarding the expression of CD86
and CD80 molecules on ECs, with some reports showing
their presence34 and others reporting their absence.35 These
discrepancies may reflect different culture conditions and
serial passages of cells or inherent differences between ECs
derived from different vascular beds. Furthermore, it is
possible that conflicting results regarding the tissue-specific
phenotype of ECs could arise from the use of different
isolation techniques or different selection of EC subpopulations during subculture. Moreover, Tregs inhibit the proinflammatory response of HUVECs to ox-LDL/LPS, accompanied by a reduction in the upregulation of NF-␬B activation.
All the data described demonstrated that Treg-treated ECs
confer a quiescent EC state with reduced expression of
costimulatory, adhesion, and chemokine molecules, indicating that Tregs may exert their protective effects against
atherogenesis in part through inducing an immune-inhibitory
phenotype of ECs to a greater extent than nontreated ECs. In
recent years, KLF2 has emerged as a master regulator of
endothelial quiescence, anti-inflammatory and antithrombotic
properties, and vascular tone by activating atheroprotective
and inhibiting atherogenic transcription.36 KLF2 acts as a
central transcriptional switch point between the quiescent and
activated states of the adult EC.37,38 Our study identified
KLF2 as being inhibited by proinflammatory stimuli, such as
ox-LDL/LPS, and inverse regulated by Tregs. Although the
He et al
Tregs May Modulate the Activation and Function of Huvecs
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molecular basis for Treg regulation of KLF2 remains unknown in HUVECs, recent studies by Kumar et al39 implicate
p65 and histone deacetylases in repressing the expression of
this factor. Whether similar mechanisms are operative in
Treg-mediated suppression of HUVEC activation is unclear.
However, the interesting possibility is raised that KLF2 and
NF-␬B may function in a mutually antagonistic manner and
that the balance of these 2 factors may dictate the degree of
cellular activation.
Several mechanisms of Treg-mediated suppression have
been proposed, including secretion by the Tregs of immunosuppressive cytokines, cell contact– dependent suppression,
and functional modification or killing of activated protein C.7
To investigate the mechanisms of Treg-mediated suppression
of ECs, TW experiments and neutralizing antibodies were
used. By disrupting physical contact between HUVECs and
Tregs (TW), the suppression of proinflammatory cytokine
expression was only partly reversed, suggesting that cell-tocell contact was required in Treg-mediated suppression.
Activated-fixed and viable Tregs almost had a similar regulatory capacity as their normal viable counterpart. Moreover,
blocking CTLA-4 on Tregs significantly decreased their
suppression effects on activated ECs. Therefore, these experiments demonstrated that the suppression function of Tregs
involved direct contact-dependent interactions with ECs and
was, in part, mediated by CTLA-4. This underlines and
extends prior findings on regulatory function, demonstrating
that surface molecules induced after activation of Tregs are
responsible for the regulatory capacity of these cells.
CTLA-4 belongs to the same family as CD28 and binds to
the same ligands, CD80 and CD86. Recent reports have
shown that the interaction of CTLA-4 with CD80/CD86,
expressed by activated protein Cs, may modulate immune
responses.40 This raises the possibility that anti–CTLA-4
mAbs may disrupt Treg function by preventing a CTLA-4 –
mediated signal through CD80/CD86 expressed on dendritic
cells. The data show that binding of a CTLA-4 –Ig fusion
protein to the surface of dendritic cells induces expression of
indoleamine 2,3 dioxygenase, leading to the depletion of
tryptophan and the inhibition of T-cell function.41 CTLA-4
expressed by Tregs has similar effects, suggesting that this
interaction may be important for the suppressive activity of
these cells.42 Our findings that activated Tregs are able to
down modulate CD86 and proinflammatory cytokine expression on ECs impaired by ox-LDL in a CTLA-4 – dependent
manner are in line with these data, raising the possibility that it
is ligation of CD86 on ECs by CTLA-4 expressed on Tregs that
is crucial for Treg-mediated suppression of inflamed ECs.
However, the facts that VCAM-1, MCP-1, and IL-6 levels
were still markedly reduced compared with those in HUVECs
alone in the absence of cell-to-cell contact and that the
combination of neutralizing antibodies to IL-10 and TGF-␤
completely abrogated suppression indicated that soluble factors may play a role in immune suppression mediated by
Tregs. Thus, it is possible that, although cell-to-cell contact
contributes to suppression, it is dependent on Treg-derived
cytokines, which regulate/induce mechanisms directly responsible for blocking HUVEC cell functions. Our data
suggest that the mechanisms involving cell-to-cell contact
2629
and also requiring cytokines contribute to suppression mediated by Tregs.
In conclusion, our study shows the following results. (1)
Activated Tregs are able to down modulate costimulatory
molecules CD86 and proinflammatory cytokine VCAM-1,
MCP-1, and IL-6 expression on HUVECs impaired by
ox-LDL/LPS. (2) Tregs downregulate proinflammatory factor
NF-␬B activation and induce resistance to suppression of
anti-inflammatory factor KLF2 in HUVECs exposed to oxLDL/LPS. (3) Tregs may exert their protective effect against
atherogenesis in part through inducing an immune-inhibitory
phenotype of ECs involving CTLA-4 – dependent cell-to-cell
contact and also requiring soluble factors, by IL-10 and
TGF-␤. This newly discovered ability of Tregs may help us to
understand the mechanisms of Tregs in atherosclerosis processes and may also provide a novel tool to manipulate
atherosclerosis development.
Acknowledgments
We thank all the anonymous reviewers for their helpful suggestions
on the quality improvement of our manuscript.
Sources of Funding
This study was supported by grant 30670855 from the National
Natural Science Foundation of China (Dr Li).
Disclosures
None.
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CD4+CD25+Foxp3+ Regulatory T Cells Protect the Proinflammatory Activation of
Human Umbilical Vein Endothelial Cells
Shaolin He, Ming Li, Xuming Ma, Jing Lin and Dazhu Li
Arterioscler Thromb Vasc Biol. 2010;30:2621-2630; originally published online October 7,
2010;
doi: 10.1161/ATVBAHA.110.210492
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
Greenville Avenue, Dallas, TX 75231
Copyright © 2010 American Heart Association, Inc. All rights reserved.
Print ISSN: 1079-5642. Online ISSN: 1524-4636
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Supplement Material: extended methods
HUVECs culture
HUVECs were isolated from human umbilical veins that were cannulated, perfused
with Hanks' solution to remove blood, and then incubated with 1% collagenase for 15
minutes at 37°C. After removal of collagenase (Sigma, USA), cells were cultured in
Ml99 medium (Gibco, USA) supplemented with 20% foetal calf serum (Gibco, USA),
100 ug/ml heparin (Sigma, USA), 50 ug/ml endothelial cell growth supplement
(Gibco, USA), 25 mM Hepes buffer, 2 mM L-glutamine, 100 U/ml penicillin, and100
ug/ml streptomycin, and grown at 37°C on tissue culture plates coated with 0.1%
gelatin. Cells were passaged at confluence by splitting1:4 and were used within the
first eight passages1. Use of human umbilical veins and the following blood from
normal donors were approved by the Ethics Committee of Tongji Medical College,
Huazhong University of Science and Technology.
Preparation of LDL and copper-oxidized LDL
Blood for lipoprotein isolation was collected in EDTA (1 mg/mL) from normal
lipidemia donors after 12 hours of fasting. LDL (density=1.03 to 1.063 g/L) was
isolated from the plasma after density adjustment with KBr, by preparative
ultracentrifugation at 50 000 rpm/min for 22 hours, using type 50 rotor. They were
dialyzed against phosphate buffered saline (PBS) containing 0.3 mM EDTA, sterilized
by filtration through a 0.22 mm filter, and stored under nitrogen gas at 4°C. Protein
content was determined by the method of Lowry et al. Copper oxidation of LDL was
performed by incubation of post-dialyzed LDL (1 mg of protein/ml in EDTA-free
PBS) with copper sulfate (10 mM) for 24 hours at 37°C. Lipoprotein oxidation was
confirmed by analysis of thiobarbituric acid-reactive substances (36.8±2.5 nmol of
malondialdehyde equivalents/mg protein, mean±S.D. n=5), and relative electrophoretic mobility was 4.0±0.3 (mean±S.D. n=5)2, 3
Isolation and purification of Tregs
Isolation of Human peripheral blood mononuclear cells (PBMCs) which obtained
from normal volunteers was performed using Ficoll-Hypaque density gradient
centrifugation following the manufacturer’s protocol (Human lymphocyte separation
medium from Chinese Academy of Medical Sciences Institute of Biomedical
Engineering, China). CD4+T cells were isolated from total PBMCs by negative
selection using LD column (Miltenyi Biotec, Germany). Purified CD4+T cells
incubated with anti-human CD25 magnetic beads (Miltenyi Biotec, Germany), and
separated into CD4+CD25+ and CD4+CD25- fractions by positive selection using MS
column (Miltenyi Biotec, Germany).The positively selected CD25+ cell fractions were
separated again over an MS column to achieve higher purities. As assessed by using
fluorescence-activated cell sorting (FACS) (Becton-Dickinson, Oxnard, NJ), purities
of CD4+CD25+Tcells and CD4+CD25-T cells were >93% and>99%, respectively. To
further confirm the identity of these freshly Tregs, the primers (5'-ACA CCA CCC
ACC GCC ACT-3' and 5'-TCG GAT GCC ACA GAT GAA GC-3') were used to
measure the expression of Foxp3 mRNA, the intracellular Foxp3 staining was
performed with the APC-anti-human Foxp3 staining set (bioscience, USA) according
to the manufacturer’s instructions4.
Co-culture of T-effector and Tregs
CD4+CD25- (Tcon) and CD4+CD25+T cells (Treg) (104 cells/well) were cultured in
RPMI medium supplemented with 10% fetal calf serum at different Tcon: Treg ratios
(0:1, 1:0, 1:1, 2:1, 4:1 and 8:1) in the presence of anti-CD3 mAbs (50 ng/ml) in
96-well plates (Nun, Germany). All cells were cultured in a final volume of 200 µL in
the presence of irradiated 104 T cell–depleted accessory cells (APC) per well. After 48
hours, [3H] thymidine (1 µCu/well) was added for 16 hours before proliferation was
assayed by scintillation counting (MicroBeta1450 Liquid Scintillation Counter, Perkin
Elmer, USA). Percent inhibition of proliferation was determined from the following
formula: (1–3[H]-thymidine uptake (cap) of co-culture of Treg with Tcon divided by
cpm of Tcon alone)×100%.Triplicate wells were used in all suppression experiments5.
Flow cytometry for detection of HLA DR, CD86, CD80 and VCAM-1
After the incubation period, HUVECs from different groups were incubated with
human leukocyte antigen DR (HLA DR)-PE antibody solution, CD86-PE antibody
solution,CD80-PE antibody solution and VCAM-1-PE antibody solution (eBioscience,
USA) at 4°C for 0.5 h, respectively. And then we washed them two times with PBS
which contains 2% bovine serum albumin (BSA) and 0.1% NaN 3. For isotype control,
PE-conjugated human anti-IgG1 antibodies (eBioscience, USA) were used. Cells were
re-suspended in 500 μL PBS with 1% paraformal-dehyde and immediately processed
using FACS LSRⅡ(Becton Dickinson, USA). After correction for unspecific binding
(isotype control), the percentage of positive cells were analyzed by the Cell Quest
program (Becton Dickinson, USA).
Enzyme-linked immunosorbent assay (ELISA) for detection of MCP-1, IL-6,
IL-10 and TGF-β
Supernatants from different groups were collected and kept frozen at -80°C until the
cytokine levels (MCP-1, IL-6, IL-10 and TGF-β) were determined by ELISA assays
according to the manufacturer’s instructions (R&D Systems, USA). The results were
compared with a standard curve. Each assay was carried out in triplicate for each
sample. Absorbance was measured at 450 nm by means of a spectrophotometer.
Real-time RT-PCR for detection of VCAM-1, MCP-1, IL-6 and KLF2
Total RNA from different groups of HUVECs were isolated using Trizol reagent
(Invitrogen, USA) according to manufacturer’s instruction, respectively. One
microgram of total RNA was reverse transcripted using RNA PCR Kit (Takara
Biotechnology, Japan) and the resulting cDNA was used as a PCR template. The
mRNA levels were determined by real-time PCR with Applied Biosystems Step One
Real-Time PCR System (Applied Biosystem, USA) according to the manufacturer’s
instructions. GAPDH was used as endogenous control. Primers for human VCAM-1
(5'-CAT CCA CAA AGC TGC AAG AA-3'and 5'-CCT GGA TTC CCT TTT CCA
GT-3');MCP-1(5'-CTC ATA GCA GCC ACC TTC ATT C-3' and 5'-CAA GTC TTC
GGA GTT TGG GTT T-3');IL-6(5'-CAA ATT CGG TAC ATC CTC GAC GGC-3' and
5'-GGT TCA GGT TGT TTT CTG CCA GTG C-3');KFL2 (5'-GCA CGCAC ACAG
GTGAGAAG-3' and 5'- ACCAGTCACAGTTTGGGAGGG-3') and GAPDH(5'-CCA
CCC ATG GCA AAT TCC ATG GCA-3' and 5'-TCT AGA CCG CAG GTC AGG
TCC ACC-3').PCR reaction mixture contained the SYBR Green I (Takara
Biotechnology, Japan), cDNA, and the primers. Relative gene expression level (the
amount of target, normalized to endogenous control gene) was calculated using the
comparative Ct method formula 2 -ΔΔCt.
Electrophoretic mobility shift assay (EMSA) for detection of NF-κB
For the preparation of nuclear extracts, HUVECs from different groups were gently
lysed in a NP-40 containing sucrose buffer while the nuclei remain intact. After a
washing step, the nuclei are suspended in a hypotonic "low salt buffer": the nuclei
swell. Then a "high salt buffer" is added slowly: the nucleoplasm is extracted into the
buffer while the nuclear envelop stays intact and retains the genomic DNA. The
extract is separated from the nuclear envelop/DNA by centrifugation. Light Shift TM
Chemiluminescent EMSA kit (Pierce, USA) was used to detect DNA-protein
interaction. The sequences of NF-κB consensus oligonucleotides were: forward
5'-AGTTGAGGGGACTTTCCCAGGC-3'; reverse 5'-GCCTGGGAAAGTCCCCTC
AACT-3'. For the EMSA, the binding reactions were performed for 20 minutes in
10mmol/L Tris-HCl (pH 7.5), 50 mmol/L KCl, 5 mmol/L MgCl2, 1 mmol/L dithiothreitol, 50 ng/uL poly (dI-dC), 0.05% NP-40, 2.5% glycerol, biotin 3’-end -labeled
double-stranded oligonucleotide, and nuclear protein extract. Samples were
electrophoresed on a native polyacrylamide gel and then transferred to a nylon
membrane. The biotin end-labeled DNA was detected by chemiluminescence. To
check if the observed shifted bands are specific for NF-κB, competition tests are run:
additionally to the labeled NF-κB probe a 200-fold excess of non-labeled ("cold")
oligonucleotide is added in.
Transwell experiments
Transwell (0.4 um pore size, Corning, Acton, MA) experiments were performed by
culturing HUVECs (1×106 cells/well) in the lower well and Tregs (5×105 cells/well)
with anti-CD3 mAbs in the inserts. After 48 hours of co-culture, the top compartments
(inserts) were removed, and ox-LDL (40 ug/ml) was added for 24h. After the
incubation period, HUVECs were collected for further experiments.
Fixation of Tregs
For fixation experiments Tregs were isolated and divided into four fractions. One part
was activated with anti-CD3 mAbs for 48h and fixed with 2% paraformaldehyde for 1
h at 4°C (activated-fixed Tregs); the second part was fixed with 2% paraformaldehyde for 1 h at 4°C without activation by ox-LDL (resting-fixed Tregs); the third part
was stimulated with anti-CD3mAbs for 48h (viable Tregs); and the last part was left
untreated (resting Tregs). Thereafter, fixated cells were washed extensively and used
in regulation assays together with the untreated fraction. This procedure prevents any
cytokines production from Tregs yet maintains their cell contact dependent
suppressive functions6.
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Supplementary Information: legend for figures I
Figure I Tregs down-regulate mRNA expression of VCAM-1, MCP-1 and IL-6 in
HUVECs impaired by ox-LDL/LPS.
Co-culture system was set up as described in Figure legend 2. Total RNA from
HUVECs were collected and VCAM-1, MCP-1, IL-6 mRNA were analyzed by
Real-Time PCR. GAPDH was used as endogenous control (A). To explore whether
there was a threshold effect for Tregs mediated expression of VCAM-1, MCP-1 and
IL-6 in mRNA levels in HUVECs, HUVECs (1×106 cells/well) were cultured without
or with various numbers of anti-CD3 mAbs activated Tregs (1×105, 2.5×105, 5×105
cells/well), then with or without(control) ox-LDL for an additional 24h, VCAM-1,
MCP-1 and IL-6 mRNA in HUVECs were measured (B). Data are presented as
mean±SEM of triplicate wells, and are representative of at least three independent
experiments. (* is indicated for V.S. control; # is indicated for V.S. no T; + is indicated
for CD25+ V.S.CD25-.*: p<0.05, **: p<0.01, ***: p<0.001; #: p<0.05, ##: p<0.01,
###: p<0.001; +: P<0.05, ++: P<0.01, +++: P<0.001)
Figure I