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.
References:
1. Wagner AH, Gebauer M, Pollok-Kopp B, Hecker M. Cytokine-inducible CD40
expression in human endothelial cells is mediated by interferon regulatory factor-1.
Blood.2002; 99: 520-525.
2. Holven KB, Aukrust P, Holm T, Ose L, Nenseter MS. Folic acid treatment reduces
chemokine release from peripheral blood mononuclear cells in hyperhomocysteinemic subjects. Arterioscler Thromb Vasc Biol. 2002; 22:699-703.
3. Ming Li, Jian Chen, Yu-Shu Li, Yi-Bai Feng, Qiu-Tang Zeng. Folic acid reduces
chemokine MCP-1 release and expression in rats with hyperhomocystinemia.
Cardiovasc Pathol. 2007; 16:305-309.
4. Dieckmann D, Plotter H, Berthold S, Berger T, Schuler G. Ex vivo isolation and
characterization of CD4+CD25+T cells with regulatory properties from human
blood. J Exp Med. 2001; 193:1303-1310.
5. Livings MK, Sangregorio R, Roncarolo MG. Human CD25+CD4+T regulatory
cells suppress naive and memory T cell proliferation and can be expanded in vitro
without loss of function. J Exp Med. 2001; 193:1295-1302.
6. Bharat A, Fields RC, Trulock EP, Patterson GA, Mohanakumar T. Induction of
IL-10 suppressors in lung transplant patients by CD4+CD25+ regulatory T cells
through CTLA-4 + signaling. J Immunol.2006; 177:5631-5638.
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