Induction of indoleamine 2,3-dioxygenase in vascular smooth muscle cells by interferon-gamma contributes to medial immunoprivilege.
ABSTRACT Atherosclerosis and graft arteriosclerosis are characterized by leukocytic infiltration of the vessel wall that spares the media. The mechanism(s) for medial immunoprivilege is unknown. In a chimeric humanized mouse model of allograft rejection, medial immunoprivilege was associated with expression of IDO by vascular smooth muscle cells (VSMCs) of rejecting human coronary artery grafts. Inhibition of IDO by 1-methyl-tryptophan (1-MT) increased medial infiltration by allogeneic T cells and increased VSMC loss. IFN-gamma-induced IDO expression and activity in cultured human VSMCs was considerably greater than in endothelial cells (ECs) or T cells. IFN-gamma-treated VSMCs, but not untreated VSMCs nor ECs with or without IFN-gamma pretreatment, inhibited memory Th cell alloresponses across a semipermeable membrane in vitro. This effect was reversed by 1-MT treatment or tryptophan supplementation and replicated by the absence of tryptophan, but not by addition of tryptophan metabolites. However, IFN-gamma-treated VSMCs did not activate allogeneic memory Th cells, even after addition of 1-MT or tryptophan. Our work extends the concept of medial immunoprivilege to include immune regulation, establishes the compartmentalization of immune responses within the vessel wall due to distinct microenvironments, and demonstrates a duality of stimulatory EC signals versus inhibitory VSMC signals to artery-infiltrating T cells that may contribute to the chronicity of arteriosclerotic diseases.
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ABSTRACT: Kynurenine is a potent endothelium-derived vasodilator. Its synthesis from tryptophan is stimulated by interferon γ and may represent an important compensatory pathway for the regulation of vascular function in inflammatory conditions. We assessed associations of urine kynurenine to tryptophan ratio (KTR) levels to incident major coronary events (MCEs), acute myocardial infarction (AMI), and ischaemic stroke and mortality in patients with suspected stable coronary artery disease (CAD). A total of 3224 patients (mean age 62 years, 69% men) underwent urine and blood sampling prior to elective coronary angiography and were subsequently followed up for median 55 months. A total of 8.4% experienced an MCE, 7.8% suffered an AMI, and 7.6% died. In age- and gender-adjusted analyses, the hazard ratios [HRs; 95% confidence intervals (CI)] of MCE, AMI, and all-cause mortality were 1.43 (1.29-1.59), 1.44 (1.29-1.59), and 1.38 (1.23-1.54) per standard deviation increment of the (log-transformed) urinary KTR, respectively. These estimates were only minimally attenuated after adjustment for potential confounders. The addition of the urine KTR to a model of conventional risk factors significantly improved goodness of fit, discrimination, and risk classification for these clinical endpoints. No association was seen between the urine KTR and the risk of incident ischaemic stroke. A novel urinary inflammation marker, KTR, is strongly associated with adverse prognosis in patients with suspected stable CAD. Underlying pathomechanisms should be further elucidated.European Heart Journal 07/2013; · 14.72 Impact Factor
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ABSTRACT: A metabonomics approach based on liquid chromatography/quadrupole time-of-flight mass spectrometry (LC-Q-TOF/MS) was utilized to obtain potential biomarkers of coronary heart disease (CHD) patients and investigate the ZHENG types differentiation in CHD patients. The plasma samples of 20 CHD patients with phlegm syndrome, 20 CHD patients with blood-stasis syndrome, and 16 healthy volunteers were collected in the study. 26 potential biomarkers were identified in the plasma of CHD patients and 19 differential metabolites contributed to the discrimination of phlegm syndrome and blood-stasis syndrome in CHD patients (VIP > 1.5; P < 0.05) which mainly involved purine metabolism, pyrimidine metabolism, amino acid metabolism, steroid biosynthesis, and arachidonic acid metabolism. This study demonstrated that metabonomics approach based on LC-MS was useful for studying pathologic changes of CHD patients and interpreting the differentiation of ZHENG types (phlegm and blood-stasis syndrome) in traditional Chinese medicine (TCM).Evidence-based complementary and alternative medicine : eCAM. 01/2014; 2014:385102.
Induction of Indoleamine 2,3-Dioxygenase in Vascular Smooth
Muscle Cells by Interferon-? Contributes to
Madison C. Cuffy,* Amanda M. Silverio,* Lingfeng Qin,* Yinong Wang,* Raymond Eid,*
Gerald Brandacher,†Fadi G. Lakkis,2‡§Dietmar Fuchs,?Jordan S. Pober,§
and George Tellides3*
Atherosclerosis and graft arteriosclerosis are characterized by leukocytic infiltration of the vessel wall that spares the media.
The mechanism(s) for medial immunoprivilege is unknown. In a chimeric humanized mouse model of allograft rejection,
medial immunoprivilege was associated with expression of IDO by vascular smooth muscle cells (VSMCs) of rejecting human
coronary artery grafts. Inhibition of IDO by 1-methyl-tryptophan (1-MT) increased medial infiltration by allogeneic T cells
and increased VSMC loss. IFN-?-induced IDO expression and activity in cultured human VSMCs was considerably greater
than in endothelial cells (ECs) or T cells. IFN-?-treated VSMCs, but not untreated VSMCs nor ECs with or without IFN-?
pretreatment, inhibited memory Th cell alloresponses across a semipermeable membrane in vitro. This effect was reversed
by 1-MT treatment or tryptophan supplementation and replicated by the absence of tryptophan, but not by addition of
tryptophan metabolites. However, IFN-?-treated VSMCs did not activate allogeneic memory Th cells, even after addition of
1-MT or tryptophan. Our work extends the concept of medial immunoprivilege to include immune regulation, establishes the
compartmentalization of immune responses within the vessel wall due to distinct microenvironments, and demonstrates a
duality of stimulatory EC signals versus inhibitory VSMC signals to artery-infiltrating T cells that may contribute to the
chronicity of arteriosclerotic diseases.
The Journal of Immunology, 2007, 179: 5246–5254.
erosclerosis results from atheromata that accumulate over decades,
while an accelerated form of arteriosclerosis may occur within
months to years in transplanted hearts termed graft arteriosclerosis.
Immunohistological analyses of arteriosclerotic lesions have re-
vealed that the leukocytic infiltrate of the arterial wall is not uni-
form. Infiltration by T cells and macrophages predominates in the
intima and adventitia, whereas the media is relatively spared
(1–4). Similar findings of a relatively bland media have also
been noted in experimental models of atherosclerosis and graft
arteriosclerosis (5–7). The mechanism(s) for medial immuno-
privilege is unknown, although it has been proposed that elastic
laminae found in that arterial layer may prevent leukocyte traf-
rteriosclerosis, the leading cause of mortality and mor-
bidity worldwide, is characterized by inflammation, in-
jury, and remodeling of the vessel wall. Coronary ath-
In other more classical sites of immune privilege in the body,
initial notions of passive physical barriers have been supplanted by
more recently discovered active biological processes. Medawar de-
scribed the brain and anterior chamber of the eye as immunolog-
ically privileged sites due to an absence of lymphatics (preventing
afferent immune responses) and blood vessels (preventing efferent
immune responses), respectively (9). More recently, the immuno-
privileged status of the brain and eye has been ascribed to immune
deviation due to a number of factors in the local microenviron-
ment, including neuropeptides, TGF -?, and Fas ligand (10, 11).
Multiple cooperative systems also sanction the immune privilege
of the fetus cohabiting within the mother. A unique mechanism
that contributes to the immunoprivilege of the placenta is the ex-
pression of IDO by trophoblast cells (12). IDO, an IFN-?-induc-
ible, intracellular enzyme, catalyzes the first and rate-limiting step
in oxidative catabolism of the essential amino acid, tryptophan
along the kynurenine pathway (13). Treatment of pregnant mice
with 1-methyl-tryptophan (1-MT),4a pharmacologic agent that in-
hibits IDO activity, causes T cell-mediated rejection of allogeneic,
but not syngeneic, fetuses (14). The immunomodulatory effects of
IDO result from tryptophan depletion in the microenvironment
which prevents T cell proliferation, promotes T cell apoptosis, in-
duces T cell ignorance, anergy, or deviation, and generates regu-
latory T cells (15).
In the present study, we investigated whether medial immunoprivi-
lege in graft arteriosclerosis results from an anti-inflammatory factor
produced by the vessel wall. We find that the IFN-?-inducible
*Interdepartmental Program in Vascular Biology and Transplantation and the Depart-
ments of Surgery,‡Internal Medicine, and§Immunobiology, Yale University School
of Medicine, New Haven, Connecticut 06510 and the†Department of General and
Transplant Surgery and?Division of Biological Chemistry, Biocentre, Innsbruck Med-
ical University, Innsbruck, Austria
Received for publication April 6, 2007. Accepted for publication August 12, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by the National Institutes of Health (PO1 HL70295) and
by the government of the State of the Austrian Tyrol. M.C.C. received a fellowship
award from the Thoracic Surgery Foundation for Research and Education.
2Current address: Thomas E. Starzl Transplantation Institute, University of Pitts-
burgh, BST-W1542, 200 Lothrop Street, Pittsburgh, PA 15261
3Address correspondence and reprint requests to Dr. George Tellides, 295 Congress
Avenue, Boyer Center for Molecular Medicine 454, New Haven, CT 06510. E-mail
4Abbreviations used in this paper: 1-MT, 1-methyl-tryptophan; ?-SMA, ?-smooth
muscle actin; EC, endothelial cell; TrpRS, tryptophanyl-tRNA synthetase; VSMC,
vascular smooth muscle cell.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
The Journal of Immunology
expression of IDO by human vascular smooth muscle cells
(VSMCs) inhibits allogeneic T cell activation, proliferation, and
accumulation in vitro and in vivo.
Materials and Methods
Segments of human epicardial coronary arteries from explanted hearts of
cadaveric organ donors or cardiac transplant recipients were interposed
into the infrarenal aortae of female, 8- to 12-wk-old, non-leaky (serum IgG
?1 ?g/ml) SCID/beige mice (Taconic Farms) using an end-to-end micro-
surgical anastomotic technique as described (7). Human subject protocols
were approved by the Yale Human Investigations Committee and the New
England Organ Bank and animal procedure protocols were approved by the
Yale Animal Care and Use Committee. At 1 wk postoperatively, certain
animals received an adoptive transfer of 3 ? 108human PBMCs i.p. which
were obtained by apheresis of healthy volunteers and isolated by density
centrifugation. In selected experiments, mice received either 1-methyl-DL-
tryptophan pellets s.c. that released 200 mg over 10 day periods from 1 to
4 wk postoperatively or similar doses of placebo pellets (Innovative Re-
search of America). Retro-orbital blood samples were collected at 2 wk
after reconstitution and analyzed for human CD3?T cells and mouse
CD45?leukocytes as described (7). In other experiments, mice received
Ad5.CMV-human IFN-? or Ad5.CMV-LacZ (Qbiogene) at 1 ? 109
plaque-forming units i.v. at 1 wk postoperatively and circulating human
IFN-? was confirmed by ELISA (R&D Systems) after 2 wk (data not
Artery grafts were procured at 5 wk postoperatively (4 wk after PBMC or
cytokine treatment) and analyzed by immunohistochemistry using mouse
anti-human CD45RO, ?-smooth muscle actin (?-SMA; DakoCytomation),
and IDO (Chemicon International). Binding of secondary Ab (Jackson Im-
munoresearch) was detected with peroxidase/3-amino-ethyl carbazole kits
(Vector Laboratories). Cell counting of nuclei surrounded by positive im-
munostaining was performed under high magnification and averaged from
5 cross-sections for each graft. The areas of vascular compartments were
measured by computer-assisted microscopy and image software as previ-
ously described (7).
Human endothelial cells (ECs) were isolated by enzymatic harvesting from
umbilical cord veins and serially cultured in M199 medium (containing
L-tryptophan at 49 ?mol/L) supplemented with 20% FBS, 2 mmol/L L-
glutamine, 100 U/ml penicillin, 100 ?g/ml streptomycin (all from Invitro-
gen), 50 ?g/ml fibroblast growth factor-1 (Collaborative Research), and
100 ?g/ml porcine intestinal heparin (Sigma-Aldrich). Human aortic or
coronary artery VSMCs were isolated by explant outgrowth and serially
cultured in M199 medium supplemented with 20% FBS, L-glutamine, and
antibiotics. No phenotypic differences were detected between the two types
of VSMCs and vascular cell cultures were used at passage 3 to 4.
Human CD4?T cells were isolated by positive selection using Dyna-
beads (Dynal Biotech) and further purified by depleting recently activated
T cells and naive T cells with anti-mouse IgG Dynabeads and mouse anti-
human HLA-DR and CD45RA Abs (BD Pharmingen) at 10 ?g/ml. Iso-
lated cells were ?95% CD45RO?/CD4?T cells by FACS analysis (data
not shown). T cells were cultured in RPMI 1640 medium (containing L-
tryptophan at 24.5 ?mol/L) supplemented with 10% FBS, L-glutamine, and
Coculture experiments were performed by placing 2 ? 105ECs or VSMCs
in gelatin-coated wells of 24-well culture plates and where indicated
treated with IFN-? (Biosource International) at 100 ng/ml for 3 days,
washed in medium, and both IFN-?-treated ECs and VSMCs were con-
firmed to express class II MHC Ags before every experiment by FACS
expression by VSMCs. Immunohistochemical analyses of human coronary
artery grafts were performed 4 wk after no treatment (left panels) or allo-
geneic PBMC reconstitution (right panels) of SCID/beige mouse recipients
using Abs to CD45RO (A), ?-SMA (B), and IDO (C) or isotype-matched,
irrelevant Ab (C inset). Positive immunostaining is a crimson/brown color.
Representative photomicrographs of graft cross-sections are shown with
arrows marking the internal (top) and external (bottom) elastic laminae at
the boundaries of the media. The bar represents 100 ?m for all panels.
Intimal, medial, and adventitial CD45RO?cells (D) were counted and
normalized to the area of each compartment and medial ?-SMA?cells (E)
were counted per cross-section (x-sec) of human coronary artery grafts 4
wk after no treatment (open bars) or allogeneic PBMC reconstitution (filled
bars) of hosts. Data are means ? SEM, n ? 6, ?, p ? 0.05 vs paired control
grafts (ANOVA for T cell counts and t test for VSMC counts).
Medial sparing by allogeneic T cells is associated with IDO
TABLE I. TRP and KYN plasma levelsa
No PBMCPBMCNo PBMC ?1-MTPBMC ?1-MT
85.3 ? 4.3
0.45 ? 0.07
5.30 ? 0.72
81.4 ? 6.9
0.49 ? 0.07
6.03 ? 0.80
79.3 ? 5.8
0.54 ? 0.11
6.70 ? 1.23
75.7 ? 3.6
0.68 ? 0.10
9.16 ? 1.54
aSCID/beige mice bearing human coronary artery grafts were reconstituted or not with 3 ? 108human PBMC i.p. after 1
wk and treated or not with 1-MT at 20 mg/day for an additional 4 wk. Tryptophan (TRP) and kynurenine (KYN) plasma levels
were determined at 5 wk postoperatively. Data represent mean ? SEM (n ? 3–5 in each group). Comparisons were by ANOVA,
and differences between the groups did not reach statistical significance.
5247The Journal of Immunology
analysis (BD Biosciences) using a FITC-labeled mouse anti-human DR Ab
(Immunotech). T cells were labeled with 250 nM CFSE (Molecular Probes)
for 20 min, and 1 ? 106CD45RO?/CD4?T cells in 1 ml of supplemented
RPMI 1640 medium was added to wells containing vascular cells. The
cultures were maintained in 5% CO2at 37°C for up to 9 days. T cell
proliferation was assessed by FACS analysis of CFSE dilution after coun-
terstaining with PE-labeled mouse anti-human CD4 Ab (Immunotech Sys-
tem). IL-2 supernatant levels were measured by ELISA (eBioscience) ac-
cording to the manufacturer’s instructions. Certain coculture experiments
were performed in advanced RPMI 1640 medium (an enriched formulation
that enables cell growth in low serum concentrations) that was custom-
ordered tryptophan-free (Invitrogen) and supplemented with 0.5% FBS and
different doses of L-tryptophan (Sigma-Aldrich). Alternatively, the cocul-
tures were maintained in conventional RPMI 1640 medium supplemented
with 10% FBS and different doses of L-tryptophan, L-kynurenine, 3-hy-
droxy-DL-kynurenine, or 3-hydroxyanthranilic acid (Sigma-Aldrich).
loss. CD45RO (A), ?-SMA (B), and IDO (C) expression were analyzed by
immunohistochemistry in human coronary artery grafts of SCID/beige
mouse recipients 4 wk after PBMC reconstitution and treatment with either
placebo (left panels) or the IDO inhibitor, 1-MT (right panels). A similar
analysis for ?-SMA expression in artery grafts from unreconstituted hosts
was also performed (B insets). The bar represents 100 ?m for all panels.
Medial CD45RO?cells (D) and ?-SMA?cells (E) were counted in pla-
cebo-treated (open symbols) or 1-MT-treated (filled symbols) PBMC-re-
constituted animals. Data are means ? SEM, n ? 6, ?, p ? 0.01 vs paired
control grafts (t test).
Inhibition of IDO increases medial infiltration and VSMC
IDO expression was analyzed by immunohistochemistry in human coro-
nary artery grafts of SCID/beige mouse recipients 4 wk after i.v. infection
with Ad-LacZ (left panel) or Ad-IFN-? (right panel). The bar represents
100 ?m for both panels. B, IDO protein expression was also analyzed by
Western blotting of cultured VSMCs treated with IFN-? for various times
and at different doses and compared with the expression of ?-actin loading
control. IDO (C) and TrpRS transcripts (D), normalized to GAPDH
mRNA, were determined by real-time RT-PCR in cultured ECs (open bars)
and VSMCs (closed bars) after treatment with IFN-? at various doses for
6 h. Levels of tryptophan (TRP; E and F), kynurenine (KYN; G and H),
and a ratio of KYN/TRP (I and J) were determined by HPLC from the
supernatants of cultured ECs (open bars) and VSMCs (closed bars) treated
with IFN-? at 30 ng/ml for various times (left panels)) or at different doses
for 48 h (right panels). Data represent single values and are representative
of three independent experiments.
IFN-? induces IDO expression and activity in VSMCs. A,
5248 VSMCs INHIBIT T CELL ALLORESPONSES VIA IDO
In experiments using the Transwell system, 2 ? 105ECs or VSMCs
were placed in gelatin-coated 0.4-?m pore size membrane inserts (BD
Biosciences) above the cell cocultures in an additional 0.5 ml of medium.
In certain Transwell experiments, 1-methyl-D-tryptophan or L-tryptophan
(Sigma-Aldrich), adjusted to pH 7.4, were added at 200 ?mol/L on day 1,
and 24.5 ?mol/L daily, respectively.
Tryptophan and kynurenine concentrations of plasma samples and culture
supernatants were determined by HPLC. Tryptophan was monitored by its
native fluorescence at 285 nm excitation and 360 nm emission wave-
lengths, and kynurenine was detected by UV absorption at 365 nm wave-
length in the same chromatographic run.
IFN-?-treated VSMCs were lysed in radioimmune precipitation assay lysis
buffer (20 mM Tris (pH 7.5), 1% Nonidet P-40, and Roche Complete
protease inhibitor mixture). Equal amounts of protein per sample were
separated by SDS-PAGE, transferred electrophoretically to a nitrocellulose
membrane (Bio-Rad), and immunoblotted with primary Abs to ?-actin or
IDO (Chemicon) followed by HRP-conjugated secondary Abs (Jackson
ImmunoResearch). Detection of the bound Ab by ECL (Pierce Biotech-
nology) was performed according to the manufacturer’s instructions.
Total RNA was isolated from IFN-?-treated and DNase-treated cells using
NucleoSpin RNA II kits (Clontech Laboratories). Bulk reverse transcrip-
tion with random hexamer primers was performed according to the Mul-
tiscribe RT system protocol (Applied Biosystems). RT-PCR were prepared
with TaqMan 2 ? PCR Master mix and predeveloped assay reagents for
IDO, TrpRS, and GAPDH (Applied Biosystems). An iCycler and its sys-
tem interface software (Bio-Rad) were used to run samples and analyze
data. All cDNA samples were run in duplicate and a DNase-treated RNA
sample processed without the reverse transcriptase enzyme was used as the
negative control. The expression level of each target was normalized to that
Student’s t test and one-way ANOVA were performed using the Prism
software program (GraphPad Software). Differences with p ? 0.05 were
considered to indicate statistical significance.
Medial Sparing by Allogeneic T cells is Associated with IDO
Expression by VSMCs
We have previously reported an experimental model of graft ar-
teriosclerosis of human coronary arteries interposed into the infra-
renal aortae of SCID/beige mouse recipients reconstituted with
allogeneic human PBMCs (7). The alloimmune-mediated arterial
injury and remodeling is characterized by intimal and adventitial
accumulation of effector T cells with relative sparing of the
media (Fig. 1A). Enumeration of graft CD45RO?cells confirmed
a 5-fold greater density of memory T cells in the adventitia than
the intima, which in turn had a 5-fold heavier inflammatory infil-
trate than the media (Fig. 1D). The mildly inflamed media did not
demonstrate a significant loss of VSMCs (Fig. 1, B and E) and the
total area of the media remained unchanged (7) despite the variable
medial thinning that occurred in association with the PBMC-induced
allogeneic memory Th cells. Allogeneic untreated (?)
or IFN-?-pretreated (?) ECs and VSMCs that either
had undetectable or uniform expression of MHC class II
Ags, respectively (A) were cocultured with CFSE-la-
beled CD4RO?/CD4?T cells. After 9 days, the cells
were labeled with CD4-PE and analyzed by flow cy-
tometry (B). The %CFSElow, or proliferating, memory
Th cells are in the upper left quadrant. Supernatants
were removed after 2 days of coculture and analyzed by
ELISA for IL-2 (C) and by HPLC for tryptophan (TRP;
D), kynurenine (KYN; E), and a ratio of KYN/TRP (F).
Data are means ? SEM; n ? 6 for ELISA and n ? 3 for
HPLC; ?, p ? 0.001 all vs EC?control;†, p ? 0.01
VSMC?vs EC?; and§, p ? 0.001 VSMC?vs VSMC?
IFN-?-treated VSMCs do not activate
5249The Journal of Immunology
outward vascular remodeling (increase in vessel diameter). We ex-
amined whether the media expressed immunoregulatory molecules
that play a role in other immunoprivileged sites. Graft infiltration
by allogeneic T cells was associated with the induction of IDO
expression, particularly within the media (Fig. 1C), although the
diffuse pattern of IDO immunostaining in frozen sections did not
allow for further analysis of cellular expression details. Graft re-
jection and local up-regulation of IDO in the vessel wall did not
perturb systemic levels of tryptophan and kynurenine in the xeno-
geneic hosts (Table I).
Inhibition of IDO increases medial infiltration and VSMC loss
To determine whether IDO was necessary for medial immuno-
privilege, we treated pairs of SCID/beige mice that received adja-
cent segments of human coronary arteries and the same allogeneic
human PBMCs with either a placebo or 1-MT. Host reconstitution
by human T cells was not affected by 1-MT compared with pla-
cebo (6.8 ? 1.7 vs 5.0 ? 0.9% human CD3?cells/mouse CD45?
cells, respectively, p ? 0.4790). Treatment with 1-MT increased
medial infiltration and decreased the expression of the VSMC
marker, ?-SMA (Fig. 2, A and B). VSMC loss did not occur in
1-MT-treated recipients in the absence of PBMCs (Fig. 2B insets).
Cell counting verified a significant increase in medial T cell infil-
tration and VSMC loss resulting from 1-MT treatment (Fig. 2, D
and E). There was also a trend to a higher density of intimal T cells
in 1-MT-treated animals compared with placebo-treated controls,
but the differences were more modest than that of medial infiltra-
tion (26 vs 83% increase, respectively) and did not reach statistical
significance (data not shown). IDO expression within the graft was
not modulated by 1-MT (Fig. 2C), and circulating levels of tryp-
tophan and kynurenine did not differ between the treatment groups
IFN-? induces IDO expression and activity in VSMCs
We further investigated the regulation and activity of IDO in hu-
man vascular cells. Exposure to the T cell-derived cytokine,
IFN-?, in the absence of allogeneic PBMCs, was sufficient to in-
duce the expression of IDO by graft VSMCs in vivo (Fig. 3A).
IFN-? also induced the expression of IDO protein and mRNA in
coronary artery or aorta VSMCs in a time- and dose-dependent
fashion (Fig. 3, B and C). Quantification of transcripts by real-time
RT-PCR demonstrated that the IFN-?-mediated induction of IDO
in VSMCs was considerably greater than in umbilical vein ECs.
Similarly, the up-regulation of tryptophanyl-tRNA synthetase
(TrpRS), an enzyme required for tryptophan incorporation in pro-
tein synthesis, was also far greater in VSMCs than ECs (Fig. 3D).
There was minimal, if any, induction of IDO and lesser up-regu-
lation of TrpRS in peripheral blood CD4?Th cells compared with
ECs (data not shown). We confirmed that the greater induction of
IDO expression in VSMCs than in ECs correlated with orders of
magnitude greater IDO activity as measured by tryptophan deple-
tion, kynurenine production, and a ratio of metabolite to precursor
(Fig. 3, E–J).
IFN-?-treated VSMCs do not activate allogeneic memory
T helper cells
We compared the capacity of cultured vascular cells to activate
allogeneic human T cells. We have previously reported that IFN-
?-treated ECs that express MHC class II molecules, but not IFN-
?-treated, MHC class II Ag-expressing VSMCs, induced CD4?T
cell proliferation as assessed by [3H]thymidine uptake (16). We
confirmed these results using untreated or IFN-?-pretreated vas-
cular cells (Fig. 4A) and CFSE-labeled, memory CD45RO?/CD4?
T cells in a coculture system. A subset of alloreactive CD45RO?/
CD4?T cells proliferated in response to IFN-?-pretreated, MHC
class II Ag-expressing ECs as determined by a progressive in-
crease in the CFSElowpopulation of T cells after 7, 8 (data not
shown), and 9 days (Fig. 4B). In contrast, untreated ECs, untreated
VSMCs, and IFN-?-pretreated VSMCs, which express comparable
levels of MHC class II molecules as IFN-?-pretreated ECs, did not
activate allogeneic memory Th cells (Fig. 4B). Similarly, IFN-?-
pretreated ECs, but not VSMCs, stimulated IL-2 production by
CD45RO?/CD4?T cells after 2 days of coculture (Fig. 4C). The
lack of IL-2 secretion and T cell proliferation by IFN-?-pretreated
VSMCs was associated with greater tryptophan depletion and
kynurenine production than similarly treated ECs (Fig. 4, D–F).
IFN-?-treated VSMCs inhibit memory T helper cell activation
by allogeneic ECs
We next examined whether IFN-? induces an inhibitor of T cell
activation by VSMCs using a Transwell system. IFN-?-pretreated
VSMCs suspended within a semipermeable membrane insert
markedly inhibited the proliferation of CD45RO?/CD4?T cells
cocultured with IFN-?-pretreated, allogeneic ECs (Fig. 5, A and
allogeneic ECs. Untreated (?) or IFN-?-pretreated (?) ECs or VSMCs
were placed within semipermeable membrane inserts overlying CFSE-la-
beled CD4RO?/CD4?T cells cocultured with allogeneic untreated (?) or
IFN-?-pretreated (?) ECs. After 9 days, the cells under the Transwells
were labeled with CD4-PE and analyzed by flow cytometry (A and B).
Supernatants were removed after 2 days from the Transwell system and
analyzed for IL-2 (C) and kynurenine/tryptophan (KYN/TRP) ratios (D).
Data are means ? SEM; n ? 5; ?, p ? 0.001 all vs EC?control,†, p ?
0.001 VSMC?vs EC?, and§, p ? 0.001 VSMC?vs VSMC?(ANOVA).
IFN-?-treated VSMCs inhibit memory Th cell activation by
5250 VSMCs INHIBIT T CELL ALLORESPONSES VIA IDO
B). In contrast, untreated VSMCs and untreated or IFN-?-pre-
treated ECs did not have a suppressive effect on T cells. IFN-?-
pretreated VSMCs above the Transwell membrane also inhibited
IL-2 production by CD45RO?/CD4?T cells cocultured with IFN-
?-pretreated, allogeneic ECs below (Fig. 5C). Inhibition of T cell
activation by IFN-?-treated VSMCs was associated with signifi-
cant tryptophan depletion and kynurenine production (Fig. 5D).
Suppressor activity of VSMCs is dependent on
We then tested if induction of IDO activity by IFN-? was neces-
sary for the contact-independent immunosuppressive effect of
VSMCs. Strikingly, addition of 1-MT reversed the inhibitory effect
across a semipermeable membrane of IFN-?-pretreated VSMCs on
the proliferation of CD45RO?/CD4?T cells cocultured with IFN-
?-pretreated, allogeneic ECs (Fig. 6, A and B). There was no effect
of 1-MT on T cell proliferation when either no cells or untreated
VSMCs were placed within the Transwell inserts. Similarly, the
presence of 1-MT specifically reversed the IFN-?-dependent,
VSMC-mediated inhibition of IL-2 production by CD45RO?/
CD4?T cells cocultured with IFN-?-pretreated, allogeneic ECs
(Fig. 6C). As expected, 1-MT treatment diminished the depletion
of tryptophan and the production of kynurenine by IFN-?-pre-
treated VSMCs in the Transwell system (Fig. 6D). Tryptophan
supplementation produced similar results to 1-MT treatment in
specifically reversing the suppressor activity of IFN-?-treated
VSMCs on memory Th cell activation by allogeneic MHC class II
Ag-expressing ECs (Fig. 6E).
Tryptophan depletion is not sufficient for T cell anergy to
allogeneic VSMCs, but prevents T cell alloresponses to ECs
We also investigated whether IFN-?-inducible IDO activity was
responsible for the absence of T cell alloresponses to MHC class
II Ag-expressing VSMCs. Treatment with 1-MT or supplementa-
tion with L-tryptophan did not result in proliferation of CD45RO?/
CD4?T cells cocultured with IFN-?-pretreated, allogeneic
VSMCs (Fig. 7, A and B).
Finally, we examined if tryptophan depletion or metabolite pro-
duction was more important in IDO-mediated immunosuppression
using the coculture system. Tryptophan supplementation increased
the proliferation and IL-2 secretion of alloreactive, memory Th
cells in a dose-dependent fashion in tryptophan-deficient custom
medium (Fig. 7, C and D). In contrast, kynurenine treatment did
not significantly affect T cell clonal expansion or cytokine produc-
tion in tryptophan-replete conventional medium (Fig. 7, E and F).
Similarly, other tryptophan metabolites, such as 3-hydroxykynure-
nine and 3-hydroxyanthranilic acid, did not influence the activation
of CD45RO?/CD4?T cells cocultured with IFN-?-pretreated, al-
logeneic ECs (data not shown).
We describe the regulation of adaptive immune responses by vas-
cular cells through IFN-?-inducible expression of IDO in human
VSMCs that prevents T cell activation and clonal expansion in
response to allogeneic ECs in vitro. This bidirectional interaction
inhibits the accumulation of T cells within the medial compartment
in vivo in a humanized model of graft arteriosclerosis that is de-
pendent on IFN-? responses (7). We have identified an anti-
inflammatory factor expressed by VSMCs that we had predicted
from earlier work (5, 16). However, because IDO inhibition was
not sufficient to enable T cell activation in response to IFN-?-
treated VSMCs in coculture, an additional contact-dependent in-
hibitory molecule may be expressed on the cell surface of VSMCs
or alternatively VSMCs lack a nonredundant, unidentified co-
stimulatory molecule. We have not as yet investigated for possible
immune deviation, although we have previously reported that
dent on IFN-?-induced IDO. No cells, untreated (?), or
IFN-?-pretreated (?) VSMCs were placed within semi-
permeable membrane Transwell inserts overlying
CFSE-labeled CD4RO?/CD4?T cells cocultured with
allogeneic untreated (?) or IFN-?-pretreated (?) ECs
in the absence (open bars) or presence (filled bars) of
1-MT. After 9 days, the cells at the bottom of the wells
were labeled with CD4-PE and analyzed by flow cy-
tometry (A and B). Supernatants were also removed af-
ter 2 days and analyzed for IL-2 (C) and kynurenine/
tryptophan (KYN/TRP) ratios (D). Similarly, the cells
were cultured across Transwells in the absence (open
bars) or presence (filled bars) of L-tryptophan (TRP) and
analyzed for T cell proliferation as evidenced by CFSE
dilution after 9 days (E). Data are means ? SEM; n ?
5; ?. p ? 0.01 all vs EC?control;†, p ? 0.05 VSMC?
vs EC?;§, p ? 0.01 VSMC?vs VSMC?; and#, p ?
0.01 1-MT vs vehicle (ANOVA).
Suppressor activity of VSMCs is depen-
5251The Journal of Immunology
CD4?T cells initially exposed to IFN-?-pretreated VSMCs sub-
sequently proliferated with a similar pattern and magnitude to ECs
from the same donor vs freshly isolated T cells and that serologic
neutralization of TGF-? did not relieve the suppressive effects of
VSMCs in coculture with T cells (16).
Our results suggest that the inhibition of T cell alloresponses to
ECs by IDO-expressing VSMCs is predominantly due to trypto-
phan deprivation rather than generation of toxic metabolites. T
cells were not activated by allogeneic ECs in the presence of min-
imal concentrations of tryptophan (?0.5 ?mol/L from 0.5% serum
supplementation) that was similar to the level of tryptophan de-
pletion generated by IFN-?-treated VSMCs in vitro. In contrast,
three-fold higher levels of kynurenine (30 ?mol/L) than that pro-
duced by IFN-?-treated VSMCs had no effect on T cell allore-
sponses. Others have reported toxic effects to T cells by much
higher concentrations (?100 ?mol/L) of tryptophan metabolites
(17, 18) that are considerably higher than the levels we measured
in vitro and in vivo and exceed the available concentration of the
precursor, tryptophan in the blood. However, a similar criticism of
our work is that circulating levels of tryptophan are not as low as
we find in vitro and it cannot be excluded that different concen-
trations of tryptophan and its metabolites exist in intracellular or
local extracellular pools that are not in equilibrium with plasma
levels. We also did not exclude an immunosuppressive effect of
tryptophan metabolites in combination with tryptophan depletion.
The paradigm of IDO-mediated suppression of T cells responses
by tryptophan depletion has been extended from the original de-
scription in trophoblasts to macrophages and dendritic cells (19–
21). Recently, the role of tryptophan deprivation in immune reg-
ulation by dendritic cells was questioned in favor of the generation
of toxic tryptophan metabolites (22). Our data in VSMCs supports
the concept of adaptive immunity regulation by tryptophan
We selected 1-MT for our studies as it is a known inhibitor of
IDO in vivo and in vitro (14, 19). The possibility that VSMC loss
in vivo was caused by nonspecific toxic effects of 1-MT was elim-
inated as the inhibitor had no effect on the artery grafts in the
absence of allogeneic T cells. Pharmacologic inhibition of IDO
also had no effect on host reconstitution with human PBMCs. It is
unlikely that 1-MT has another target besides IDO as it does not
induce abortion of allogeneic fetuses in IDO-deficient mice, al-
though it elicits such in IDO-sufficient mice (12). Unexpectedly,
allogeneic pregnancies of IDO-deficient mice had similar out-
comes to those of IDO-sufficient mice (12). This discrepancy high-
lights the differences between transient blocking experiments and
permanent gene targeting and implies compensatory redundant im-
munosuppressive mechanisms in the placenta.
Depletion of tryptophan in the microenvironment inhibits T cell
activity without exerting overt negative effects on IDO-expressing
regulatory cells (15). There are several possible reasons for the
differential sensitivity to deprivation of an essential amino acid.
First, activated T cells undergo massive clonal expansion and fre-
quently dividing cells require a higher rate of protein synthesis.
Second, the rate of transmembrane transport is a limiting step in
tryptophan metabolism (23) and a putative high-affinity transporter
of tryptophan, defined only as a biochemical activity at present, is
T cell anergy to allogeneic VSMCs, but prevents T cell
alloresponses to ECs. Untreated (?) or IFN-?-pre-
treated (?) allogeneic ECs and VSMCs were cocultured
with CFSE-labeled CD4RO?/CD4?T cells in the ab-
sence or presence of 1-MT (A) or L-tryptophan (B) and
analyzed by flow cytometry after 9 days. Similar anal-
yses of allogeneic EC-T cell cocultures were also per-
formed in tryptophan-deficient medium supplemented
with 10% or 0.5% serum and different concentrations of
L-tryptophan (C) or in tryptophan-replete medium sup-
plemented with 10% serum and different concentrations
of kynurenine (D). IL-2 levels were also measured at 2
days after L-tryptophan (E) and kynurenine (F) supple-
mentation. Data are means ? SEM; n ? 3–8; ?, p ?
0.01 all vs EC?control (ANOVA).
Tryptophan depletion is not sufficient for
5252VSMCs INHIBIT T CELL ALLORESPONSES VIA IDO
expressed by APCs that may preferentially take up available amino
acid under suboptimal extracellular concentrations (24). Third,
tryptophan incorporation into protein biosynthesis by the amino-
acyl-tRNA synthetase, TrpRS may also be induced by IFN-? and
compensate for a reduction in intracellular tryptophan (25). We
found a direct correlation between IDO and TrpRS transcript in-
duction by IFN-? in vascular cells and CD4?T cells and others
have noted a similar differential regulation of IDO and TrpRS by
IFN-? in nonhemopoietic and myeloid vs lymphoid cell lines (26).
Finally, paracrine IDO activity may induce different signaling ef-
fects in target cells, such as activation of the stress kinase, general
control nonderepressible-2 in T cells (27).
The inducible expression of IDO has been described in diverse
cell types and IDO dysregulation has been implicated in various
animal models of disease and several clinical disorders (25). In
studies related to microbial infection, IFN-?-inducible IDO activ-
ity was detected at significantly higher levels in human VSMCs
than in ECs or PBMCs (28) and the IFN-?-dependent resistance to
certain pathogens in human vascular cells was dependent on IDO
activity (29–32). In transplantation-related studies, IFN-?-induced
IDO expression and activity was highest in umbilical vein ECs and
barely detectable in saphenous vein ECs or somatic artery ECs
(33). In contrast to our findings, Beutelspacher et al. reported that
addition of 1-MT to allogeneic T cell-umbilical vein EC cell co-
cultures did increase cellular proliferation (33); however their sys-
tem differs from ours in a number of conditions, including the use
of unfractionated T cells, pooled ECs from multiple donors, and
assessment of proliferation by [3H]thymidine incorporation. The T
cell suppressor activity by IFN-?-treated ECs in this study was
judged submaximal as overexpression of IDO in ECs further di-
minished T cell proliferation and also induced T cell apoptosis and
anergy. Our findings of the relatively low expression and activity
of IDO in ECs, compared with VSMCs, may explain why we see
that stimulatory functions of umbilical vein ECs predominate over
inhibitory interactions and result in net activation of allogeneic T
cells (16). The paradigm of IDO overexpression has been success-
fully used in animal transplantation models to prolong pancreatic
islet, lung, and corneal allograft survival (34–36), even though
inhibition of IDO had no effect on allograft survival (36). We have
recently described increased IDO activity as a marker of IFN-?
responses in patients with coronary atherosclerosis or acute rejec-
tion of allografts (37, 38), however the role of IDO in vascular
inflammation was not determined in these or other studies. Our
findings in human VSMCs, ECs, and arteries may not necessarily
apply to murine systems due to species differences in vascular cell
interactions with T cells. MHC class II Ag-expressing human ECs
can directly activate allogeneic memory CD4?T cells (39),
whereas IFN-?-treated murine ECs cannot (40). Conversely, mu-
rine VSMCs appear to be immunogenic and can activate T cells to
produce IFN-? and mediate vasculitis (41, 42).
The concept of medial immunoprivilege has been previously
described in the context of host defense against pathogens unlike
the classical definition of immune privileged sites in terms of al-
lograft rejection. Infection with ?-herpesvirus 68 or cytomegalo-
virus causes vasculitis of elastic arteries in mice that is more severe
and chronic in the absence of IFN-? responses (43, 44). The per-
sistent infection of VSMCs is due to inefficient clearance of virus
associated with a failure of T cells and macrophages to enter the
medial compartment and the investigators postulated that this may
reflect a fundamental property of elastic laminae to restrict traf-
ficking of leukocytes (8). Furthermore, genetic absence of IFN-?
receptors in vascular cells, but not leukocytes, and serologic neu-
tralization of IFN-? increased medial infiltration and necrosis (8).
These findings were interpreted as demonstrating a protective role
of IFN-? in vascular infection and, interestingly, IDO has been
shown to inhibit the replication of herpes simplex virus and cyto-
megalovirus (31, 45). Our results suggest an additional possible
explanation of loss of an IFN-?-inducible anti-inflammatory factor
in medial VSMCs that normally inhibits the accumulation of T
It is important to note that medial immunoprivilege is a relative
phenomenon. Transmural arterial inflammation and medial necro-
sis can occur in robust acute rejection episodes (46), and the di-
agnosis of transmural arteritis portends a poor outcome in acute
rejection of cardiac and renal allografts (47, 48). Similarly, pan-
arteritic infiltration and medial destruction is characteristic of cer-
tain vasculitides (49, 50). It is not surprising that the media is
capable of recruiting leukocytes under certain conditions as
VSMCs may be induced by cytokines to express many immuno-
genic and proinflammatory molecules (5, 16). We hypothesize that
medial immunoprivilege becomes manifest when a balance is
achieved between the limited resistance of the media to inflam-
mation vs the indolent IFN-?-producing immune responses char-
acteristic of atherosclerosis and graft arteriosclerosis. Although,
other proinflammatory factors have been reported to induce vas-
cular inflammation and the expression of IDO (25), we believe that
the evidence for a pathogenetic role for IFN-? in atherosclerosis
and graft arteriosclerosis is particularly compelling (51). In keep-
ing with our hypothesis for preferential IDO-mediated suppression
of T cells by VSMCs, we have found a relatively uniform trans-
mural infiltration of CD68?macrophages in human coronary ar-
tery grafts in SCID/beige mice reconstituted with human periph-
In conclusion, our work extends the understanding of medial
immunoprivilege from immune isolation to immune regulation and
supports the concept of distinct immunological responses within
separate vascular compartments and microenvironments. The
chronicity of arteriosclerotic diseases may in part result from the
duality of stimulatory EC signals versus inhibitory VSMC signals
to artery-infiltrating T cells. Finally, enhancement of the natural
resistance of the vascular wall to inflammation may represent a
novel strategy for treatment of atherosclerosis and graft
The authors have no financial conflict of interest.
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5254VSMCs INHIBIT T CELL ALLORESPONSES VIA IDO