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The Journal of Immunology, 2007, 179, 5246 -5254
Copyright © 2007 by The American Association of Immunologists, Inc.
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Induction of Indoleamine 2,3-Dioxygenase in Vascular Smooth Muscle Cells by Interferon-{gamma} Contributes to Medial Immunoprivilege1

Madison C. Cuffy*, Amanda M. Silverio*, Lingfeng Qin*, Yinong Wang*, Raymond Eid*, Gerald Brandacher§, Fadi G. Lakkis2,{dagger},{ddagger}, Dietmar Fuchs, Jordan S. Pober{ddagger} and George Tellides3,*

* Interdepartmental Program in Vascular Biology and Transplantation and the Departments of Surgery, {dagger} Internal Medicine, and {ddagger} 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 Medical University, Innsbruck, Austria


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Arteriosclerosis, the leading cause of mortality and morbidity worldwide, is characterized by inflammation, injury, and remodeling of the vessel wall. Coronary atherosclerosis 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 revealed that the leukocytic infiltrate of the arterial wall is not uniform. Infiltration by T cells and macrophages predominates in the intima and adventitia, whereas the media is relatively spared (1, 2, 3, 4). Similar findings of a relatively bland media have also been noted in experimental models of atherosclerosis and graft arteriosclerosis (5, 6, 7). The mechanism(s) for medial immunoprivilege is unknown, although it has been proposed that elastic laminae found in that arterial layer may prevent leukocyte trafficking (8).

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 described the brain and anterior chamber of the eye as immunologically privileged sites due to an absence of lymphatics (preventing afferent immune responses) and blood vessels (preventing efferent immune responses), respectively (9). More recently, the immunoprivileged status of the brain and eye has been ascribed to immune deviation due to a number of factors in the local microenvironment, including neuropeptides, TGF -beta, 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 expression of IDO by trophoblast cells (12). IDO, an IFN-{gamma}-inducible, 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),4 a pharmacologic agent that inhibits 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, induces T cell ignorance, anergy, or deviation, and generates regulatory T cells (15).

In the present study, we investigated whether medial immunoprivilege in graft arteriosclerosis results from an anti-inflammatory factor produced by the vessel wall. We find that the IFN-{gamma}-inducible 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
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Artery grafting

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 µg/ml) SCID/beige mice (Taconic Farms) using an end-to-end microsurgical 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 x 108 human 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 Research 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-{gamma} or Ad5.CMV-LacZ (Qbiogene) at 1 x 109 plaque-forming units i.v. at 1 wk postoperatively and circulating human IFN-{gamma} was confirmed by ELISA (R&D Systems) after 2 wk (data not shown).

Graft analysis

Artery grafts were procured at 5 wk postoperatively (4 wk after PBMC or cytokine treatment) and analyzed by immunohistochemistry using mouse anti-human CD45RO, {alpha}-smooth muscle actin ({alpha}-SMA; DakoCytomation), and IDO (Chemicon International). Binding of secondary Ab (Jackson Immunoresearch) was detected with peroxidase/3-amino-ethyl carbazole kits (Vector Laboratories). Cell counting of nuclei surrounded by positive immunostaining 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 previously described (7).

Cell isolation

Human endothelial cells (ECs) were isolated by enzymatic harvesting from umbilical cord veins and serially cultured in M199 medium (containing L-tryptophan at 49 µmol/L) supplemented with 20% FBS, 2 mmol/L L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (all from Invitrogen), 50 µg/ml fibroblast growth factor-1 (Collaborative Research), and 100 µg/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 Dynabeads (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 µg/ml. Isolated 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 µmol/L) supplemented with 10% FBS, L-glutamine, and antibiotics.

Cell culture

Coculture experiments were performed by placing 2 x 105 ECs or VSMCs in gelatin-coated wells of 24-well culture plates and where indicated treated with IFN-{gamma} (Biosource International) at 100 ng/ml for 3 days, washed in medium, and both IFN-{gamma}-treated ECs and VSMCs were confirmed to express class II MHC Ags before every experiment by FACS 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 x 106 CD45RO+/CD4+ T cells in 1 ml of supplemented RPMI 1640 medium was added to wells containing vascular cells. The cultures were maintained in 5% CO2 at 37°C for up to 9 days. T cell proliferation was assessed by FACS analysis of CFSE dilution after counterstaining with PE-labeled mouse anti-human CD4 Ab (Immunotech System). IL-2 supernatant levels were measured by ELISA (eBioscience) according 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 cocultures were maintained in conventional RPMI 1640 medium supplemented with 10% FBS and different doses of L-tryptophan, L-kynurenine, 3-hydroxy-DL-kynurenine, or 3-hydroxyanthranilic acid (Sigma-Aldrich).

In experiments using the Transwell system, 2 x 105 ECs or VSMCs were placed in gelatin-coated 0.4-µm 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 µmol/L on day 1, and 24.5 µmol/L daily, respectively.

HPLC

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 wavelengths, and kynurenine was detected by UV absorption at 365 nm wavelength in the same chromatographic run.

Western blotting

IFN-{gamma}-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 beta-actin or IDO (Chemicon) followed by HRP-conjugated secondary Abs (Jackson ImmunoResearch). Detection of the bound Ab by ECL (Pierce Biotechnology) was performed according to the manufacturer’s instructions.

Quantitative PCR

Total RNA was isolated from IFN-{gamma}-treated and DNase-treated cells using NucleoSpin RNA II kits (Clontech Laboratories). Bulk reverse transcription with random hexamer primers was performed according to the Multiscribe RT system protocol (Applied Biosystems). RT-PCR were prepared with TaqMan 2 x PCR Master mix and predeveloped assay reagents for IDO, TrpRS, and GAPDH (Applied Biosystems). An iCycler and its system 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 of GAPDH.

Statistical analyses

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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Medial Sparing by Allogeneic T cells is Associated with IDO Expression by VSMCs

We have previously reported an experimental model of graft arteriosclerosis of human coronary arteries interposed into the infrarenal 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 infiltrate 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 outward vascular remodeling (increase in vessel diameter). We examined 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 rejection and local up-regulation of IDO in the vessel wall did not perturb systemic levels of tryptophan and kynurenine in the xenogeneic hosts (Table I).


Figure 1
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  		FIGURE 1. Medial sparing by allogeneic T cells is associated with IDO expression by VSMCs. Immunohistochemical analyses of human coronary artery grafts were performed 4 wk after no treatment (left panels) or allogeneic PBMC reconstitution (right panels) of SCID/beige mouse recipients using Abs to CD45RO (A), {alpha}-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 µm for all panels. Intimal, medial, and adventitial CD45RO+ cells (D) were counted and normalized to the area of each compartment and medial {alpha}-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).

 

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  		Table I. TRP and KYN plasma levelsa

 
Inhibition of IDO increases medial infiltration and VSMC loss

To determine whether IDO was necessary for medial immunoprivilege, we treated pairs of SCID/beige mice that received adjacent 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 placebo (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, {alpha}-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 infiltration 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 infiltration (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 tryptophan and kynurenine did not differ between the treatment groups (Table I).


Figure 2
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  		FIGURE 2. Inhibition of IDO increases medial infiltration and VSMC loss. CD45RO (A), {alpha}-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 {alpha}-SMA expression in artery grafts from unreconstituted hosts was also performed (B insets). The bar represents 100 µm for all panels. Medial CD45RO+ cells (D) and {alpha}-SMA+ cells (E) were counted in placebo-treated (open symbols) or 1-MT-treated (filled symbols) PBMC-reconstituted animals. Data are means ± SEM, n = 6, *, p < 0.01 vs paired control grafts (t test).

 
IFN-{gamma} induces IDO expression and activity in VSMCs

We further investigated the regulation and activity of IDO in human vascular cells. Exposure to the T cell-derived cytokine, IFN-{gamma}, in the absence of allogeneic PBMCs, was sufficient to induce the expression of IDO by graft VSMCs in vivo (Fig. 3A). IFN-{gamma} 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-{gamma}-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 protein synthesis, was also far greater in VSMCs than ECs (Fig. 3D). There was minimal, if any, induction of IDO and lesser up-regulation 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 depletion, kynurenine production, and a ratio of metabolite to precursor (Fig. 3, E–J).


Figure 3
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  		FIGURE 3. IFN-{gamma} induces IDO expression and activity in VSMCs. A, IDO expression was analyzed by immunohistochemistry in human coronary artery grafts of SCID/beige mouse recipients 4 wk after i.v. infection with Ad-LacZ (left panel) or Ad-IFN-{gamma} (right panel). The bar represents 100 µm for both panels. B, IDO protein expression was also analyzed by Western blotting of cultured VSMCs treated with IFN-{gamma} for various times and at different doses and compared with the expression of beta-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-{gamma} 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-{gamma} 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-{gamma}-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-{gamma}-treated ECs that express MHC class II molecules, but not IFN-{gamma}-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-{gamma}-pretreated vascular 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-{gamma}-pretreated, MHC class II Ag-expressing ECs as determined by a progressive increase in the CFSElow population of T cells after 7, 8 (data not shown), and 9 days (Fig. 4B). In contrast, untreated ECs, untreated VSMCs, and IFN-{gamma}-pretreated VSMCs, which express comparable levels of MHC class II molecules as IFN-{gamma}-pretreated ECs, did not activate allogeneic memory Th cells (Fig. 4B). Similarly, IFN-{gamma}-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-{gamma}-pretreated VSMCs was associated with greater tryptophan depletion and kynurenine production than similarly treated ECs (Fig. 4, D–F).


Figure 4
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  		FIGURE 4. IFN-{gamma}-treated VSMCs do not activate allogeneic memory Th cells. Allogeneic untreated (–) or IFN-{gamma}-pretreated (+) ECs and VSMCs that either had undetectable or uniform expression of MHC class II Ags, respectively (A) were cocultured with CFSE-labeled CD4RO+/CD4+ T cells. After 9 days, the cells were labeled with CD4-PE and analyzed by flow cytometry (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; {dagger}, p < 0.01 VSMC+ vs EC+; and §, p < 0.001 VSMC+ vs VSMC– (ANOVA).

 
IFN-{gamma}-treated VSMCs inhibit memory T helper cell activation by allogeneic ECs

We next examined whether IFN-{gamma} induces an inhibitor of T cell activation by VSMCs using a Transwell system. IFN-{gamma}-pretreated VSMCs suspended within a semipermeable membrane insert markedly inhibited the proliferation of CD45RO+/CD4+ T cells cocultured with IFN-{gamma}-pretreated, allogeneic ECs (Fig. 5, A and B). In contrast, untreated VSMCs and untreated or IFN-{gamma}-pretreated ECs did not have a suppressive effect on T cells. IFN-{gamma}-pretreated VSMCs above the Transwell membrane also inhibited IL-2 production by CD45RO+/CD4+ T cells cocultured with IFN-{gamma}-pretreated, allogeneic ECs below (Fig. 5C). Inhibition of T cell activation by IFN-{gamma}-treated VSMCs was associated with significant tryptophan depletion and kynurenine production (Fig. 5D).


Figure 5
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  		FIGURE 5. IFN-{gamma}-treated VSMCs inhibit memory Th cell activation by allogeneic ECs. Untreated (–) or IFN-{gamma}-pretreated (+) ECs or VSMCs were placed within semipermeable membrane inserts overlying CFSE-labeled CD4RO+/CD4+ T cells cocultured with allogeneic untreated (–) or IFN-{gamma}-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, {dagger}, p < 0.001 VSMC+ vs EC+, and §, p < 0.001 VSMC+ vs VSMC– (ANOVA).

 
Suppressor activity of VSMCs is dependent on IFN-{gamma}-induced IDO

We then tested if induction of IDO activity by IFN-{gamma} was necessary for the contact-independent immunosuppressive effect of VSMCs. Strikingly, addition of 1-MT reversed the inhibitory effect across a semipermeable membrane of IFN-{gamma}-pretreated VSMCs on the proliferation of CD45RO+/CD4+ T cells cocultured with IFN-{gamma}-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-{gamma}-dependent, VSMC-mediated inhibition of IL-2 production by CD45RO+/CD4+ T cells cocultured with IFN-{gamma}-pretreated, allogeneic ECs (Fig. 6C). As expected, 1-MT treatment diminished the depletion of tryptophan and the production of kynurenine by IFN-{gamma}-pretreated VSMCs in the Transwell system (Fig. 6D). Tryptophan supplementation produced similar results to 1-MT treatment in specifically reversing the suppressor activity of IFN-{gamma}-treated VSMCs on memory Th cell activation by allogeneic MHC class II Ag-expressing ECs (Fig. 6E).


Figure 6
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  		FIGURE 6. Suppressor activity of VSMCs is dependent on IFN-{gamma}-induced IDO. No cells, untreated (–), or IFN-{gamma}-pretreated (+) VSMCs were placed within semipermeable membrane Transwell inserts overlying CFSE-labeled CD4RO+/CD4+ T cells cocultured with allogeneic untreated (–) or IFN-{gamma}-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 cytometry (A and B). Supernatants were also removed after 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; {dagger}, p < 0.05 VSMC+ vs EC+; §, p < 0.01 VSMC+ vs VSMC–; and #, p < 0.01 1-MT vs vehicle (ANOVA).

 
Tryptophan depletion is not sufficient for T cell anergy to allogeneic VSMCs, but prevents T cell alloresponses to ECs

We also investigated whether IFN-{gamma}-inducible IDO activity was responsible for the absence of T cell alloresponses to MHC class II Ag-expressing VSMCs. Treatment with 1-MT or supplementation with L-tryptophan did not result in proliferation of CD45RO+/CD4+ T cells cocultured with IFN-{gamma}-pretreated, allogeneic VSMCs (Fig. 7, A and B).


Figure 7
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  		FIGURE 7. Tryptophan depletion is not sufficient for T cell anergy to allogeneic VSMCs, but prevents T cell alloresponses to ECs. Untreated (–) or IFN-{gamma}-pretreated (+) allogeneic ECs and VSMCs were cocultured with CFSE-labeled CD4RO+/CD4+ T cells in the absence or presence of 1-MT (A) or L-tryptophan (B) and analyzed by flow cytometry after 9 days. Similar analyses of allogeneic EC-T cell cocultures were also performed in tryptophan-deficient medium supplemented with 10% or 0.5% serum and different concentrations of L-tryptophan (C) or in tryptophan-replete medium supplemented 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) supplementation. Data are means ± SEM; n = 3–8; *, p < 0.01 all vs EC– control (ANOVA).

 
Finally, we examined if tryptophan depletion or metabolite production 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 production in tryptophan-replete conventional medium (Fig. 7, E and F). Similarly, other tryptophan metabolites, such as 3-hydroxykynurenine and 3-hydroxyanthranilic acid, did not influence the activation of CD45RO+/CD4+ T cells cocultured with IFN-{gamma}-pretreated, allogeneic ECs (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We describe the regulation of adaptive immune responses by vascular cells through IFN-{gamma}-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 dependent on IFN-{gamma} 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-{gamma}-treated VSMCs in coculture, an additional contact-dependent inhibitory molecule may be expressed on the cell surface of VSMCs or alternatively VSMCs lack a nonredundant, unidentified costimulatory molecule. We have not as yet investigated for possible immune deviation, although we have previously reported that CD4+ T cells initially exposed to IFN-{gamma}-pretreated VSMCs subsequently proliferated with a similar pattern and magnitude to ECs from the same donor vs freshly isolated T cells and that serologic neutralization of TGF-beta 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 tryptophan deprivation rather than generation of toxic metabolites. T cells were not activated by allogeneic ECs in the presence of minimal concentrations of tryptophan (<0.5 µmol/L from 0.5% serum supplementation) that was similar to the level of tryptophan depletion generated by IFN-{gamma}-treated VSMCs in vitro. In contrast, three-fold higher levels of kynurenine (30 µmol/L) than that produced by IFN-{gamma}-treated VSMCs had no effect on T cell alloresponses. Others have reported toxic effects to T cells by much higher concentrations (>100 µmol/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 concentrations 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 description in trophoblasts to macrophages and dendritic cells (19, 20, 21). Recently, the role of tryptophan deprivation in immune regulation 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 catabolism.

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 eliminated 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, although it elicits such in IDO-sufficient mice (12). Unexpectedly, allogeneic pregnancies of IDO-deficient mice had similar outcomes to those of IDO-sufficient mice (12). This discrepancy highlights the differences between transient blocking experiments and permanent gene targeting and implies compensatory redundant immunosuppressive 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 frequently 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 expressed by APCs that may preferentially take up available amino acid under suboptimal extracellular concentrations (24). Third, tryptophan incorporation into protein biosynthesis by the aminoacyl-tRNA synthetase, TrpRS may also be induced by IFN-{gamma} and compensate for a reduction in intracellular tryptophan (25). We found a direct correlation between IDO and TrpRS transcript induction by IFN-{gamma} in vascular cells and CD4+ T cells and others have noted a similar differential regulation of IDO and TrpRS by IFN-{gamma} in nonhemopoietic and myeloid vs lymphoid cell lines (26). Finally, paracrine IDO activity may induce different signaling effects 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-{gamma}-inducible IDO activity was detected at significantly higher levels in human VSMCs than in ECs or PBMCs (28) and the IFN-{gamma}-dependent resistance to certain pathogens in human vascular cells was dependent on IDO activity (29, 30, 31, 32). In transplantation-related studies, IFN-{gamma}-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 cocultures did increase cellular proliferation (33); however their system 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-{gamma}-treated ECs in this study was judged submaximal as overexpression of IDO in ECs further diminished 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 successfully used in animal transplantation models to prolong pancreatic islet, lung, and corneal allograft survival (34, 35, 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-{gamma} responses in patients with coronary atherosclerosis or acute rejection 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-{gamma}-treated murine ECs cannot (40). Conversely, murine VSMCs appear to be immunogenic and can activate T cells to produce IFN-{gamma} 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 allograft rejection. Infection with {gamma}-herpesvirus 68 or cytomegalovirus causes vasculitis of elastic arteries in mice that is more severe and chronic in the absence of IFN-{gamma} responses (43, 44). The persistent 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 trafficking of leukocytes (8). Furthermore, genetic absence of IFN-{gamma} receptors in vascular cells, but not leukocytes, and serologic neutralization of IFN-{gamma} increased medial infiltration and necrosis (8). These findings were interpreted as demonstrating a protective role of IFN-{gamma} in vascular infection and, interestingly, IDO has been shown to inhibit the replication of herpes simplex virus and cytomegalovirus (31, 45). Our results suggest an additional possible explanation of loss of an IFN-{gamma}-inducible anti-inflammatory factor in medial VSMCs that normally inhibits the accumulation of T cells.

It is important to note that medial immunoprivilege is a relative phenomenon. Transmural arterial inflammation and medial necrosis can occur in robust acute rejection episodes (46), and the diagnosis of transmural arteritis portends a poor outcome in acute rejection of cardiac and renal allografts (47, 48). Similarly, panarteritic infiltration and medial destruction is characteristic of certain 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 immunogenic 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 inflammation vs the indolent IFN-{gamma}-producing immune responses characteristic of atherosclerosis and graft arteriosclerosis. Although, other proinflammatory factors have been reported to induce vascular inflammation and the expression of IDO (25), we believe that the evidence for a pathogenetic role for IFN-{gamma} in atherosclerosis and graft arteriosclerosis is particularly compelling (51). In keeping with our hypothesis for preferential IDO-mediated suppression of T cells by VSMCs, we have found a relatively uniform transmural infiltration of CD68+ macrophages in human coronary artery grafts in SCID/beige mice reconstituted with human peripheral blood CD34+ hemopoietic stem cells (unpublished observations).

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 arteriosclerosis.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.

1 This 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. Back

2 Current address: Thomas E. Starzl Transplantation Institute, University of Pittsburgh, BST-W1542, 200 Lothrop Street, Pittsburgh, PA 15261 Back

3 Address correspondence and reprint requests to Dr. George Tellides, 295 Congress Avenue, Boyer Center for Molecular Medicine 454, New Haven, CT 06510. E-mail address: george.tellides{at}yale.edu Back

4 Abbreviations used in this paper: 1-MT, 1-methyl-tryptophan; {alpha}-SMA, {alpha}-smooth muscle actin; EC, endothelial cell; TrpRS, tryptophanyl-tRNA synthetase; VSMC, vascular smooth muscle cell. Back

Received for publication April 6, 2007. Accepted for publication August 12, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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