Published online before print September 22, 2006
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,1
* Immunotherapy Center and Departments of
Biochemistry and Molecular Biology,
Medicine, and
Pediatrics, Medical College of Georgia, Augusta, Georgia, USA
1 Correspondence: Immunotherapy Center, CN-4141, Medical College of Georgia, Augusta, GA 30912, USA. E-mail: dmunn{at}mail.mcg.edu
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Key Words: indoleamine 2,3-dioxygenase • antigen-presenting cells • tolerance
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, IFN-
, and other proinflammatory signals [1
2
3
]. The IDO system, whose evolutionary origins extend back to invertebrates [4
], has been hypothesized to block the replication of certain intracellular pathogens and viruses [5
6
7
8
]. In addition, IDO has been shown to function as a potent regulatory mechanism in the immune system (reviewed in ref. [9
]). IDO has been implicated in maternal tolerance toward the allogeneic fetus [10
, 11
], control of T cell proliferation and anergy by APC [12
13
14
15
], and regulation of antitumor immunity [16
17
18
]. Transfection of recombinant IDO cDNA into tumors, cell lines, or tissue grafts confers the ability to inhibit T cell responses in vivo [16
, 19
20
21
]. Thus, IDO-mediated tryptophan catabolism is emerging as an important pathway in immune regulation. In humans, IDO can be expressed by human monocyte-derived macrophages (MDM) and by certain monocyte-derived dendritic cells. When active IDO enzyme is expressed by these APC, it allows them to inhibit T cell proliferation in vitro [17 , 22 23 24 25 ]. However, IDO is an intracellular enzyme, and tryptophan is not membrane-permeant; therefore, tryptophan must be transported across the plasma membrane of APC to reach IDO. In placental trophoblast cells, the transmembrane transport step has been shown to constitute the rate-limiting step in tryptophan catabolism by IDO [26 ]. Therefore, in the case APC, an efficient tryptophan transport system would appear to be a critical requirement for effective IDO-mediated immune regulation.
Human MDM rapidly degrade tryptophan in vitro in response to signals from T cells, which in turn, possess a tryptophan-sensitive, cell-cycle checkpoint that prevents their proliferation in low tryptophan [22 ]. However, this checkpoint only operates at low levels of tryptophan (below 1–2 µM [15 ]). Although MDM are capable of depleting extracellular tryptophan into this range in vitro [22 ], it has been unclear how MDM would be able to take up tryptophan efficiently at such low levels. The known amino acid transport systems that accept tryptophan have substrate affinities (Michaelis constant, Km values) in the range of 10 µM–1 mM [27 , 28 ]. Therefore, conventional transport systems would be inefficient at nanomolar concentrations of tryptophan. Moreover, all of the known transport systems accept multiple amino acids besides tryptophan [27 , 28 ]. As these other substrates would be present at concentrations orders of magnitude greater than tryptophan, they should act as virtually complete, competitive inhibitors of tryptophan uptake. We therefore asked whether there existed a specialized transport activity with high affinity and selectivity for tryptophan in MDM.
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Isolation and culture of monocytes, T cells, and THP1 cell line
Human MDM were selected to study tryptophan transport, as we have shown previously that they display high levels of IDO activity and tryptophan uptake and are potently suppressive for T cells [22
]. Peripheral blood monocytes and T cells were isolated from healthy volunteer donors by leukocytapheresis and counterflow centrifugal elutriation as described previously [22
] following appropriate, informed consent under a protocol approved by our Institutional Review Board (Medical College of Georgia, Augusta). Monocytes (>95% purity by cell surface markers [24
, 29
]) were cultured as described previously [22
] for 7 days in the presence of M-CSF (200 U/ml). The concentration of tryptophan in the culture medium was 25 uM. T cells (>80% pure, balance B cells, <2% monocytes) were activated using immobilized anti-CD3 mAb [Clone OKT3, American Type Culture Collection (ATCC), Manassas, VA] plus soluble anti-CD28 (PharMingen, San Diego, CA). The THP1 monocytic leukemia cell line and the Jurkat T cell line were obtained from ATCC.
Amino acid uptake measurements
Single-cell suspensions were obtained by treating MDM monolayers with 2 mM EDTA in HBSS for 15 min at room temperature. T cells and cell lines were grown as suspension cultures. Cells were washed twice in Tris-choline buffer (150 mM choline chloride, 10 mM Tris, pH 7.4) and suspended in Tris-choline at 1–2 x 106 cells/ml, and 100 µl/well was added to 96-well microtiter plates. Radiolabeled amino acids were then added to the wells, and uptake was measured over 10 min at room temperature.
The 2-min time-point was treated as the initial uptake rate for purposes of kinetic analysis. Preliminary validation experiments (not shown) indicated that uptake times below 2 min were unreliable as a result of low signal-to-noise ratio, and uptake became nonlinear beyond 15 min. Uptake was linear between 2 and 10 min, and comparable Km values were obtained at 5 and 10 min. Comparable Km values were obtained in the presence or absence of cyclohexamide (4 µg/ml), thus ruling out any distorting effect of protein synthesis during the assay period. Uptake of tryptophan was reduced by >90% at 4°C (using 10 min and 30 min time-points), indicating that accumulation of cell-associated tryptophan was consistent with transmembrane transport rather than surface-receptor binding.
At the end of the assay period, cells were harvested onto glass-fiber filters using a Tomtec 96-well parallel harvester and washed vigorously for 30 s with PBS solution (140 mM NaCl, 10 mM sodium phosphate buffer, pH 7.4). Nonspecific binding of radioactivity to the filters (based on wells containing radiolabeled substrate without cells) was typically <10% of the total signal and was subtracted from each data point. All assays were performed in triplicate. Where the standard deviation between replicates was <10%, error bars have been omitted from the figures for clarity. In all assays, the uptake rate (V) was normalized to cell number and expressed as pmol · 106 cells–1· 10 min–1. Normalization of uptake to protein content gave identical conclusions but was considered less informative, as protein content can change markedly over the course of MDM differentiation, independently of other functional attributes [29 ].
Analysis of saturation kinetics
Uptake kinetics was analyzed using Eadie-Hofstee transformation (V/[S] vs. V, where [S] is the concentration of labeled substrate) to determine the kinetic parameters for each transport system. Data from the two concentration ranges of tryptophan (<1 µM and >8 µM) were then analyzed separately using double-reciprocal (Lineweaver-Burk, 1/[S] vs. 1/V) plots to determine Km and maximum velocity (Vmax) for the low-affinity and high-affinity transport systems. In all cases, comparable results were also obtained if Km and Vmax were estimated from the Eadie-Hofstee plots. Analysis of the data over the entire range of tryptophan concentrations was also carried out using a model consisting of two independent transport systems functioning in parallel [30
]. Kinetic parameters were calculated on a minimum of four to six points and were considered reliable if the correlation coefficient of the regression line was >0.95.
For competitive inhibition studies, the affinity constant of the inhibitor for the transporter (Ki) value for each competitor was derived by fitting the measured inhibition curve to the formula shown in Equation 1, as described in ref. [31
]: Vi = Vmax · [S]/Km · (1+[I]/Ki) + [S] (Eq. 1), where [I] is the concentration of unlabeled inhibitor, Vi is the observed rate of uptake in the presence of inhibitor, and Km and Vmax have their customary meanings. This equation, representing a single transport system, was used for the determination of Ki values, as under the experimental conditions used in these studies (i.e., 0.125 µM tryptophan), the high-affinity transport system was primarily (
90%) responsible for the observed uptake. The affinity constants of the low-affinity and high-affinity transport systems differed so widely (100-fold) that data for each transport system closely fit a single-system model at substrate concentrations surrounding the respective Km values.
In all figures showing inhibition studies, the observed data are shown superimposed on the predicted inhibition curve calculated from Equation 1 for a single transport system, and Ki is equal to the observed IC50. The closeness of fit between the observed data and the calculated curve thus reflects the degree to which the data conform to the behavior of a single system following classical Michaelis-Menten kinetics.
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Figure 1. Sodium-independent transport of tryptophan. MDM were incubated with [3H]tryptophan (125 nM) in Tris-buffered saline solution ( ) or sodium-free buffer (isotonic choline chloride buffer,  ). Measurements began after 2 min to allow for mixing and continued until 10 min. Regression lines are superimposed on each dataset. Bars show SD of triplicate determinations. Data are from a representative experiment, and similar data were obtained in three other experiments. TRP, Tryptophan.
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Figure 2. Saturation kinetics of tryptophan transport. MDM were incubated with varying concentrations of [3H]tryptophan as described in Materials and Methods, and initial uptake rates (V) were measured as a function of substrate concentration ([S]). (A) Lineweaver-Burk plots of the uptake data, analyzed separately for low (<1 µM) and high (>8 µM) ranges of substrate concentrations. The superimposed lines show linear regression analysis of each dataset. (B) Eadie-Hofstee plot of data for tryptophan uptake over a broad concentration range (32 nM–64 µM), demonstrating the presence of high- and low-affinity transport systems. The superimposed lines represent hypothetical curves (calculated using the Michaelis-Menten equation) using the Km and Vmax values obtained for the low-affinity and high-affinity systems from the data shown in A. Data are from a representative experiment, and similar results were obtained in five other experiments. (C) Saturation kinetics for uptake at the low range of substrate concentrations (32 nM–2.25 µM) over which the high-affinity system predominated. The curve shows the least-squares fit to the observed data. The inset shows an Eadie-Hofstee transformation of the same dataset, yielding a calculated Km value of 230 nM in this experiment (regression line for the transformed data is superimposed). Data are from a representative experiment, and similar results were obtained in 17 other experiments.
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The high-affinity system is distinct from system L
The Km value of the low-affinity system was consistent with values reported for system L [32
33
34
35
36
], a widely expressed, sodium-independent transport system that accepts tryptophan and other large neutral amino acids [37
]. We therefore performed competition studies using the prototypical system L substrate, 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BCH). Figure 3A
shows that BCH inhibited the uptake of [3H]tryptophan (125 nM). The IC50 value (i.e., concentration of BCH necessary for 50% inhibition) calculated from the dose-response relationship was 30 µM. Although this value is comparable with the affinity of BCH to the classical system L, tryptophan uptake at a concentration of 125 nM is mediated primarily through the high-affinity system rather than by system L, a low-affinity system. This was evident from the findings that under these conditions, unlabeled tryptophan was able to block the uptake of [3H]tryptophan with an IC50 value of 300–400 nM. This suggests that BCH interacts not only with the classical system L but also with the high-affinity system and that although the affinities of tryptophan for the two systems differ by 100-fold, the affinities of BCH for the two systems are similar.
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Figure 3. Interaction of BCH, a canonical system L substrate, with the tryptophan uptake systems. (A) Various concentrations of BCH ( ) or unlabeled tryptophan ( ) were allowed to compete for uptake with [3H]tryptophan (125 nM). The observed data approximate well to hypothetical inhibition curves (dotted lines), which would be expected for a Ki value of 30 µM (for BCH) or 300 nM (for tryptophan). (B) Saturation kinetics for tryptophan uptake was performed in the presence (•) or absence () of 500 µM BCH. Over the concentration range, where system L predominated (>8 µM tryptophan), BCH acted as a competitive inhibitor. Regression lines are superimposed. (C) Over the range where the high-affinity system predominated (<1 µM tryptophan), BCH acted as a noncompetitive inhibitor. Data are representative of eight similar experiments using BCH, phenylalanine, or leucine as inhibitors, all with similar results.
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The high-affinity system is selective for tryptophan
Additional competition studies (Fig. 4
) showed that none of the other amino acids tested was able to compete with [3H]tryptophan for uptake via the high-affinity system. In these experiments, uptake of tryptophan was measured at a low concentration of [3H]tryptophan (200 nM, chosen to be near the Km of the high-affinity system) in the absence or presence of a 20-fold excess of 11 different unlabeled amino acids as competitors. Under these conditions, none of the amino acids other than tryptophan competed significantly with [3H]tryptophan for uptake. In contrast, unlabeled tryptophan itself competed effectively with [3H]tryptophan, showing 80% inhibition of uptake (which was the amount predicted from Equation 1 under these conditions for the high-affinity system). This suggested that the high-affinity system was unusually selective for tryptophan.
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Figure 4. The high-affinity transport system is selective for tryptophan. Competition studies were performed using a low concentration of [3H]tryptophan (200 nM) in the absence or presence of 20-fold excess of unlabeled amino acids (4 µM). Data are expressed as [3H]tryptophan uptake relative to control without the inhibitors. Data are from a representative experiment, and similar results were obtained in three other experiments. VAL, Valine; TYR, tyrosine; THR, threonine; PHE, phenylalanine; MET, methionine; LYS, lysine; LEU, leucine; ILE, isoleucine; HIS, histidine; ARG, arginine.
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Figure 5. The high-affinity system is distinct from system L. A titration of unlabeled tryptophan was allowed to compete for uptake with 125 nM [3H]leucine () or [3H]phenylalanine (), both of which are known substrates for system L. As a control, the same titration of unlabeled tryptophan was allowed to compete with 125 nM [3H]tryptophan (). Data are expressed as percentage uptake of radiolabeled amino acids compared with control (uptake without competitor). The superimposed. dotted lines show the hypothetical inhibition curves (calculated from Equation 1) expected for competition for a system with Ki= Km = 30 µM or Ki= Km = 300 nM. Data are from a representative experiment, and similar results were obtained in eight other experiments.
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Table 1. Specificity of the High-Affinity Transport System for Tryptophan Versus Closely Related Structural Analogsa
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Figure 6. Up-regulation of the high-affinity tryptophan transport system during MDM differentiation. Monocytes were allowed to differentiate in vitro for up to 7 days under the influence of M-CSF and analyzed at the time-points shown. Tryptophan uptake kinetics was analyzed, and Vmax of the high-affinity system was calculated from the regression lines of the Eadie-Hofstee plots. Data are from a representative experiment, and similar results were obtained in three other experiments performed on Day 0 (A) and five other experiments performed on Days 4–7. (C) Time course showing up-regulation of the high-affinity system (Vmax; pmol·106 cells–1·10 min–1) during MDM differentiation.
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Table 2. Analysis of the High-Affinity Tryptophan Transport System in Macrophages and T Cellsa
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All of the major mammalian amino acid transport systems identified to date have a relatively modest affinity for substrate (Km values ranging from 10 µM to several millimolar). This low affinity would be consistent with the fact that amino acids are normally abundant in vivo (plasma concentrations typically 50 µM–1 mM), as a high-affinity transport system may not be required to mediate uptake under these conditions. However, in the special case of cells expressing IDO, it becomes possible for the local concentration of tryptophan to become significantly lower than that found in plasma [38 ]. This specialized circumstance may explain the biologic importance of a high-affinity transport system. For similar reasons, the selectivity of the system for tryptophan becomes important when the concentration of tryptophan becomes low compared with other amino acids. For example, system L accepts at least 10 other amino acids [27 , 28 ], which together, would be present at millimolar concentrations. These would saturate system L and thus, act as essentially complete, competitive inhibitors of tryptophan uptake.
We have recently shown that IDO expression by APC triggers activation of the stress-kinase known as GCN2 in neighboring T cells [15 ]. Activation of the GCN2 pathway is mechanistically required for IDO-induced cell-cycle arrest and anergy [15 ]. GCN2 kinase is activated by an increase in the pool of uncharged transfer-RNA, as would occur if the T cell were deprived of tryptophan. Thus, local tryptophan depletion may serve as a mechanistic component of IDO-mediated effects in vivo. Alternatively, it has been suggested that IDO may affect T cells via production of immunomodulatory tryptophan metabolites [39 , 40 ]. These are not mutually exclusive hypotheses, and in either case, the effects of IDO would depend critically on efficient transmembrane transport of tryptophan into the APC.
Several recent reports have shown that the rate of transmembrane transport is a limiting step in tryptophan degradation by IDO. In human placental trophoblasts [41 ] and breast carcinoma cell lines [42 ], IDO activity was rate-limited by the transport step. Although transport in these particular cell types was mediated by system L (not by the high-affinity system that we now describe), they serve to emphasize the critical role of transmembrane transport for IDO activity. Indeed, as APC and T cells are in obligate, close physical contact, they effectively "compete" for tryptophan in the local microenvironment. Expression of a high-affinity transporter by the APC but not by the resting T cells might help the APC preferentially take up the available tryptophan.
The putative, high-affinity transport system that we describe is currently identified as a biochemical activity, not yet at the protein level. It is possible that the new activity could represent a novel system unrelated to existing transporters; alternatively, it might represent a variant of a known transport system with an alteration that confers enhanced affinity for tryptophan (as, for example, different light-chain subunits are known to alter the properties of system L [37 ]). Whichever is the case, we hypothesize that this system represents an important route of tryptophan uptake in IDO-expressing APC.
Received December 9, 2005; revised August 12, 2006; accepted August 16, 2006.
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-inducible indoleamine 2,3-dioxygenase cDNA Biochem. Biophys. Res. Commun. 168,1-8[CrossRef][Medline], indoleamine 2,3-dioxygenase, and tryptophan catabolism FASEB J. 5,2516-2522[Abstract]-mediated antimicrobial response: indoleamine 2,3-dioxygenase-deficient mutant host cells no longer inhibit intracellular Chlamydia spp. or Toxoplama growth J. Immunol. 150,5529-5534[Abstract] blocks the growth of Toxoplasma gondii in human fibroblasts by inducing the host cells to degrade tryptophan Proc. Natl. Acad. Sci. USA 81,908-912-induced indoleamine 2,3 dioxygenase and inducible nitric oxide synthase in the replication of human cytomegalovirus in retinal pigment epithelial cells J. Immunol. 162,957-964 Cancer Res. 53,2603-2613 in vivo Int. J. Cancer 60,115-122[Medline]This article has been cited by other articles:
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