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The Journal of Immunology, 2001, 166: 6332-6340.
Copyright © 2001 by The American Association of Immunologists

Antioxidants Inhibit Indoleamine 2,3-Dioxygenase in IFN-{gamma}-Activated Human Macrophages: Posttranslational Regulation by Pyrrolidine Dithiocarbamate1

Shane R. Thomas2,*, Houta Salahifar*, Ryuichi Mashima*, Nicholas H. Hunt{ddagger}, Des R. Richardson{dagger} and Roland Stocker3,*

* Biochemistry and {dagger} Iron Groups, The Heart Research Institute, Camperdown, New South Wales, Australia; and {ddagger} The Department of Pathology, University of Sydney, Sydney, New South Wales, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Induction of the heme-containing indoleamine 2,3-dioxygenase (IDO) by IFN-{gamma} is implicated in anti-microbial and pro-inflammatory activities of human macrophages. Antioxidants can modulate the expression of immune and inflammatory genes, and pyrrolidine dithiocarbamate (PDTC) is a frequently used antioxidant to inhibit the transcription factor NF-{kappa}B. Here we show that IFN-{gamma} treatment of human monocyte-derived macrophages (hMDMs) increased the proportion of oxidized glutathione. PDTC attenuated this increase and inhibited IDO activity, although it increased IDO protein expression and did not affect IDO mRNA expression and enzyme activity directly. Other antioxidants, 2-ME, ebselen, and t-butyl hydroquinone, inhibited IDO protein expression. Similar to PDTC, the heme biosynthesis inhibitor succinylacetone (SA) and the iron-chelator pyridoxal isonicotinoyl hydrazone inhibited cellular IDO activity without affecting protein expression, whereas addition of hemin or the heme precursor {delta}-aminolevulinic acid increased IDO activity. Also, incubation of IFN-{gamma}-activated hMDM with {delta}-[14C]-aminolevulinic acid resulted in the incorporation of label into immunoprecipitated IDO, a process inhibited by PDTC and SA. Furthermore, supplementation of lysates from PDTC- or SA-treated hMDM with hemin fully restored IDO activity to control levels, and hemin also reversed the inhibitory action of SA but not PDTC in intact cells. Together these results establish a requirement for de novo heme synthesis for IDO activity in IFN-{gamma}-activated hMDM. They show that, similar to other pro-inflammatory proteins, the activity of IDO is modulated by antioxidants though in the case of PDTC this takes place posttranslationally, in part by limiting the availability of heme for the formation of holo-IDO.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interferon-{gamma} is a key mediator of inflammation and modulates the antimicrobial and antitumor activities of monocytes and macrophages (1). For example, IFN-{gamma} primes mononuclear phagocytes for the enhanced production of reactive oxygen and nitrogen species and the expression of several enzymes with tumoricidal and bacteriocidal activities (1). Among these are inducible NO synthase (NOSII)4 (2) and indoleamine 2,3-dioxygenase (IDO). IDO initiates the degradation of tryptophan (Trp) along the kynurenine (Kyn) pathway by catalyzing the cleavage of the pyrrole ring of Trp to form N-formyl-Kyn that decomposes to Kyn and formate (3). Protoheme IX is the sole prosthetic group required for IDO activity. IDO is unique in that it can use superoxide anion radical as a substrate and cofactor (4). Whereas superoxide reductively activates IDO, NO inhibits enzyme activity (5, 6).

For reasons poorly understood at present, there is a striking species difference in the response of macrophages to IFN-{gamma} in that such treatment potently induces NOSII in murine but not human macrophages (5, 7). Conversely, such treatment strongly induces IDO in human but not murine cells (5, 8). The induction of IDO and Trp metabolism along the Kyn pathway is implicated in several physiological and pathophysiological processes. It can deplete target cells of Trp, the least abundant of all essential amino acids, and this is partially responsible for the antimicrobial, antiviral, and anti-proliferative activities of IFN-{gamma} (3, 9). Trp depletion is also involved in the inhibition of T cell proliferation by IFN-{gamma}-treated human monocyte-derived macrophages (hMDMs) (10, 11). In addition, the local production by macrophages of the Trp-derived neurotoxin, quinolinic acid (12), is linked to neurological disorders associated with systemic immune activation or CNS inflammation (13, 14, 15). Finally, IFN-{gamma}-mediated induction of IDO and Trp metabolism along the Kyn pathway has been proposed to represent an extracellular antioxidant defense aimed at preventing oxidative damage to host tissue during inflammation (16, 17, 18). Given these potential physiological and pathological roles, it is important to understand the regulation of IDO in macrophages.

The intracellular reduction and oxidation (redox) status of the cell can regulate the expression of various inflammatory genes. Pyrrolidine dithiocarbamate (PDTC) is a frequently used antioxidant to study such regulation (19, 20, 21). It is commonly thought to modulate the activity of the redox-sensitive transcription factors NF-{kappa}B, AP-1, and p53 (22, 23, 24). For example, PDTC inhibits the induction of NOSII and VCAM-1 in activated murine macrophages and endothelial cells, respectively, at the transcriptional level by inhibiting the activation of NF-{kappa}B (25, 26). PDTC and other related dithiocarbamates also exhibit anti-inflammatory activity in vivo (27, 28, 29) and this too is thought to be mediated via inhibition of NF-{kappa}B (29).

In this study we examined whether antioxidants regulate the induction of IDO in IFN-{gamma}-activated hMDM. It is shown that PDTC and other antioxidants effectively inhibit IDO activity in these cells, and that in the case of PDTC such inhibition is at the posttranslational rather than transcriptional level, by limiting the availability of heme for formation of IDO holoenzyme.


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

{delta}-Aminolevulinic acid (ALA), catalase (bovine liver), dansyl chloride, hemin, iodoacetic acid, Kyn, Triton X-100, PDTC, PBS tablets, 2-ME, nordihydroguaiaretic acid, succinylacetone (SA), and tert-butyl hydroquinone were obtained from Sigma (St. Louis, MO). Ascorbic acid, methylene blue, and 3-hydroxyanthranilic acid (3-HAA) were obtained from Aldrich (Milwaukee, WI), and L-Trp from Merck (West Point, PA). Lidocaine was obtained from ICN Pharmaceuticals (Costa Mesa, CA). Human recombinant IFN-{gamma} and TNF-{alpha} were obtained from Boehringer Mannheim (Indianapolis, IN) and {delta}-[4-14C]aminolevulinic acid hydrochloride (14C-ALA; specific activity 47.6 mCi/mmol) from NEN Life Sciences (Boston, MA). 59Fe (DuPont-NEN) was used to prepare 59Fe-saturated human transferrin (59Fe-transferrin) (30). Desferrioxamine (DFO) was obtained from CIBA-Geigy (Pendle Hill, New South Wales, Australia). Pyridoxal isonicotinoyl hydrazone (PIH) was prepared as described (31). Ebselen (2-phenyl-1,2-benzoisoselenazol) was a gift from Rhône-Poulenc-Nattermann (Cologne, Germany). Complete protease inhibitor cocktail tablets (Boehringer Mannheim) were used as recommended by the manufacturer. Human white cell concentrates (buffy coats <24-h old) and nonfasted, pooled human serum were provided by the New South Wales Red Cross Blood Transfusion Service (Sydney, Australia). All sera were heat-inactivated at 56°C for 30 min and stored at -20°C before use in cell culture.

Isolation of human monocytes and culture of hMDMs

Human monocytes (>95% purity) were isolated from buffy coats, adhered onto 12-well culture plates (Falcon) at 1–2 x 106 cells/well, and allowed to differentiate into hMDM for 9 days in RPMI 1640 medium (Sigma) supplemented with 10% (v/v) human serum, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin, as described previously (17). hMDM routinely yielded 125–250 µg cell protein/well, with comparable cell protein from well to well within the same batch of cells. Unless specified otherwise, experiments were performed with 9- to 18-day old hMDM cultured in the above mentioned medium supplemented with 100–200 µM L-Trp, and test compounds of interest were added 30 min before treatment of cells with IFN-{gamma} (500 U/ml or 25 ng/ml). Cell cultures were maintained at 37°C in a humidified atmosphere of 5% CO2 in air.

Northern blot analysis

RNA was extracted from ~10 x 106 hMDM using the acid guanidinium thiocyanate-phenol-chloroform method (32). Total RNA (20 µg) was subjected to electrophoresis under denaturing conditions on a 1% (w/v) agarose gel containing formaldehyde, transferred onto a nylon {zeta}-probe membrane (Amersham, Arlington Heights, IL), and cross-linked by UV irradiation. After prehybridization (4 h at 42°C) the RNA blot was hybridized (18 h at 42°C) with a 32P-labeled 1-kb PstI fragment of a cDNA probe specific for human IDO (provided by Luigi Varesio, Laboratorio di Biologia Molecolare, Instituto Giannina Gaslini, Genova Quarto, Italy) (33). The probe was labeled with [{alpha}-32P]dCTP (specific activity 3000 Ci/mmol; Bresatec, Adelaide, Australia) using the Megaprime DNA-labeling system according to the manufacturer’s instructions (RPN 1606; Amersham), and the Northern blots were exposed to x-ray film overnight at -80°C.

Western blot analysis

hMDM (1 x 106 cells/well) were lysed in 200 µl of 0.1% Triton X-100 (supplemented with protease inhibitors) or by subjecting the cells in PBS (200 µl supplemented with protease inhibitors) to 3 cycles of freezing and thawing. Equal amounts of protein (10–20 µg) were loaded onto a 10% acrylamide stacking gel and separated by SDS-PAGE using a 20% acrylamide separating gel and a MiniProtean II electrophoresis cell (Bio-Rad, Hercules, CA) run at 40 V for 30 min and then at 100 V for 1 h. Molecular mass rainbow markers (Bio-Rad) were used as molecular mass standards. Separated proteins were transferred onto nitrocellulose membranes (Amersham) at 75 V for 45 min using a mini-blot module (NOVEX, San Diego, CA), and the blotted membranes were blocked in 3% (w/v) skim milk powder in TBS for 2–4 h at room temperature. The blocked membrane was probed overnight at 4°C with a murine anti-human IDO mAb (provided by Osamu Takikawa, University of Wollongong, Wollongong, Australia) at a dilution of 1:1000 in 1% (w/v) skim milk and 0.1% (v/v) Tween 20 (Sigma) in TBS. The membrane was probed for 2 h at room temperature with sheep anti-mouse HRP-conjugated Ig (Bio-Rad) (1:10,000 dilution) in 1% (w/v) skim milk and 0.1% (v/v) Tween 20 in TBS. Detection of bands was performed by ECL according to the manufacturer instructions (RPN 2106; Amersham). The relative intensity of the IDO band was determined using the Gel-Doc 1000 molecular analyst program (Bio-Rad).

IDO activity assay

IDO activity was assessed by the ascorbate/methylene blue assay (34) using cell lysates of IFN-{gamma}-activated hMDM (1 x 106 cells/well) prepared by freezing and thawing as described above. Lysates were centrifuged at 14,000 x g at 4°C for 5 min, and the resulting supernatant was used. The IDO assay was performed in 50 mM potassium phosphate buffer (pH 7.2) containing 3.5 mM EDTA, 10 mM ascorbic acid, 25 µM methylene blue, 200 µM L-Trp, and 0.2 mg/ml catalase. The reaction was initiated by mixing the reaction buffer with the cell lysate (1:1, v/v) and conducted for 30–60 min at 37°C after which time 200-µl aliquots were removed and added to 50 µl of 20% (w/v) tricarboxylic acid. The samples were maintained at room temperature for up to 2 h and then stored overnight at 4°C to allow for complete conversion of N-formyl-Kyn into Kyn. IDO activity was expressed as nmol Kyn formed/h/mg cell protein. The concentrations of Trp, Kyn, or 3-HAA were determined by HPLC (17).

Immunoprecipitation of IDO

IDO mAb (1 mg/ml) was added to hMDM lysates at 1:25 (v/v), and the lysates were rotated overnight at 4°C before addition of {gamma}-bind Sepharose beads (Pharmacia, Piscataway, NJ) and further rotated for 4 h at 4°C. The beads were then washed 4–5 times with PBS. This method resulted in complete immunoprecipitation of IDO as verified by Western blotting.

Metabolic labeling of protoporphyrin IX in IDO

hMDMs were incubated for 30 min with {delta}-[14C]-ALA (0.2–0.5 µCi/well, corresponding to 2–5 µM ALA) before addition of IFN-{gamma} (500 U/ml) and cell culture for another 18–24 h. hMDM lysates were then prepared by freeze/thawing and IDO immunoprecipitated as described above, and 80% of the {gamma}-bind Sepharose beads subjected to {beta}-counting in 5 ml of Ultima-Gold scintillant (Canberra Packard, Meriden, CT). The remaining 20% of the {gamma}-bind Sepharose beads were used for Western blotting.

Uptake and efflux of 59Fe by hMDM

For iron efflux, hMDMs were labeled with 59Fe by incubation for 6 h in culture medium supplemented with 1% human serum and 59Fe-transferrin (0.1 mg/ml or 1.25 µM; specific activity 19.65–22.2 mCi/mg Fe). hMDMs were then washed four times with PBS and incubated for 20 h in culture medium supplemented with 10% human serum in the absence or presence of IFN-{gamma} (500 U/ml) and test compounds. The amount of radiolabel present in the medium and cell lysates was then measured by {gamma}-scintillation counting. For iron uptake experiments, hMDM were cultured in medium supplemented with 10% human serum and 59Fe-labeled transferrin (0.1 mg/ml or 1.25 µM; specific activity 19.65–22.2 mCi/mg Fe) in the absence or presence of IFN-{gamma} (500 U/ml) and test compounds. After 24 h, cells were washed four times with PBS, and the amount of radiolabel incorporated into hMDM lysates was determined by {gamma}-scintillation counting.

HPLC analysis of reduced (GSH) and oxidized glutathione (GSSG)

Glutathione levels in cellular samples were measured as previously described (35). Briefly, cells were harvested with EDTA (1 mM) and lidocaine (4 mg/ml) solution followed by centrifugation (1000 x g for 5 min). Cell pellets were resuspended in 0.5 ml of 10% perchloric acid in 0.2 M boric acid with 10 µM {gamma}-glutamyl glutamate added as the internal standard. Cells were sonicated on ice, and the lysates were centrifuged (1000 x g, 5 min, 4°C). Aqueous iodoacetic acid (60 µl) and KOH/borate solution (200 µl) were added to 250 µl supernatant to adjust the pH to 9. The samples were then incubated for 20 min at room temperature (to ensure complete reaction of free thiols with iodoacetic acid), derivatized with dansyl chloride in acetone (300 µl) followed by incubation overnight in the dark at room temperature. Unreacted dansyl chloride was removed by the addition of chloroform (500 µl) and centrifugation (10,000 x g, 3 min). The concentration of dansyl derivatives present in an aliquot (20 µl) of the supernatant was determined by HPLC via fluorescence detection (LC240 detector; Perkin-Elmer, Norwalk, CT) with excitation at 335 nm and emission at 515 nm. Dansyl derivatives were eluted on a LC-NH2 analytical column (4.6 x 250 mm, 5 µm; Supelco, Bellefonte, PA) at a flow rate of 1 ml/min (S-200 HPLC pump; Perkin-Elmer) using two mobile phases: A, methanol-H2O (80:20, v/v); B, methanol-H2O-acetate (640:50:325, v/v/v) and the following gradient program: 20% B, 0–10 min; 20–80% B, 10–30 min; 30–46% B, 46–48 min. Quantification of GSH and GSSG was performed by area comparison with known concentrations of authentic standards and standardization to the internal standard. Statistical differences in the data was examined using a one-way ANOVA followed by Fisher’s protected least significant difference test using SYSTAT 8.0 software (SPSS, Chicago, IL). Significance was accepted at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IFN-{gamma} induces the expression of IDO in hMDM

Treatment of hMDM with IFN-{gamma} resulted in the time-dependent induction of IDO. This was indicated by the parallel increase in IDO protein (Mr {approx} 42–45 kDa) (Fig. 1Go, A and D) and IDO activity in cell lysates (Fig. 1GoB) and intact cells (Fig. 1GoC), the latter reflected by the increased Trp consumption and accumulation of Kyn and 3-HAA in the culture medium. IDO induction was dose-dependent and maximal at IFN-{gamma} concentrations of >=250 U/ml (data not shown). Kyn and 3-HAA detected in the culture medium due to their secretion accounted for 60–65 and 7–15%, respectively, of the Trp consumed. The remaining 20–30% of Trp consumed is converted to anthranilic (8) and quinolinic acids (36). IDO protein and activity continued to increase after Trp was consumed completely (Fig. 1Go, A and B). This coincided with the onset of a decrease in Kyn and continued accumulation of 3-HAA (Fig. 1GoC), indicating that hMDM may take up some of the Kyn formed to metabolize it further down the Kyn pathway.



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  		FIGURE 1. Time course of IFN-{gamma}-mediated induction of IDO in hMDM. Cells were incubated in the presence of IFN-{gamma} (500 U/ml or 25 ng/ml). At the indicated time, cell lysates were prepared and analyzed for IDO protein (A) and IDO activity (B), and the medium was analyzed for Trp ({diamondsuit}), Kyn ({circ}), and 3-HAA ({triangleup}) (C). Results are the mean ± SE of three independent experiments (A and C) and a representative of two experiments (B). The result in D shows a Western blot representative of three experiments.

 
Antioxidants inhibit IDO activity induced by IFN-{gamma} in hMDM

Several genes that encode inflammatory proteins are subject to redox control (25, 26), raising the possibility that IFN-{gamma}-mediated induction of IDO may be regulated similarly. Indeed, several structurally and functionally different antioxidants inhibited IDO activity in IFN-{gamma}-activated hMDM (Table IGo). Inhibition was not due to toxicity as >85% of the hMDM remained viable at the end of the experiments, as assessed by trypan blue exclusion and lactate dehydrogenase release (data not shown). At the concentrations used, the antioxidants inhibited IDO-mediated Trp metabolism in intact cells by 80–90% and IDO activity in cell lysates by 55–66%.


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  		Table I. Inhibition of IDO activity in IFN-{gamma}-activated human MDM by different antioxidants1

 
PDTC acts as an antioxidant in IFN-{gamma}-activated hMDM

PDTC has been reported to act as an anti- (37, 38) or pro-oxidant (39, 40) depending on the experimental conditions used. To directly test whether PDTC acted as an antioxidant in IFN-{gamma}-activated hMDM, we examined its effect on the cellular glutathione redox status. Glutathione is the single major redox-active molecule in cells, and its redox status can be used as an index of the cellular redox potential (41). We used an HPLC assay that measures both the reduced and oxidized forms of glutathione. Table IIGo shows that treatment of hMDM with IFN-{gamma} alone increased GSH, GSSG, and total glutathione levels. It also significantly increased the proportion of glutathione present as GSSG, indicating an increase in the cellular oxidation status. PDTC significantly inhibited the increases in GSSG, total glutathione as well as the cellular oxidation status, demonstrating an overall antioxidant action of the dithiocarbamate.


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  		Table II. Glutathione concentrations and redox status in hMDM treated with IFN-{gamma} and PDTC1

 
PDTC inhibits IDO activity in IFN-{gamma}-activated hMDM at the posttranslational level

PDTC is widely used as an antioxidant that inhibits the transcription of various immune and inflammatory genes via inhibition of NF-{kappa}B (25, 26). Fig. 2GoA demonstrates that PDTC dose dependently inhibited IDO activity in hMDM activated with 500 U/ml IFN-{gamma}, with an IC50 of 6.5–12.5 µM. IDO mRNA was not detected in nonactivated hMDM (Fig. 2GoB), similar to nonstimulated human monocytes (33). However, after 18 h of incubation with IFN-{gamma}, formation of a ~2-kb IDO mRNA was detected (Fig. 2GoB), together with an additional minor, high molecular mass RNA form (arrow in Fig. 2GoB) that may represent a precursor IDO mRNA (33) or alternative splice variant. Despite inhibiting Trp metabolism by ~90%, PDTC at 125 µM did not inhibit the IFN-{gamma}-induced expression of IDO mRNA (Fig. 2GoB) or IDO protein (Fig. 3Go). In fact, compared with cells treated with IFN-{gamma} alone PDTC consistently increased the amount of IDO protein expressed by ~30% (Fig. 3Go). Similar results with PDTC were also obtained when IDO was induced in hMDM with a suboptimal dose of IFN-{gamma} (10 U/ml) plus TNF-{alpha} (250 U/ml) (data not shown). Importantly, PDTC (75 µM) also inhibited IDO activity by >80% in freshly isolated human monocytes treated with IFN-{gamma} (data not shown), demonstrating that this effect of the dithiocarbamate was not limited to in vitro differentiated cells. In contrast to PDTC, 2-ME, ebselen, and t-butyl hydroquinone all inhibited the expression of IDO protein (Fig. 3Go). Therefore, although antioxidants appear to generally inhibit IFN-{gamma}-induced IDO activity in hMDM, and some antioxidants decreased IDO protein, PDTC appeared to act at the posttranslational level.



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  		FIGURE 2. PDTC decreases IDO activity but not IDO mRNA levels in IFN-{gamma}-activated hMDM. Cells incubated for 30 min in the absence and presence of the indicated concentration of PDTC were treated with IFN-{gamma} (500 U/ml) and then cultured for 18–24 h. Kyn (•) and Trp ({blacksquare}) in the medium and IDO mRNA in hMDM (B) were then determined. Results in A are expressed as a percentage of the amount of Kyn accumulated or Trp consumed in hMDM treated with IFN-{gamma} alone and represent the mean ± SE of four independent experiments. The 100% values for hMDM treated with IFN-{gamma} alone were 38 ± 9 µM Kyn accumulated and 61 ± 11 µM Trp consumed. The result in B shows a Northern blot representative of three independent experiments.

 


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  		FIGURE 3. Effect of different antioxidants on the expression of IDO protein in IFN-{gamma}-activated hMDM. Cells were incubated for 30 min in the absence (control, C) and presence of 125 µM PDTC, 35 µM t-butyl hydroquinone, 25 µM ebselen, or 125 µM 2-ME before treatment with IFN-{gamma} (500 U/ml) and further culture for 18–24 h. After such time IDO protein was determined by Western blotting. Results in A are expressed as a percentage of the amount of IDO protein detected in hMDM treated with IFN-{gamma} alone and represent the mean ± SE of four to six independent experiments. The result in B shows a representative Western blot for the results shown in A.

 
We next addressed the mechanism(s) by which PDTC inhibited IDO activity at the posttranslational level. When added to lysates prepared from IFN-{gamma}-activated hMDM, PDTC had no effect: IDO activity in the presence of 100 µM PDTC was 98.5 ± 3.5% (mean ± SE, n = 3) of the corresponding control. PDTC also had no effect on the activity of recombinant human IDO (data not shown), demonstrating that it did not inhibit IDO directly.

Posttranslational regulation of IDO could involve interference with the availability of heme, the sole prosthetic group required for IDO activity. Although not commonly appreciated in studies on gene regulation, PDTC can chelate transition metals including cadmium, zinc, nickel, mercury, and copper (27, 42). Although considered to be an ineffective iron-chelator (42), we considered that PDTC may limit the availability of iron for the synthesis of heme. Consistent with this, two different iron chelators, DFO and PIH, inhibited Trp metabolism in IFN-{gamma}-activated hMDM (Fig. 4GoA) similar to PDTC. Inclusion of equimolar concentrations of FeCl3 with either chelator before addition to the cells significantly abrogated the inhibitory action (Fig. 4GoA). Unfortunately, the effect of FeCl3 addition on PDTC-mediated inhibition of IDO activity could not be tested as the PDTC-iron complex was toxic to hMDM (data not shown), as reported previously for thymocytes and copper ions (42). Similar to PDTC, PIH inhibited IDO without inhibiting protein expression, whereas DFO inhibited protein expression by ~50% (Fig. 4Go, B and C).



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  		FIGURE 4. Iron chelators inhibit IDO activity in IFN-{gamma}-activated hMDM. Cells were incubated for 30 min in the absence and presence of 150 µM DFO, 150 µM PIH, 150 µM DFO-iron complex (DFO+Fe), or PIH-iron complex (PIH+Fe) before treatment with IFN-{gamma} (500 U/ml) and further culture for 18–24 h. Kyn produced ({blacksquare}) and Trp consumed () in the medium (A) and IDO protein in hMDM (B) were then determined. Chelator/iron complexes were prepared by mixing equimolar amounts of chelator and FeCl3 before addition to the cells. Results in A and B represent the mean ± SE of three independent experiments and are expressed as a percentage of the values obtained with hMDM treated with IFN-{gamma} alone. 100% values for hMDM treated with IFN-{gamma} alone were 49 ± 9.5 µM Kyn accumulated and 95 ± 15 µM Trp consumed. The result in C shows a representative Western blot for the result shown in B.

 
DFO and PIH markedly enhanced the efflux of iron from IFN-{gamma}-activated hMDM preloaded with 59Fe, whereas PDTC had a comparatively minor effect (Fig. 5GoA). The extent of iron effluxed after 20 h of incubation corresponded to 58, 39, and 20% of the cellular 59Fe for PIH, DFO, and PDTC, respectively. This compared with 14% 59Fe efflux noted for hMDM treated with IFN-{gamma} alone or nonactivated hMDM. Therefore, PIH, DFO, and PDTC increased iron efflux above hMDM treated with IFN-{gamma} alone 4.1-, 2.8-, and 1.4-fold, respectively (Fig. 5GoA). As IFN-{gamma} increases transferrin receptor expression in hMDM (43), PDTC may decrease intracellular iron by inhibiting its uptake. Indeed, IFN-{gamma} treatment increased the uptake of 59Fe by hMDM cultured in the presence of 59Fe-transferrin by ~25%, and PDTC inhibited this process by 42% (Fig. 5GoB). Together, these results establish a regulatory role for cellular iron in IFN-{gamma}-mediated induction of IDO activity in hMDM and that PDTC decreases iron uptake that may contribute to the inhibition of IDO activity.



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  		FIGURE 5. Effect of PDTC on iron efflux and uptake in IFN-{gamma}-activated hMDM. A, Cells in RPMI 1640 supplemented with 1% serum and 0.1 mg/ml of 59Fe-transferrin were incubated for 5–6 h, washed, and then incubated further in fresh medium supplemented with 10% serum in the absence or presence of IFN-{gamma} (500 U/ml) and 125 µM PDTC, 150 µM PIH, or 150 µM DFO. After 20 h the 59Fe in the medium was determined by {gamma}-scintillation counting. B, Cells cultured in RPMI 1640 were supplemented with 10% human serum, L-Trp (150 µM), and 0.1 mg/ml of 59Fe-saturated human transferrin in the absence or presence of IFN-{gamma} (500 U/ml) and 125 µM PDTC. After 24 h the 59Fe in the cell lysates was determined. The 100% value for untreated, control cells was 62,900 ± 20,200 dpm. Results for A and B represent the mean ± SE of three independent experiments.

 
De novo synthesis of heme is important for IDO activity in IFN-{gamma}-activated hMDM

Addition of hemin to cell lysates prepared from IFN-{gamma}-activated hMDM increased IDO activity ~2-fold (Fig. 6GoA). Similarly, addition of hemin or the heme biosynthesis pathway precursor, ALA, to intact IFN-{gamma}-activated hMDM increased Trp metabolism by 40–70% (Fig. 6GoB), indicating that cellular heme is limiting for IDO activity and that a significant portion of IDO expressed in these cells is present as apoprotein.



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  		FIGURE 6. Increasing the availability of heme increases IDO activity. A, Cells were treated with IFN-{gamma} (500 U/ml) and cultured for 24 h. Cell lysates were then prepared and incubated on ice for 2 h in the absence or presence of 5 µM hemin, before determination of IDO activity. Results are the mean ± SE of seven independent experiments. B, Cells were incubated for 18–24 h in the absence or presence of 5 µM hemin or ALA before the medium was analyzed for Kyn ({blacksquare}) and Trp (). Results are expressed as a percentage of the values obtained with hMDM treated with IFN-{gamma} alone and represent the mean ± SE of three independent experiments. The 100% values were 29 ± 7 µM Kyn accumulated and 48 ± 10 µM Trp consumed.

 
To examine the role of de novo synthesis of protoporphyrin IX for IDO activity and whether PDTC interfered with this pathway, we used SA (which inhibits protoporphyrin IX synthesis) and compared its effect with that of PDTC. Like PDTC, SA (250 µM) added 30 min before IFN-{gamma} had no marked effect on IDO protein expression, yet inhibited Trp metabolism in IFN-{gamma}-activated hMDM by 80–90% (Fig. 7Go). Cell lysates obtained from such treated cells exhibited decreased IDO activity when compared with corresponding control lysates (Fig. 8Go). Also similar to PDTC, SA did not inhibit IDO activity when added to lysates of IFN-{gamma}-activated hMDM (100 ± 6% compared with control, mean ± SE, n = 3). However, addition of hemin to lysates prepared from PDTC- or SA-treated hMDM restored IDO activity to control levels (Fig. 8Go), indicating that PDTC and SA inhibited IDO activity by interfering with the formation of holoenzyme rather than apoprotein.



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  		FIGURE 7. SA inhibits IDO activity, but not IDO protein expression. Cells were incubated in the absence and presence of 250 µM SA for 30 min before treatment with IFN-{gamma} (500 U/ml) and culture for another 18–24 h. Kyn and Trp in the medium, and IDO protein in the cells were then determined (A). Results in A are expressed as a percentage of the values obtained with hMDM treated with IFN-{gamma} alone and represent the mean ± SE of five to six independent experiments. The result in B shows a representative Western blot for the IDO protein data shown in A.

 


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  		FIGURE 8. Hemin restores IDO activity when added to cell lysates of PDTC- and SA-treated hMDM. Cells were treated with PDTC (100 µM), SA (250 µM), or nontreated for 30 min before addition of IFN-{gamma} (500 U/ml) and cultured for another 24 h. Cell lysates were then prepared and incubated in the absence ({blacksquare}) or presence () of 5 µM hemin on ice for 2 h, after which IDO activity was assessed. Results are expressed as a percentage of IDO activity measured in the corresponding nonsupplemented or hemin-supplemented lysates prepared from hMDM treated with IFN-{gamma} alone. The results represent the mean ± SE of four and seven independent experiments for SA and PDTC, respectively. One hundred percent IDO activity was 22.8 ± 4 or 47.6 ± 8 nmol Kyn/h/mg protein for lysates from IFN-{gamma}-activated hMDM nonsupplemented or supplemented with hemin, respectively. IDO activity in lysates of cells not treated with IFN-{gamma} was <5% of that measured in lysates from IFN-{gamma}-activated hMDM, whether hemin was added or not.

 
Synthesis of heme requires the formation of protoporphyrin IX into which iron is incorporated. We next compared the effect of PDTC and SA on the incorporation of protoporphyrin IX into the IDO protein. For this, hMDM were cultured in the presence of {delta}-[14C]-ALA and the extent of radiolabel incorporated into immunoprecipitated IDO was measured. This approach has been used previously to provide evidence that neuronal NO synthase incorporates newly synthesized heme (44). Fig. 9Go shows that compared with nonactivated hMDM immunoprecipitated IDO from IFN-{gamma}-treated cells contained increased amounts of the radiolabel, indicative of incorporation of newly synthesized protoporphyrin IX. More importantly, SA and PDTC inhibited incorporation of radiolabel into IDO by 95 and 56%, respectively, indicating that PDTC inhibited IDO activity in part by limiting the availability of newly synthesized protoporphyrin IX. Addition of hemin, but not ALA, partially reversed the inhibitory activity of SA on IDO in IFN-{gamma}-activated hMDM (Fig. 10GoA), demonstrating that hemin added to intact cells can be available for the formation of holo-IDO. However, despite this, addition of hemin or ALA to IFN-{gamma}-activated hMDM treated with PDTC did not reverse its inhibitory activity (Fig. 10GoB). Together these results suggest that PDTC acts by inhibiting the incorporation of heme into apo-IDO rather than by inhibiting heme synthesis.



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  		FIGURE 9. Effect of SA and PDTC on the incorporation of protoporphyrin IX into IDO in IFN-{gamma}-activated hMDM. Cells were treated with {delta}-[14C]-ALA (0.2–0.5 µCi/well; 2.5–5 µM) in the absence (C) or presence of IFN-{gamma} (500 U/ml), with or without PDTC (100 µM) or SA (250 µM), and cultured for 18–24 h. The 14C-label associated with immunoprecipitated IDO was then determined by {beta}-scintillation counting. Results are expressed as the amount of 14C-radioactivity (dpm) per IDO protein present, as assessed by Western blotting (data not shown). Results are mean ± SE of seven independent experiments. The 100% value for IDO immunoprecipitated from IFN-{gamma}-treated hMDM was 2800 ± 700 dpm.

 


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  		FIGURE 10. Addition of hemin to IFN-{gamma}-activated hMDM partially abrogates the inhibitory activity of SA but not PDTC. Cells were nontreated or treated with 250 µM SA (A) or 125 µM PDTC (B) for 30 min before the addition of IFN-{gamma} (500 U/ml) and, where indicated, hemin (5 µM) or ALA (5 µM). After 24 h of incubation, the medium was analyzed for Kyn or Trp. Results in A are expressed as a percentage of the values obtained with hMDM treated with IFN-{gamma} alone and represent the mean ± SE of three independent experiments. The 100% values were 29 ± 7 µM Kyn accumulated and 51 ± 8 µM Trp consumed.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study provides novel insights into the regulation of IDO activity in IFN-{gamma}-activated hMDM. Our results show that IDO is regulated by antioxidants and that iron and de novo heme synthesis are required for IDO activity, and suggest that the commonly used antioxidant PDTC inhibits IDO activity at the posttranslational level by limiting the availability of heme for the enzyme.

IDO is induced during inflammatory conditions in vivo and, where examined, IFN-{gamma} represents the primary inducer (reviewed in Ref. 18). Inflammation is associated with activation of phagocytes and increased formation of reactive oxygen and nitrogen species that can alter cellular redox status. Numerous studies have reported that expression of genes encoding proteins relevant to inflammation are subject to redox control (19, 20, 21, 45). In light of this and the fact that induction of IDO and Trp metabolism along the Kyn pathway may represent an antioxidant defense, we hypothesized that induction of IDO in hMDM is subject to redox control (18). In support of this hypothesis, this study shows, for the first time, that structurally and functionally different antioxidants inhibit IDO activity, although they differentially affected the expression of IDO protein. We chose to focus on how PDTC inhibited IDO activity, as it is a frequently used antioxidant in studies examining redox control of cellular responses (19, 20).

Redox control by PDTC is generally thought to occur at the level of transcription and frequently involves the transcription factors NF-{kappa}B and/or AP-1 (19, 20, 21, 45). Although there is a potential for redox control of IDO transcription (18) and PDTC acts as an antioxidant under the experimental conditions used here, this study demonstrates that in IFN-{gamma}-activated hMDM, PDTC acts at the posttranslational level, as it inhibited cellular enzyme activity but not IDO mRNA or protein expression. A previous study demonstrated that PDTC inhibits the expression of cyclooxygenase-2 in IL-1{beta}-activated rat mesangial cell at the posttranscriptional level (46). The novel information provided in this study is that in IFN-{gamma}-activated hMDM PDTC inhibits the availability of heme for incorporation into apo-IDO. Thus, addition of hemin to lysates from PDTC-treated cells restored IDO activity and PDTC inhibited the incorporation on protoporphyrin IX into the IDO protein. The precise mechanism underlying the action of PDTC remains to be established. PDTC could inhibit protoporphyrin IX biosynthesis, incorporation of iron into protoporphyrin IX, and/or insertion of heme into apo-IDO. Inhibition of protoporphyrin IX synthesis at best only partially explains the inhibitory action of PDTC (Fig. 9Go). More likely, PDTC acts by interfering with the incorporation of functional heme into apo-IDO as addition of hemin to intact IFN-{gamma}-activated cells partially reversed inhibition of IDO activity by SA but not that exhibited by PDTC (Fig. 10Go). There is evidence (47) for a role of H2O2 in the binding of heme to thyroperoxidase in thyroid cells, suggestive of redox control of heme incorporation into hemoproteins.

These experiments established the importance of de novo heme synthesis for IFN-{gamma}-induced IDO activity in hMDM. Thus, addition of hemin or the heme precursor, ALA, increased IDO-mediated Trp metabolism, whereas the heme synthesis inhibitor SA strongly, and PDTC partially, inhibited the formation of holo-IDO and enzyme activity. Furthermore, addition of hemin to lysates of IFN-{gamma}-activated hMDM increased IDO activity 2-fold, indicating that approximately half of the IDO in these cells is present as apo-protein. In addition, inhibition of IDO activity by SA was reversed by added hemin, and iron chelators inhibited IDO activity, consistent with a requirement of iron for heme biosynthesis.

Previous studies have established the importance of heme availability and iron metabolism in the regulation of cytokine-induced heme proteins, including NOSII (48, 49) where heme is required for formation of the active dimer. However, to date few studies have examined the role of heme for IDO activity. It was reported that addition of iron or heme inhibited whereas iron chelators increased IDO activity in the human monocytic leukemia cell line, THP-1 (50, 51). This study shows that supplementing primary human macrophages with heme increases and the iron chelators DFO and PIH inhibit IDO activity. The reasons for this apparent discrepancy are not known, although it may reflect differences between cell lines and primary human macrophages.

DFO can increase NOSII activity at the transcriptional level in activated murine macrophages (52). Whether the observed inhibition of IDO protein expression by the iron chelator (Fig. 4Go) was due to transcriptional regulation requires further investigation. In contrast to DFO, PIH did not inhibit IDO protein expression, possibly because the two chelators affect different cellular iron pools important for IDO activity. For example, compared with DFO, PIH has greater access to mitochondrial iron (31, 53) that may be required for de novo heme synthesis, and this could explain how the compound regulates IDO posttranslationally.

Iron homeostasis is subject to redox control (54) and hence could be modulated by PDTC. In this study, IFN-{gamma} increased the uptake of 59Fe-transferrin by hMDM, and PDTC inhibited this by ~40%, even though it did not markedly induce iron efflux (Fig. 5Go). Therefore, in IFN-{gamma}-activated hMDM PDTC may generally modulate iron metabolism by decreasing its availability. This did not appear to be a result of iron chelation, as PDTC was ineffective in promoting iron efflux compared with PIH and DFO, and previous studies have shown that PDTC is not an effective iron chelator in cell culture systems (42). Therefore, these results indicate that PDTC inhibits IDO activity in IFN-{gamma}-activated hMDM by interfering with the incorporation of heme rather than removal of iron.

PDTC could conceivably inhibit IDO by interfering with the posttranslational processing of apo-IDO, as described for myeloperoxidase and NOSII (48, 49). In these studies precursor apo-myeloperoxidase and monomeric NOSII were immunoreactive yet catalytically inactive due to a deficiency in heme. These precursor proteins exhibited different molecular mass than their respective mature active forms. With IDO, only one major band of 45 kDa, the molecular mass of mature IDO, was seen consistently on Western blots, independent of the treatment. As this protein was functionally active upon addition of hemin, the results of Fig. 7Go suggest that inhibition of posttranslational processing of IDO by PDTC is unlikely.

At present, little is known about translational and posttranslational events involved in IFN-{gamma}-mediated induction of IDO. As an alternative to interfering with heme synthesis or incorporation, PDTC could conceivably inhibit IDO activity by preventing transport of apo-IDO to the cellular site where heme is incorporated. This may explain why addition of hemin to IFN-{gamma}-activated hMDM in culture did not reverse the inhibitory activity of PDTC, whereas its addition to cell lysates fully restored IDO activity to control levels.

Previous studies have shown that dithiocarbamates can exhibit both anti- (37, 38) and pro-oxidant activity (39, 40). We show here (Table IIGo) that PDTC decreased cellular GSSG and the proportion of glutathione present as GSSG, indicative of an antioxidant action. How precisely this is involved in the regulation of IDO requires further investigation, although studies by others (55) have shown that changes in cellular GSH regulates the sensitivity of tumor cells to growth inhibition by IFN-{gamma}, which is dependent on Trp degradation by IDO. This study demonstrates, for the first time, that IFN-{gamma} treatment of hMDM increases both their concentration of glutathione and their oxidation status, the latter perhaps reflecting oxidative stress associated with the pro-inflammatory activities of IFN-{gamma}. Previous studies by others (41) have shown that in vitro differentiation of cells other than monocytes is accompanied by an increase in the cellular oxidation status, raising the possibility that the redox status in hMDM is different than that in monocytes. However, such a putative difference is unlikely to explain the observed inhibition of IFN-{gamma}-induced activation of IDO, as PDTC was effective in both hMDM and freshly isolated monocytes.

Our results with 2-ME are in accordance with a previous study demonstrating that this thiol inhibits Trp metabolism in IFN-{gamma}-treated human tumor cell lines (55). Although inhibition of IDO by 2-ME was in part due to decreased synthesis of IDO protein, posttranslational control analogous to that exhibited by PDTC may also be involved, as addition of heme to lysates of 2-ME-treated hMDM significantly reversed inhibition of IDO (data not shown).

In conclusion, this study establishes important roles for iron, de novo heme synthesis, and redox reactions in the regulation of IDO activity in IFN-{gamma}-activated hMDM. Our study establishes PDTC as a pharmacological and posttranslational inhibitor of IDO. As PDTC has been used clinically to treat HIV patients (28) and exhibits anti-inflammatory activity in vivo (29), inhibition of IDO activity and Trp metabolism along the Kyn pathway may contribute to the in vivo anti-inflammatory action of PDTC. Whether PDTC inhibits other IFN-{gamma}-activated responses and/or whether PDTC in general inhibits heme enzymes at the posttranslational level is worthy of investigation.


   Acknowledgments
 
We thank Dr. Takikawa, University of Wollongong, Australia for the generous gift of anti-human IDO mAb, Dr. Varesio, Instituto Giannina Gaslini, Genova, Italy for the cDNA probe for human IDO, and Professor Roger Dean for his constructive discussions and for carefully reading the manuscript.


   Footnotes
 
1 This study was funded in part by a Harry Windsor Biomedical Postgraduate Research Scholarship and a C. J. Martin Post-Doctoral Research Fellowship by the National Health and Medical Research Council (to S.R.T.), by the Community Health and Anti-Tuberculosis Association, and the National Health and Medical Research Council Grants 951002 and 119230 (to R.S.). D.R.R. thanks the National Health and Medical Research Council and the Australian Research Council, for grant support. Back

2 Current address: Boston University School of Medicine, The Whitaker Cardiovascular Institute, Room W506, Boston, MA 02118. Back

3 Address correspondence and reprint requests to Professor Roland Stocker, The Heart Research Institute, 145 Missenden Road, Camperdown, New South Wales 2050 Australia. Back

4 Abbreviations used in this paper: NOSII, inducible NO synthase; ALA, aminolevulinic acid; DFO, desferrioxamine; 3-HAA, 3-hydroxyanthranilic acid; IDO, indoleamine 2,3-dioxygenase; Kyn, kynurenine; hMDM, human monocyte-derived macrophage; PDTC, pyrrolidine dithiocarbamate; PIH, pyridoxal isonicotinoyl hydrazone; SA, succinylacetone; Trp, tryptophan; GSH, reduced glutathione; GSSG, oxidized glutathione. Back

Received for publication September 15, 2000. Accepted for publication March 8, 2001.


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