Mechanisms regulating the expression of indoleamine 2,3-dioxygenase during decidualization of human endometrium

Yoshiki Kudo1,4, Tetsuaki Hara1, Takafumi Katsuki1, Aya Toyofuku1, Yuki Katsura1, Osamu Takikawa2, Tsuneo Fujii3 and Koso Ohama1

1 Department of Obstetrics and Gynecology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, 2 Department of Pharmacology, Hokkaido University School of Medicine, Sapporo and 3 Department of Obstetrics and Gynecology, National Kure Medical Center, Hiroshima, Japan

4 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi 1-2-3, Hiroshima 734-8551, Japan. e-mail: yoshkudo@hiroshima-u.ac.jp


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Expression of the tryptophan catabolizing enzyme, indoleamine 2,3-dioxygenase, in the mouse placenta has been shown to be critical in preventing immunological rejection of the fetal allograft. To clarify the physiological importance of indoleamine 2,3-dioxygenase in human pregnancy, we have studied how the expression of this enzyme changes during decidualization of human endometrium at both the cell and tissue level. METHODS and RESULTS: The level of indoleamine 2,3-dioxygenase mRNA expression (determined by RT–PCR) was higher in decidual than in endometrial tissue. Uterine decidual tissue in ectopic pregnancy similarly showed increased mRNA expression. Immunohistochemistry demonstrated that indoleamine 2,3-dioxygenase protein immunoreactivity was found in glandular epithelium and in stromal cells. The intensity of this immunoreactivity was increased in decidualized tissue. In a cell culture model, the level of indoleamine 2,3-dioxygenase mRNA was suppressed specifically by progesterone-induced decidualization of isolated endometrial stromal cells. Indoleamine 2,3-dioxygenase protein abundance (determined by Western blot) was also decreased by progesterone-induced decidualization. However interferon-{gamma}, a potent stimulator of indoleamine 2,3-dioxygenase gene expression, increased the level of indoleamine 2,3-dioxygenase mRNA and protein in both non-decidualized and in decidualized cells. Indoleamine 2,3-dioxygenase activity (determined by measuring the concentration of tryptophan and its indoleamine 2,3-dioxygenase catabolite, kynurenine) was also decreased by progesterone-induced decidualization but enhanced following interferon-{gamma} treatment. Expression of other interferon-{gamma} inducible genes (STAT1 and tryptophanyl-tRNA synthetase) showed the same pattern as that of indoleamine 2,3-dioxygenase in tissue samples, but was not changed by decidualization in the cell culture model. CONCLUSIONS: These data suggest that despite suppression by progesterone, indoleamine 2,3-dioxygenase expression in endometrial stromal cells may increase during decidualization due to stimulation by interferon-{gamma} secreted by infiltrating leukocytes.

Key words: decidualization/endometrium/indoleamine 2,3-dioxygenase/interferon-{gamma}


    Introduction
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 Abstract
 Introduction
 Materials and methods
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The enzyme indoleamine 2,3-dioxygenase is widely expressed in a variety of tissues of mammals (Hirata and Hayaishi, 1972Go; Cook et al., 1980Go; Yoshida et al., 1980Go; Yamazaki et al., 1985Go). It catalyses the oxidative cleavage of the pyrrole ring of the indole nucleus of various indoleamine derivatives (e.g. tryptophan, 5-hydroxytryptophan, tryptamine and serotonin) upon the insertion of two atoms of molecular oxygen (Yoshida and Hayaishi, 1987Go). It is primarily induced by interferon-{gamma}, but other proinflammatory stimulants are also effective (Taylor and Feng, 1991Go). By reducing the availability of tryptophan, indoleamine 2,3-dioxygenase is thought to regulate processes as diverse as intracellular infections (Daubener and MacKenzie, 1999Go), growth of malignant cells (Burke et al., 1995Go) and immune cell function (Munn et al., 1999Go; Hwu et al., 2000Go). One tissue with particularly high activity is the human placenta (Yamazaki et al., 1985Go). Munn et al. (1998Go) formulated the hypothesis that placental expression of indoleamine 2,3-dioxygenase at the maternal–fetal interface prevents immunological rejection of the fetal allograft. They suggested that T cells are inhibited by a mechanism involving indoleamine 2,3-dioxygenase-dependent localized depletion of tryptophan at the site of placentation. The role of this mechanism (discovered in mouse) in the human, and the extent to which defective activation of this process is responsible for disorders of pregnancy such as recurrent miscarriage, are currently unknown. We have previously studied the physiological significance of indoleamine 2,3-dioxygenase in human pregnancy (Kudo and Boyd, 2000Go, 2001; Kudo et al., 2000Go, 2001). Using placental villous and decidual explants from the first trimester, we showed that the same mechanism is available at the maternal–fetal interface of human pregnancy as in mouse (Kudo et al., 2004Go). Thus, indoleamine 2,3-dioxygenase-mediated localized depletion of tryptophan in human pregnancy can regulate proliferation of human peripheral blood mononuclear cells at the maternal–fetal interface. It has also been suggested that indoleamine 2,3-dioxygenase-mediated tryptophan depletion induces apoptosis of extravillous trophoblast cells, thereby limiting invasion into the uteroplacental arteries (Reister et al., 2001Go). Immunohistochemical staining for indoleamine 2,3-dioxygenase was found in syncytiotrophoblast, extravillous cytotrophoblast and macrophages in the villous stroma. Staining was also seen in the glandular epithelium, stromal cells and extravillous trophoblast cells of the decidual tissue (Kudo et al., 2004Go). Sedlmayr et al. (2002Go) also found indoleamine 2,3-dioxygenase strongly expressed in the glandular epithelium with some positive cells in the decidual stroma. They also demonstrated that endometrial glandular and surface epithelial cells showed increasing indoleamine 2,3-dioxygenase expression during the course of the menstrual cycle. To clarify the physiological potential of indoleamine 2,3-dioxygenase at the human maternal–fetal interface, we have now studied the expression of this enzyme during decidual change both in intact human endometrium tissue and in a cellular model of endometrial stromal cells.


    Materials and methods
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 Materials and methods
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Tissue collection and processing
Samples of human endometrium were obtained by punch biopsy after hysterectomy of patients with fibroma (proliferative phase, n = 5; secretory phase, n = 5). Samples of first trimester decidua were obtained directly from therapeutic first trimester terminations (7–8 weeks, n = 5). Uterine decidual tissue samples from women with tubal ectopic pregnancy were obtained by curettage of uterine contents after surgical intervention for ectopic pregnancy (6–7 weeks, n = 3). Freshly dissected tissues were fixed in neutral buffered formalin (NBF), embedded in paraffin and 4 µm sections were cut onto adhesive slides and dried overnight at 37°C in an oven. These studies were approved by the Ethics Committee of the Graduate School of Biomedical Sciences, Hiroshima University.

Culture of endometrial stromal cells
Endometrial stromal cells were prepared from freshly obtained human endometrium by a method modified from that described previously (Maruyama et al., 1999Go). Endometrial samples were histologically diagnosed as late proliferative phase according to a previously described method (Noyes et al., 1950Go). Tissue samples were washed with phosphate-buffered saline (PBS) and minced into small pieces (>1 mm3). The tissues were then incubated for 2 h at 37°C in a 1:1 mixture of Dulbecco’s modified Eagles’ medium (DMEM) and Ham’s F-12 medium (F-12) containing 0.25% collagenase, 0.002% deoxyribonuclease I, 100 IU/ml penicillin and 100 IU/ml streptomycin and 10% dextran-coated charcoal (DCC)-treated fetal bovine serum (FBS). After enzyme treatment, cell clumps were dispersed by pipetting. Most of the endometrial stromal cells that were present as single cells or small aggregates were filtered through a 70 µm cell strainer (Falcon 2350; Becton Dickinson Co., USA). The filtrate was washed three times, and the number of viable cells was counted by Trypan Blue dye exclusion. The isolated endometrial stromal cells were cultured at 37°C in DMEM/F-12 (1:1) supplemented with 10% DCC-treated FBS, 100 IU/ml penicillin and 100 IU/ml streptomycin in a humidified atmosphere of 5% CO2 and 95% air for 2–3 days to the stage of sub-confluence. The cells were then cultured with 10–9 mol/l 17{beta}-estradiol (E2) and/or 10–7 mol/l progesterone or vehicle for 6 days with the medium being changed every 48 h. Other additions are as described in the table and figure legends. Cultures were conducted in triplicate for each set of experiments to assess reproducibility. Cells were then used for immunocytochemistry, and total RNA and protein extraction as described below. The conditioned medium was collected and used for high-performance liquid chromatography (HPLC) analysis.

Purity of isolated endometrial stromal cells
The homogeneity of the stromal cell preparation was confirmed by means of immunostaining for vimentin (stromal cell; >97%), cytokeratin (epithelial cell; <2%) and CD45 (leukocyte common antigen; <2%) using standard technique.

Immunohistochemistry and immunocytochemistry
Sections were microwaved in Tris–EDTA antigen retrieval solution for 15 min (3x5 min cycles). The slides were left to cool for 20 min, before washing for 5 min in Tris-buffered saline (TBS). Sections were heated for 12 min in a conventional oven at 58–60°C, before being dewaxed in xylene and rehydrated in alcohol and finally washed in double-distilled (dd)H2O for 5 min. Endogenous peroxidase activity was blocked in 0.03% H2O2. For immunocytochemistry, cultured cells were fixed with neat acetone. Sections were blocked with neat fetal calf serum for 10 min to minimize non-specific background staining. The anti-indoleamine 2,3-dioxygenase monoclonal antibody (mAb1; which is highly specific to human indoleamine 2,3-dioxygenase and does not react with human tryptophan 2,3-dioxygenase; Takikawa et al., 1988Go) was used at a dilution of 1:50 (2 µg/ml) with mouse IgG as the negative control. Sections were incubated at room temperature for 1 h. Detection of antibody binding was done using goat anti-mouse Ig conjugated to peroxidase-labelled dextran polymer (EnVision+). After the substrate–chromogen solution (DAB substrate–chromogen) had been applied, the sections were rinsed in ddH2O for 5 min, counterstained in haematoxylin for ~10 s, washed in ddH2O and mounted with Aquamount.

RNA extraction and RT–PCR analysis
Indoleamine 2,3-dioxygenase, tryptophanyl-tRNA synthetase, STAT1 and prolactin gene expression were analysed by semi-quantitative RT–PCR using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal standard as described previously (Kudo et al., 2000Go). The primers used in the RT–PCR were as follows: indoleamine 2,3-dioxygenase, forward, 5'-TGCTAAACATCTGCCTGATC-3' and backward, 5'-GGAGCAATTGACTCAAATCA-3'; tryptophanyl-tRNA synthetase, forward, 5'-AGCTCAACTGCCCAGCGTGACC-3' and backward, 5'-CAGTCAGCCTTGTAATCCTCCCCC-3'; STAT1, forward, 5'-AAGGTGGCAGGATGTCTCAGTG-3' and backward, 5'-TGGTCTCGTGTTCTCTGTTCTG-3'; prolactin, forward, 5'-CGAAGACAAGGAGCAAGC-3' and backward, 5'-AAG CAGAAAGGCGAGACT-3'; GAPDH, forward, 5'-CGGGAAGC TTGTGATCAATGG-3' and backward, 5'-GGCAGTGATGG CATGGACTG-3'. The expected sizes of the PCR products were 144 bp for indoleamine 2,3-dioxygenase, 314 bp for tryptophanyl-tRNA synthetase, 564 bp for STAT1, 288 bp for prolactin and 358 bp for GAPDH. To control for DNA contamination, reactions were run without RNA or with RNA in the absence of the reverse transcriptase and revealed no amplified product (data not shown). The PCR conditions were: 94°C for 3 min, 60°C for 1 min and 72°C for 2 min; then 25 cycles (for indoleamine 2,3-dioxygenase, tryptophanyl-tRNA synthetase and STAT1), 22 cycles (for prolactin) and 20 cycles (for GAPDH) of 94°C for 1 min, 60°C for 1 min and 72°C for 2 min; followed by a 10 min final extension at 72°C. The amount of template cDNA and the number of cycles were determined experimentally so that quantitative comparison could be made during the exponential phase of the amplification process for both target and reference gene. PCR products were separated on a 2% agarose gel which was stained with ethidium bromide. The intensity of either the target gene or GAPDH band for each sample was quantified using scanning densitometry and the ratio of the target gene to GAPDH was used as a normalized measure of the target gene.

Western blot analysis
Harvested cells were washed twice with ice-cold PBS, suspended in 1 ml of ice-cold PBS containing 50 µl/g tissue protease inhibitor mixture and disrupted by sonication for 30 s in an ice bath at a power of 25 W. The homogenate was centrifuged at 15 000 g for 15 min at 4°C. The resultant supernatant (extract) was stored at –70°C. The extracts were mixed with Laemmli sample buffer (Laemmli, 1970Go) and boiled for 5 min before loading. Samples (20 µg of protein for each lane) were separated by electrophoresis under reducing conditions on 12% (w/v) sodium dodecyl sulphate (SDS)–polyacrylamide gels and then transferred to nitrocellulose membrane. After blocking by incubation in TBS containing 2% (w/v) bovine serum albumin (BSA) for 1 h at room temperature, the membrane was soaked overnight at 4°C in TBS containing indoleamine 2,3-dioxygenase monoclonal antibody (0.75 µg/ml) and 1% (w/v) BSA. The membrane was rinsed and washed three times for 5 min in TBS containing 0.1% (v/v) Tween 20 (TBS-T), incubated with anti-mouse IgG peroxidase-linked antibody (1:5000 dilution) in TBS-T for 1 h at room temperature and then rinsed and washed three times for 5 min in TBS-T followed by one wash in TBS for 5 min. Proteins were determined by ECL detection. The intensity of the band for each sample was quantified using an image documentation and analysis system.

HPLC analysis of L-tryptophan catabolism
The medium conditioned by culture with endometrial stromal cells was well mixed by vortexing with one-tenth volume of ice-cold 2.4 mol/l perchloric acid. The mixture was chilled on ice for 15 min and centrifuged at 10 000 g for 3 min. The clear protein-free supernatant was used for HPLC analysis of tryptophan and kynurenine concentrations. The HPLC system consisted of a Shimadzu LC-10AD pump and a Shimadzu SPD-10A variable wavelength detector (Kyoto, Japan) with a Spherisorb S5-ODS1 column, 4.6x150 mm (Waters, USA). The mobile phase consisted of 40 mmol/l citrate buffer (pH 2.25), 50% methanol and 0.4 mmol/l SDS, which were used after filtration through a 0.45 µm membrane filter and degassed using a vacuum aspirator. A 20 µl volume of protein-free extract was injected onto the column, chromatographed at a flow rate of 2.0 ml/ml and detected at 365 nm for kynurenine and at 280 nm for tryptophan. The minimum amount of kynurenine and tryptophan reproducibly detected was 20 pmol and calibration was linear up to 10 nmol of kynurenine or of tryptophan.

Protein estimation
Protein concentration of the cell extract was determined by the method of Lowry et al. (1951Go) using BSA as a standard.

Statistical analysis
Differences between two groups were analysed using an ANOVA and results were considered statistically significant at P < 0.05.

Chemicals
EnVision+ and DAB substrate–chromogen were purchased from Dako Corporation (USA), anti-mouse IgG peroxidase-linked antibody and ECL detection system were from Amersham Biosciences (USA). QuickPrep Total RNA Extraction Kit was purchased from Amersham Biosciences, deoxyribonuclease I (DNase I), Moloney murine leukaemia virus (M-MLV) reverse transcriptase, oligo(dT)12–18 primer, deoxynucleotide 5'-triphosphate and Taq DNA polymerase were from Gibco BRL (USA). Tissue protease inhibitor mixture and interferon-{gamma} were obtained from Sigma–Aldrich Chemical (USA) and tissue culture supplements were from Gibco BRL. All chemicals were of the highest purity commercially available.


    Results
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 Materials and methods
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 References
 
Indoleamine 2,3-dioxygenase expression in tissue samples of uterine endometrium and decidua
The relative expression of mRNA encoding indoleamine 2,3-dioxygenase in endometrium of proliferative and secretory phase, and in decidua of normal pregnancy and in the uterine decidual tissue of ectopic pregnancy was examined by RT–PCR (Figure 1). Indoleamine 2,3-dioxygenase mRNA expression level showed a 1.8-fold increase in secretory compared to proliferative phase endometrium and was further increased in decidua from either normal or ectopic pregnancy. Decidua of ectopic pregnancy had a lower (non-significant) mRNA expression level than that of normal pregnancy. GAPDH mRNA was constant for each sample. In order to study the localization of indoleamine 2,3-dioxygenase in human endometrium and decidua, sections were used to localize indoleamine 2,3-dioxygenase antigenicity. Proliferative phase endometrium showed immunoreactive indoleamine 2,3-dioxygenase in the glandular epithelium with faint staining of the stromal cells (Figure 2a). In the secretory phase there was enhanced staining of the stromal cells and of the glandular epithelium (Figure 2c). Some indoleamine 2,3-dioxygenase positive cells in the stroma are probably leukocytes. In first trimester decidua there was strong staining of the decidual stromal cells and of the glandular epithelium (Figure 2e). In uterine decidual tissue from ectopic pregnancy, nearly the same intensity of immunoreactivity was seen in the decidual stromal cells and in the glandular epithelium (Figure 2g).




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Figure 1. Relative abundance of mRNA encoding indoleamine 2,3-dioxygenase, tryptophanyl-tRNA synthetase and STAT1 in endometrial and in decidual tissue. Total RNA was extracted from endometrium of proliferative (Prolif) and secretory (Secret) phase, and from decidua of normal (Decidua) and ectopic (Ectopic) pregnancy. (A) RT–PCR. The relative abundance of indoleamine 2,3-dioxygenase mRNA (a), tryptophanyl-tRNA synthetase mRNA (b), STAT1 mRNA, (c) and glyceraldehyde phosphate 3-dehydrogenase (GAPDH) mRNA (d) were analysed by RT–PCR as described in Materials and methods. The results presented are from a single representative experiment with three different samples for each stage. (B) Relative quantification of indoleamine 2,3-dioxygenase mRNA, tryptophanyl-tRNA synthetase (Trp-tRNA synthetase) mRNA and STAT1 mRNA. The intensity of either the target gene or the GAPDH band was quantified by using a gel documentation and analysis system of PCR products and the ratio of the two was used as a normalized relative abundance value of each target gene. Data represent the mean ± SD of experiments performed with five proliferative and five secretory phase endometrial tissues, and five decidual tissues of normal first trimester and three uterine decidual tissues of ectopic pregnancy expressed as percentage of control (i.e. values for proliferative phase endometrium). *Significantly different from values for proliferative phase endometrium (P < 0.05). {dagger}Significantly different from values for secretory phase endometrium (P < 0.05). {ddagger}Significantly different from values for decidua of normal pregnancy (P < 0.05).

 


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Figure 2. Immunohistochemical localization of indoleamine 2,3-dioxygenase in human endometrium and decidua. Day 11 endometrium stained for indoleamine 2,3-dioxygenase (a) and MsIgG control (b). Day 25 endometrium stained for indoleamine 2,3-dioxygenase (c) and MsIgG control (d). First trimester decidua of normal pregnancy stained for indoleamine 2,3-dioxygenase (e) and MsIgG control (f). First trimester decidua of ectopic pregnancy stained for indoleamine 2,3-dioxygenase (g) and MsIgG control (h). Magnification: x40. Scale bars = 100 µm.

 
Indoleamine 2,3-dioxygenase expression during decidual change of endometrial stromal cells
The expression of indoleamine 2,3-dioxygenase during decidual change of cultured endometrial stromal cells following progesterone treatment was studied in order to observe the processes seen in the intact tissue at the level of one particular cell type in isolation. Decidualization of endometrial stromal cells was confirmed by their pavement-like morphology and by increased expression of prolactin mRNA (Tabanelli et al., 1992Go); thus following either progesterone or progesterone + E2 treatment, the level of expression of prolactin mRNA expression level was markedly increased. In contrast, the level of indoleamine 2,3-dioxygenase mRNA was suppressed (Figure 3). E2 alone had no effect on the level of indoleamine 2,3-dioxygenase or on prolactin mRNA. Figure 4 shows analysis by Western blot of indoleamine 2,3-dioxygenase protein levels in extracts of cells treated with progesterone and/or E2 or vehicle. The expected band, corresponding to the predicted molecular weight of indoleamine 2,3-dioxygenase, was found at 45 kDa. Indoleamine 2,3-dioxygenase protein level was also decreased in progesterone, or progesterone and E2-treated cells compared to that in control (vehicle alone) or E2-treated ones. Indoleamine 2,3-dioxygenase function was studied by measuring tryptophan and its degradation product kynurenine in the conditioned medium of cells treated with progesterone and/or E2 or vehicle (Table I). Both tryptophan degradation and kynurenine appearance correlated well with the level of indoleamine 2,3-dioxygenase protein determined by Western blot.




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Figure 3. Relative abundance of mRNA encoding prolactin, indoleamine 2,3-dioxygenase, tryptophanyl-tRNA synthetase and STAT1 in cultured endometrial stromal cells. Total RNA was extracted from cells cultured for 6 days with 10–7 mol/l progesterone and/or 10–9 mol/l E2 or vehicle (Nil), followed by further incubation in each medium with or without 1000 IU/ml interferon-{gamma} for 36 h. (A) RT–PCR. The relative abundance of prolactin mRNA (a), indoleamine 2,3-dioxygenase mRNA (b), tryptophanyl-tRNA synthetase mRNA (c), STAT1 mRNA (d) and glyceraldehyde phosphate 3-dehydrogenase (GAPDH) mRNA (e) were analysed by RT–PCR as described in Materials and methods. The results presented are from a single representative experiment. (B) Relative quantification of prolactin mRNA, indoleamine 2,3-dioxygenase mRNA, tryptophanyl-tRNA synthetase (Trp-tRNA synthetase) mRNA and STAT1 mRNA. The intensity of either the target gene or the GAPDH band was quantified by using a gel documentation and analysis system of PCR products and the ratio of the two was used as a normalized relative abundance value of each target gene. Data represent the mean ± SD of four separate experiments using four different samples, expressed as percentage of control (i.e. values for cells cultured with vehicle alone without interferon-{gamma} treatment). *Significantly different from control (P < 0.05). {dagger}Significantly different from values for cells cultured with vehicle alone with interferon-{gamma} treatment (P < 0.05).

 


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Figure 4. Protein expression level of indoleamine 2,3-dioxygenase in cultured endometrial stromal cells. Cells were cultured for 6 days with 10–7 mol/l progesterone and/or 10–9 mol/l E2 or vehicle (Nil) and medium was changed every 48 h, followed by further incubation in each medium with or without 1000 IU/ml interferon-{gamma} for 36 h. Indoleamine 2,3-dioxygenase protein levels in the cellular extracts were analysed as described in Materials and methods. (A) Western blot (under reducing conditions). The results presented are from a single representative experiment. (B) Quantification of indoleamine 2,3-dioxygenase protein levels. The intensity of each band was quantified by using an image documentation and analysis system. Data represent the mean ± SD of four separate experiments using four different samples, expressed as percentage of control (i.e. values for cells cultured with vehicle alone without interferon-{gamma} treatment). *Significantly different from control.

 

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Table I. Tryptophan catabolism by indoleamine 2,3-dioxygenase
 
Involvement of interferon-{gamma} in indoleamine 2,3-dioxygenase expression during decidual change
It is apparent that the findings on indoleamine 2,3-dioxygenase expression obtained in the cell model of decidualization differ from those obtained using whole tissue. It seems likely that tissue factors within intact tissue are responsible for this discrepancy. The effects of interferon-{gamma} on indoleamine 2,3-dioxygenase expression in cultured endometrial stromal cells were therefore studied since this cytokine stimulates indoleamine 2,3-dioxygenase gene expression (Taylor and Feng, 1991Go). Moreover, production of interferon-{gamma} by infiltrating leukocytes in decidua has been previously demonstrated (Ho et al., 1996Go; von Rango et al., 2003Go). In the experimental protocol, expression of mRNA encoding both tryptophanyl-tRNA synthetase and STAT1 were used as positive controls in both the tissue and the cell culture models. STAT1 is required for interferon-{gamma}-dependent transcription (Chon et al., 1996Go) and tryptophanyl-tRNA synthetase expression is induced by interferon-{gamma} through the same pathway as indoleamine 2,3-dioxygenase (Tolstrup et al., 1995Go). As expected, interferon-{gamma} treatment produced marked stimulation of indoleamine 2,3-dioxygenase, tryptophanyl-tRNA synthetase and STAT1 mRNA expression both in non-decidualized (E2 or vehicle treated) and in decidualized (progesterone or progesterone and E2 treated) cells (Figure 3). Indoleamine 2,3-dioxygenase protein expression was also stimulated by interferon-{gamma} both in non-decidualized and in decidualized cells, as demonstrated by both Western blot (Figure 4) and immunocytochemistry (Figure 5). Indoleamine 2,3-dioxygenase function was also enhanced following interferon-{gamma} treatment (Table I) since a further decrease in tryptophan and increase in kynurenine concentration was observed. The expression of other interferon-{gamma} inducible genes (i.e. tryptophanyl-tRNA synthetase and STAT1) during the course of the menstrual cycle and decidual change showed the same pattern as that of indoleamine 2,3-dioxygenase in the tissue sample (Figure 1). In contrast, in the endometrial stromal cell culture, progesterone and/or E2 had no effect on the expression level of mRNA encoding these interferon-{gamma} inducible genes in either the presence or the absence of interferon-{gamma} (Figure 3).



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Figure 5. Immunocytochemistry of indoleamine 2,3-dioxygenase in cultured endometrial stromal cells. Cells were cultured for 6 days with 10–9 mol/l E2 (a, b, c) or 10–7 mol/l progesterone and 10–9 mol/l E2 (d, e, f), followed by further incubation in each medium with or without 1000 IU/ml interferon-{gamma} for 36 h. Interferon-{gamma}-untreated non-decidualized cells stained for indoleamine 2,3-dioxygenase (a), interferon-{gamma}-treated non-decidualized cells stained for indoleamine 2,3-dioxygenase (b) and MsIgG control (c). Interferon-{gamma}-untreated decidualized cells stained for indoleamine 2,3-dioxygenase (d), interferon-{gamma}-treated decidualized cells stained for indoleamine 2,3-dioxygenase (e) and MsIgG control (f). Magnification: x100. Scale bars = 40 µm.

 

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 Materials and methods
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 References
 
We conducted the experiments described here to ask questions concerning the mechanism of the expression of indoleamine 2,3-dioxygenase in human endometrial stromal cells during decidualization. In the intact tissue, indoleamine 2,3-dioxygenase gene expression and immunoreactivity are enhanced during the process of decidualization. It might therefore have been predicted that indoleamine 2,3-dioxygenase expression would also have been enhanced in our cell model of decidualization. Instead, the converse was observed both at gene and at protein level. This paradox can be explained if induction of indoleamine 2,3-dioxygenase during the decidualization of endometrial stromal cells does not simply depend on their differentiation, but requires local factors. We propose that one such factor is interferon-{gamma} secreted by leukocytes infiltrating the endometrium during the process of decidualization. This conclusion arises from the following observations both from this study and by others published previously; (i) indoleamine 2,3-dioxygenase gene expression and protein level are decreased following progesterone-induced decidualization of isolated endometrial stromal cells; (ii) when interferon-{gamma} is added to endometrial stromal cells in culture, indoleamine 2,3-dioxygenase gene expression and protein level are both increased; (iii) other interferon-{gamma}-regulated genes (tryptophanyl-tRNA synthetase and STAT1) show the same pattern of altered expression as indoleamine 2,3-dioxygenase during decidual change in tissue samples; (iv) the expression of tryptophanyl-tRNA synthetase and STAT1 is not altered following progesterone-induced decidualization of cultured endometrial stromal cells; and (v) well-established data show that there are large numbers of different leukocyte populations within the stromal component of the endometrium during the luteal phase and that this infiltration of the decidua increases in the first trimester of pregnancy. These leukocyte populations are a major source of cytokines, including interferon-{gamma} (Jokhi et al., 1997Go).

The mechanism of suppression of indoleamine 2,3-dioxygenase expression (at both gene and protein level) following progesterone-induced decidualization in cultured endometrial stromal cells is unclear at present. It may be due to the direct effect of progesterone or to indirect effects, e.g. through increased intracellular cAMP concentrations (Brar et al., 1997Go). Physiologically it seems reasonable to suggest that it will be the combination of progesterone inhibition, and local cytokine stimulation, of gene transcription that will regulate indoleamine 2,3-dioxygenase activity and consequently immune cell function at the maternal–fetal interface.

It has been shown that in early pregnancy there is a particularly dense infiltration of activated T cells and natural killer cells in the decidua having a different phenotype from peripheral blood natural killer cells (King et al., 1998Go); and that decidual natural killer cells also produce a wide variety of cytokines (Jokhi et al., 1994Go). Secretion of interferon-{gamma} by these immune cells has been confirmed (Ho et al., 1996Go). It is possible to speculate that cytokines produced by these cells regulate indoleamine 2,3-dioxygenase expression and form a cytokine network at the site of placentation. The local concentration of tryptophan (controlled by the extent of indoleamine 2,3-dioxygenase expression) may in turn control the differentiation and function of natural killer cells. This is obviously of relevance to the possible immunoregulatory role of indoleamine 2,3-dioxygenase at the maternal–fetal interface (Bonney and Matzinger, 1998Go; Mellor and Munn, 1999Go).

Indoleamine 2,3-dioxygenase expression is also enhanced in the uterine decidua of women with a tubal ectopic pregnancy in which endometrial stromal cells decidualize morphologically and functionally in a way similar to those of normal pregnancy. However, in deciduas from ectopic pregnancy, there are fewer decidual T cells and a lower concentration of interferon-{gamma} (von Rango et al., 2001Go). This is consistent with the slight decrease of indoleamine 2,3-dioxygenase mRNA expression that we found in the uterine decidual tissue of ectopic compared with that of normal pregnancy (Figure 1). It is also possible that the absence of extravillous trophoblast cells (which are known to express indoleamine 2,3-dioxygenase) is responsible for decreased indoleamine 2,3-dioxygenase mRNA expression level in the uterine decidual tissue of ectopic pregnancy.

Beside indoleamine 2,3-dioxygenase expression in human placental tissue, it has been shown that indoleamine 2,3-dioxygenase expression in dendritic cells and monocytes is exaggerated in pregnant women when compared with non-pregnant controls (Steckel et al., 2003Go). This may also be relevant for the development of maternal immune tolerance.

As discussed in an earlier work (Kudo et al., 2001Go) there are still fundamental questions about the role of the immune response and its regulation in normal human pregnancy; the data provided here about indoleamine 2,3-dioxygenase expression and its regulation in the maternal tissue (the uterine endometrium) may help in the understanding of the normal maternal–fetal immune interaction.


    Acknowledgements
 
We thank Dr C.A.R.Boyd, Department of Human Anatomy and Genetics, University of Oxford for valuable comments and Research Centre for Molecular Medicine, Faculty of Medicine, Hiroshima University for the use of their facilities. We are grateful to the Grant-in-Aid for Scientific Research (15591752), Ministry of Education, Science and Culture, Japan for financial support.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on July 23, 2003; resubmitted on December 15, 2003; accepted on February 19, 2004.