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
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Abstract |
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Key words:
decidualization/endometrium/indoleamine 2,3-dioxygenase/interferon-
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Introduction |
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Materials and methods |
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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., 1999). Endometrial samples were histologically diagnosed as late proliferative phase according to a previously described method (Noyes et al., 1950
). 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 Dulbeccos modified Eagles medium (DMEM) and Hams 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 23 days to the stage of sub-confluence. The cells were then cultured with 109 mol/l 17
-estradiol (E2) and/or 107 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 TrisEDTA 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 5860°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., 1988) 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 substratechromogen solution (DAB substratechromogen) 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 RTPCR analysis
Indoleamine 2,3-dioxygenase, tryptophanyl-tRNA synthetase, STAT1 and prolactin gene expression were analysed by semi-quantitative RTPCR using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal standard as described previously (Kudo et al., 2000). The primers used in the RTPCR 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, 1970) 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. (1951) 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 substratechromogen 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)1218 primer, deoxynucleotide 5'-triphosphate and Taq DNA polymerase were from Gibco BRL (USA). Tissue protease inhibitor mixture and interferon- were obtained from SigmaAldrich Chemical (USA) and tissue culture supplements were from Gibco BRL. All chemicals were of the highest purity commercially available.
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Results |
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Discussion |
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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., 1997). 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 maternalfetal 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., 1998); and that decidual natural killer cells also produce a wide variety of cytokines (Jokhi et al., 1994
). Secretion of interferon-
by these immune cells has been confirmed (Ho et al., 1996
). 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 maternalfetal interface (Bonney and Matzinger, 1998
; Mellor and Munn, 1999
).
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- (von Rango et al., 2001
). 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., 2003). This may also be relevant for the development of maternal immune tolerance.
As discussed in an earlier work (Kudo et al., 2001) 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 maternalfetal immune interaction.
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Acknowledgements |
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Submitted on July 23, 2003; resubmitted on December 15, 2003; accepted on February 19, 2004.