Effect of Kidney-Replenishing Herb on the Indoleamine 2,3- Dioxygenase of Human Syncytiotrophoblasts Cultured in Vitro and the Balance of Helper T-Cell Cytokines

By Li, Xuelian Gui, Suiqi; Wang, Haiyan

Abstract Background. There is complicated pathogeny involved in spontaneous abortion. At present, the focus of study is on the interface between mother and fetus, the trophoblasts. Indoleamine 2,3-dioxygenase (IDO) is the first and regulatory enzyme in the major route of L-tryptophan catabolism, which induces immunosuppression of T lymphocytes. In the present study we investigated the effect of Kidney-replenishing herb on the expression and activity of IDO in human syncytiotrophoblasts cultured in vitro and the balance of helper T cell (Th) cytokines.

Methods. Syncytiotrophoblasts were cultured in vitro for 24, 48 or 72 h, with either control serum or serum made from Kidney- replenishing herb, without or with different concentrations of the IDO inhibitor 1-methyltryptophan (1-MT). Reverse transcription- polymerase chain reaction was applied to analyze the IDO mRNA transcription of syncytiotrophoblasts and Western blotting was applied to determine the expression of IDO protein in syncytiotrophoblasts. The concentration of interleukin-10 and interferon-gamma in co-culture medium of syncytiotrophoblasts and decidual T lymphocytes was determined by enzyme-linked immunosorbent assay. High-performance liquid chromatography was used to determine the concentration of kynurenine (Kyn) and tryptophan (Tyr) in the co- culture medium, and the ratio of Kyn/Try was used to assess IDO activity.

Results. IDO mRNA and protein were detected in human syncytiotrophoblasts cultured in vitro. The IDO inhibitor 1-MT caused the balance of Th cytokines to depart from type 2; when IDO activity was inhibited, Kidney-replenishing herb improved the expression of IDO mRNA and protein, promoted IDO activity and caused the balance of Th cytokines depart from type 1.

Conclusion. Kidney-replenishing herb improves the expression of IDO mRNA and protein, promotes IDO activity to an appropriate value, resumes the balance of Th cytokines and regulates maternofetal tolerance.

Keywords: Indoleamine 2,3-dioxygenase, syncytiotrophoblasts, Kidney-replenishing herb, decidual T lymphocytes, Th cytokine balance

Introduction

There is complicated pathogeny involved in spontaneous abortion, and the modulatory abnormality of incretion and immunity is estimated to account for 80-94% of such events. Why is the fetus, a homogeneous implant, not excluded by the mother? At present, the focus of study is on the interface between mother and fetus – the trophoblasts – with the aim to elucidate the immunological relationship between the trophoblasts and the lymphocytes which come from the mother. Indoleamine 2,3-dioxygenase (IDO), which is mostly found in the antigen-presenting cells of lymphoid organs, syncytiotrophoblasts and macrophages of gestation, is the first and regulatory enzyme of the kynurenine pathway (KP), which is the major route of L-tryptophan catabolism. The tryptophan catabolites produced through the KP induce immunosuppression of T lymphocytes [1,2], and may play an important role in the balance of helper T cell (Th) cytokines at the maternofetal interface and thus the maternofetal tolerance. We use Kidney-replenishing herb to treat those women with spontaneous abortion caused by the modulatory abnormality of incretion and immunity, and the outcome is satisfactory. Our previous study revealed that type 1 Th (Th1) cytokines such as interferon (IFN)-gamma and type 2 Th (Th2) cytokines such as interleukin (IL)-10 are related to spontaneous abortion. IFN-gamma is the major Th1 cytokine which could induce abortion in many complex ways [3-6]. IL-10 could restrain the expression of IFN-gamma, IL-1, tumor necrosis factor (TNF)-alpha and other cytokines, and protect the fetus from exclusion [7-9]. But each cytokine must act as one member of a network. It is the balance of Th1 and Th2 cytokines, not the absolute value of any one cytokine, which is most important to the maintenance of pregnancy [3]. Our previous study showed that the balance of IL-10 and IFN- gamma is the best indicator of the balance of Th1 and Th2 cytokines in the rat model of abortion [10].

In order to further study the modulation of Kidney-replenishing herb on maternofetal tolerance and probe into the mechanism of protecting the fetus, we studied the effect of Kidney-replenishing herb on the expression and activity of IDO in human syncytiotrophoblasts cultured in vitro and the balance of Th cytokines.

Material and methods

Tissue collection

Villus and decidual specimens were donated with informed consent by 32 healthy women undergoing elective termination of pregnancy at 6-12 weeks of gestation in the Hospital of Obstetric & Gynecology, Fudan University, Shanghai, China.

Animals

Twelve- to 14-week-old female Sprague-Dawley rats, weight 200 +- 10 g, were chosen to be bred cleanly. The animals received standard grain feed for rats, fresh water ad libitum and were housed in cages with 12 h of illumination daily.

Chemicals

Trypsin (Amresco); deoxyribonuclease I (Sigma); Dulbecco’s modified Eagle’s medium (DMEM; Gibco); fetal calf serum (FCS; Gibco); Ficoll-Paque (Pharmacia); recombinant human epidermal growth factor (rHuEGF; Peprotech); Percoll (Gibco); diethylpyrocarbonate (DEPC; Genview); phytohemogglutinin (PHA-P; Sigma); histochemical ABC kit (Huamei); RNAex Reagent, RevertAid(TM) First Strand cDNA Synthesis Kit and Taq DNA polymerase (Fermentas); BCA-100 Protein Quantitative Analysis Kit (Shenergy); Visual AP Western Blotting Reagent Set (Ab Minus) SNBC, Human IFN-gamma/IL-10 Immunoassay (R&D), mouse anti-CD3 antibodies (human T cells) (UCHT1) and mouse anti-IDO monoclonal antibody (Chemoicon); mouse anti-vimentin, cytokeratin and human chorionic gonadotropin-beta (hCG-beta) monoclonal antibodies (Zhongshan); L-tryptophan and kynurenine (Sigma); 1-methyltryptophan (1-MT; Aldrich); IDO forward primer (5′- TCC GTG AGT TTG TCC TT-3′) and reverse primer (5′-GCA TAG TAT TAG TTT GTG GC-3′), product 361 bp and beta-actin forward primer (5′- GAG CGG GAA ATC GTG CGT GAC ATT-3′) and reverse primer (5′-GAT GGA GTT GAA GGT AGT TTC GTG-3′), product 240 bp (Bioasia).

Instruments

UNICO(TM) UV-2102C ultraviolet spectrophotometer; DNA Thermal Cycler (Perkin Elmer); SCR-4 high-pressure electrophoretic instrument with H6-1 electrophoretic slot; Tanon GIS2010 GIS gel image disposal system; Waters high-performance liquid chromatograph with C-18 column (Zorbax C18, 250 mm x 4.6 mm, 5 [mu]m) and Waters 2996 PDA detector.

Preparation of serum

Based on our experiential prescription, the Kidney-replenishing herb comprising Dangshen 12 g, Tusizi 15 g, Baizhu 6 g, Baishuo 9 g, Duzhong 12 g, Sangjisheng 12 g, Sugeng 6 g and Tiaohuangqin 15 g has a satisfactory effect on patients with threatened abortion. These herbs were collected by the scholars of Shanghai Graduate School of Medication (Chinese Academy of Science), boiled in water and then filtrated, and the liquid was concentrated and precipitated with 90% ethanol, these courses were repeated three times then the deposit was collected and dissolved in ddd-H2O. Based on our previous experiments, rats of the experimental group were given liquid medicine (16 g herb/kg body weight) directly into the stomach, once a day for 5 days, and rats of the control group were given dddH^sub 2^O according to the same formula. On the sixth day, the rats were anesthetized by injection of amyl barbitone, blood was extracted from the ventral aorta, and the serum was separated and kept at – 20[degrees]C.

Isolation, culture and identification of syncytiotrophoblasts

Following previous authors [11-20], the villus tissue was collected sterilely, rinsed in D-Hanks medium, cut into smaller pieces, and digested at 37[degrees]C with 0.125% trypsin and 0.02% deoxyribonuclease I for three times, 5 min each time. The supernatants were filtered over a succession of sieves with 200 [mu]m and 60 [mu]m pore size to remove aggregates. The cells were then centrifuged at 350g for 10 min, re-suspended in DMEM, layered over a continuous Percoll gradient (30-70% in 5% steps of 2 ml each), then centrifuged at 37[degrees] C and 1200g for 20 min to separate the different cell types. Cytotrophoblast cells between the density markers of 1.049 and 1.062 g/ml were collected, suspended in DMEM containing FCS (10%), penicillin (100 U/ml) and streptomycin (100 [mu]g/ml), then plated in 24-well plates at a density of 10^sup 6^ cells/ml per well or in a 5 ml flask at 1-5 x 10^sup 6^ cells/ ml. The cells were cultured in a humidified atmosphere (95% air-5% CO2 at 37[degrees]C) with rHuEGF (10 ng/ml) to promote the congregation and amalgamation for 24, 48 or 72 h, and next fixed and immunostained for cytokeratin, vimentin and hCG-beta according to the guide of the histochemical ABC kit. The cultures obtained in this way usually consist of more than 90% syncytiotrophoblasts, as judged by morphological and immunocytochemical determination of vimentin-negative, cytokeratin-positive and hCG-beta-positive.

Isolation, culture and identification of decidual T lymphocytes

The deciduas was collected sterilely, trimmed into 1 mm pieces, milled to pass a sieve with 60 fim pore size, centrifuged at 500g for 10 min, suspended in DMEM, layered over Ficoll-Paque and centrifuged at 1200g for 20 min. Cells between the interface were collected, suspended in DMEM containing FCS (10%), penicillin (100 U/ ml) and streptomycin (100 [mu]g/ml), then plated in a 30 mm tissue culture dish at a density of 1-5 x 106 cells/ml and cultured in a humidified atmosphere for 4 h (95% air-5% C02 at 37[degrees]C). Non- adherent cells were collected, fixed and immunostained for CD3 according to the guide of the histochemical ABC kit. The cultures obtained in this way usually consist of more than 90% decidual T lymphocytes, as judged by immunocytochemical determination of CD3- positive. Co-culture of syncytiotrophoblasts and decidual T lymphocytes

Cells were co-cultured with PHA-P (5 fig/ml), serum of the herb group or control group rats (10%) and the IDO inhibitor 1-MT (0, 500 or 1000 fiM) for 24, 48 or 72 h in a humidified atmosphere (95% air- 5% C02 at 37[degrees]C). The culture medium was collected, tested immediately or kept at – 20[degrees]C.

RNA isolation and amplification of indoleamine 2,3-dioxygenase mRNA from human syncytiotrophoblasts cultured in vitro

Total RNA was prepared with RNAex Reagent, treated with RNase- free DNase and quantified spectrophotometrically. Reverse transcription products were prepared with the RevertAid(TM) First Strand cDNA Synthesis Kit in accordance with the manufacturer’s instructions. The reaction system for polymerase chain reaction (PCR) comprised 1-5 [mu]l of template DNA, 2.5 [mu]l of PCR buffer (1.5 mM MgCl^sub 2^), 0.5 [mu]l of dNTP mix, 1 [mu]l of forward primer, 1 [mu]l of reverse primer, 0.5 [mu]l of Taq DNA polymerase and DEPC in water to reach a final volume of 25 [mu]l. The mixture was incubated at 95[degrees] C for 3 min, followed by reaction at 94 C for 60 s, 47[degrees] C for 60 s and 72[degrees]C for 60 s, for a total of 30 cycles, and 72[degrees] C for 15 min. Samples were subjected to electrophoresis on 1% agarose gel, stained with ethidium bromide and photographed under ultraviolet light. As a positive control, human beta-actin RNA was reverse transcribed and amplified in parallel for each sample, and genomic DNA was used as a positive control for the PCR. As a negative control, an identical amount of RNA for each sample was amplified without being reverse- transcribed.

Western blot analysis for expression of indoleamine 2,3- dioxygenase protein in human syncytiotrophoblasts cultured in vitro

The protein was collected and quantified using the BCA-100 kit in accordance with the manufacturer’s instructions. A 10% polyacrylamide slab gel for separation and a 5% gel for concentration were used, 20 fd of sample being loaded into each well with an equal amount of sodium dodecylsulfate and subjected to electrophoresis for 1.5-2 h. Electrophoretic transfer of the protein onto a nitrocellulose membrane was done at 250 mA constant current for 1.5-2 h in ice. The membrane was then washed with Tris-buffered saline (TBS), placed in 4% skimmed milk powder-TBS for 1.5-2 h to block the non-specific binding sites, removed and washed. Next it was placed in a buffered solution of primary monoclonal IDO antibody (1:1000 dilution), left overnight at 4[degrees]C, removed and washed, then placed in secondary antibody [alkaline phosphatase (AP)- conjugated goat anti-mouse immunoglobulin G, 1:2000] for 1 h at room temperature, removed and washed. Finally, the membrane was incubated for 10-30 min at room temperature with AP substrate solution [3 ml AP substrate solution + 20 [mu]l NBT (nitroblue tetrazolium chloride) solution + 20 [mu]l BCIP (5-bromo-4-chloro-3-indoyl phosphate) solution].

Indoleamine 2,3-dioxygenase activity of syncytiotrophoblasts detected by high-performance liquid chromatography

Samples of culture medium were deproteinized with 4% trichloroacetic acid and total free tryptophan (Tyr) and kynurenine (Kyn) were assayed by high-performance liquid chromatography (HPLC) on a C-18 column eluted isocratically using a solvent of 0.1 M phosphate buffer (pH 4.0 with sodium hydroxide). The ratio of Kyn/ Try was used to assess the IDO activity.

Interleukin-10 and interferon-y in the co-culture medium of syncytiotrophoblasts and decidual T lymphocytes

Measurements were performed according to Human IFN-gamma/IL-10 Immunoassay kit.

Statistical analyses

Results were analyzed statistically by means of the t test using the SPSS 11.0 software package. Probability values of <0.05 and <0.01 were considered significant.

Results

Identification of syncytiotrophoblasts

Figures 1 and 2 are electron micrographs of cytotrophablasts and syncytiotrophoblasts. The cells collected are syncytiotrophoblasts as confirmed by immunochemistry (vimentin-negative, keratinpositive and hCG-beta-positive; Figures 3-5). Figure 6 shows that syncytiotrophoblasts are IDO-positive.

Figure 1. The electron-microscope view of cytotrophoblasts.

Figure 2. The electron-microscope view of syncytiotrophoblasts.

Indoleamine 2,3-dioxygenase mRNA expression of syncytiotrophoblasts

The IDO mRNA expression of two groups of syncytiotrophoblasts (with control serum and with herb serum) without IDO inhibitor is shown in Figures 7 and 8, where it can be seen that IDO mRNA expression increases with time of culture (p < 0.05). The IDO mRNA expression of two groups of syncytiotrophoblasts (with control serum and with herb serum) with different concentrations of the IDO inhibitor 1-MT is shown in Figures 9 and 10, where it can be seen that the expression of IDO mRNA does not change with increasing 1- MT concentration in the control group (p > 0.05) but improves in the herb group (p < 0.05).

Figure 3. Syncytiotrophoblasts are vimentin-negative ( x 200).

Figure 4. Syncytiotrophoblasts are keratin-positive ( x 200).

Figure 5. Syncytiotrophoblasts are positive for human chorionic gonadotropin-beta ( x 200).

Figure 6. Syncytiotrophoblasts are positive for indoleamine 2,3- dioxygenase ( x 200).

Indoleamine 2,3-dioxygenase protein expression of syncytiotrophoblasts

The IDO protein expression of two groups of syncytiotrophoblasts (with control serum and with herb serum) without IDO inhibitor is shown in Figures 11 and 12, where it can be seen that IDO protein expression increases with time of culture (p < 0.05). The IDO protein expression of two groups of syncytiotrophoblasts (with control serum and with herb serum) with different concentrations of the IDO inhibitor 1-MT is shown in Figures 13 and 14, where it can be seen that the expression of IDO protein does not change with increasing 1-MT concentration in control group (p > 0.05) but improves in the herb group (p < 0.05).

Figure 7. Effect of Kidney-replenishing herb on the expression of indoleamine 2,3-dioxygenase mRNA (361 bp) of syncytiotrophoblasts cultured for different times (240 bp is beta-actin mRNA).

Figure 8. Effect of Kidney-replenishing herb on the expression of indoleamine 2,3-dioxygenase mRNA (361 bp) of syncytiotrophoblasts cultured for different times. Values are means, with standard deviation shown by vertical bars. *Mean values were significantly different compared with syncytiotrophoblasts cultured for 24 h: p < 0.05; **mean values were significantly different compared with syncytiotrophoblasts cultured for 48 h: p < 0.05.

Figure 9. Effect of Kidney-replenishing herb on the expression of indoleamine 2,3-dioxygenase (IDO) mRNA (361 bp) of syncytiotrophoblasts cultured with different concentrations of the IDO inhibitor, 1-methyltryptophan (1-MT) (240 bp is beta-actin mRNA).

Figure 10. Effect of Kidney-replenishing herb on the expression of indoleamine 2,3-dioxygenase (IDO) mRNA (361 bp) of syncytiotrophoblasts cultured with different concentrations of the IDO inhibitor, 1-methyltryptophan (1-MT). Values are means, with standard deviation shown by vertical bars. * Mean values were significantly different compared with the control group without 1- MT: p < 0.05.

Figure 11. Effect of Kidney-replenishing herb on the expression of indoleamine 2,3-dioxygenase protein of syncytiotrophoblasts cultured for different times.

Figure 12. Effect of Kidney-replenishing herb on the expression of indoleamine 2,3-dioxygenase protein of syncytiotrophoblasts cultured for different times. Values are means, with standard deviation shown by vertical bars. *Mean values were significantly different compared with syncytiotrophoblasts cultured for 24 h: p < 0.05; ** mean values were significantly different compared with syncytiotrophoblasts cultured for 48 h: p<0.05.

Indoleamine 2,3-dioxygenase activity of syncytiotrophoblasts

Amounts of L-tryptophan and kynurenine in the culture medium were detected by HPLC. The IDO activity of two groups of syncytiotrophoblasts (with control serum and with herb serum) cultured with different concentrations of the IDO inhibitor 1-MT for different times is shown in Figure 15. The IDO activity of the control group was inhibited by 1-MT and related to the concentration of 1-MT {p < 0.05) but not to time of culture (p > 0.05); IDO activity of the herb group was not affected by 1-MT concentration (p > 0.05) and also unrelated to time to culture (p > 0.05).

Figure 13. Effect of Kidney-replenishing herb on the expression of indoleamine 2,3-dioxygenase (IDO) protein of syncytiotrophoblasts cultured with different concentrations of the IDO inhibitor, 1- methyltryptophan (1-MT).

Figure 14. Effect of Kidney-replenishing herb on the expression of indoleamine 2,3-dioxygenase (IDO) protein of syncytiotrophoblasts cultured with different concentrations of the IDO inhibitor, 1- methyltryptophan (1-MT). Values are means, with standard deviation shown by vertical bars. *Mean values were significandy different compared with the control group without 1-MT: p < 0.05.

Figure 15. Effect of Kidney-replenishing herb on the indoleamine 2,3-dioxygenase (IDO) activity (calculated as the ratio of kynurenine to tryptophan) of syncytiotrophoblasts cultured with different concentrations of the IDO inhibitor, 1-methyltryptophan (1- MT), for different times.

Cytokine balance of co-culture medium of syncytiotrophoblasts and decidual T lymphocytes

The content of Thl cytokine (IFN-gamma) in the coculture medium of syncytiotrophoblasts and decidual T lymphocytes, cultured with different concentrations of the IDO inhibitor 1-MT and for different times, is shown in Table I; Table II presents corresponding results for Th2 cytokine (IL-10). The balance of Thl/Th2 cytokines (shown as IFN-y/IL-10) is detailed in Table III. The Th1/Th2 cytokine balance of the control group increased with 1-MT concentration (p < 0.05) but was unrelated with time of culture (p > 0.05); while Th1/Th2 cytokine balance of the herb group was not affected by 1-MT concentration (p > 0.05) or time of culture (p > 0.05). Discussion

The pathogeny of spontaneous abortion involves disorders of both the mother and the fetus. One of the main factors is the modulatory abnormality of incretion and immunity. Our experiential prescription of Kidney-replenishing herb, consisting of Dangshen, Tusizi, Baizhu, Sugeng, Baishuo and three other kinds of herbs, affects the immunity of the mother. Our previous study proved that Kidney-replenishing herb can modulate the differentiation of the trophoblast, accelerate its growth and fusion, increase its invasion activity, favor the synthesis of hCG-beta, reduce trophoblast apoptosis induced by the ratio of TNF-alpha to IFN-gamma, and favor embedding of the zygote.

Table I. Content (pg/ml) of Thl cytokine (IFN-gamma) in co- culture supernatants of syncytiotrophoblasts and decidual T lymphocytes cultured with different concentrations of the IDO inhibitor 1-MT for different times.

Table II. Content (pg/ml) of Th2 cytokine (IL-10) in co-culture supernatants of syncytiotrophoblasts and decidual T lymphocytes cultured with different concentrations of the IDO inhibitor 1-MT for different times.

Table III. Ratio of Th1/Th2 cytokines (shown as IFN-gamma/IL-10) of co-culture supernatants of syncytiotrophoblasts and decidual T lymphocytes cultured with different concentrations of the IDO inhibitor 1-MT for different times.

IDO, a cytosolic heme protein found mostly in the antigen- presenting cells of lymphoid organs, syncytiotrophoblasts and macrophages of gestation, is a regulatory and rate-limiting enzyme in the conversion of L-tryptophan in the KP, which converts the essential amino acid L-tryptophan into N-formylkynurenine. Our previous study revealed that the expression of IDO mRNA and protein was lower in villus of inevitable abortion than in that of normal pregnancy [21].

We supposed that normal expression and activity of IDO in villus was an important factor in the maintenance of pregnancy. In the present study, which was designed to elucidate the effect of Kidney- replenishing herb on the IDO of human syncytiotrophoblast cultured in vitro and the balance of Th cytokines, we found that the expression of IDO mRNA and protein increased with the time of culture. IDO was found to modulate the balance of Th1/Th2 cytokines at the maternofetal interface and the IDO inhibitor 1-MT was found to reduce IDO activity, making the balance of Th cytokines depart from Th2, which is unfavorable for pregnancy, the effect being more obvious as the concentration of 1-MT increased.

These findings can be explained by the reports that IDO is expressed in trophoblasts and defends the conceptus against rejection by reducing the level of tryptophan, a necessary amino acid of cell proliferation, and suppressing T-cell activity [1]. IDO- expressing cells could create a local microenvironment in which levels of tryptophan are low, block the cell cycle at mid-G1 stage, make T cells prone to apoptosis especially via Fas, and regulate T- cell proliferation and activation [22,23]. IDO is inducible by cytokines such as IFN-gamma and plays a role in the balance of Th cytokines at the maternofetal interface and in the inflammation and maternal tolerance of fetal allografts. Blocking tryptophan catabolism during murine pregnancy allows maternal T cells to provoke fetal allograft rejection. Cells expressing IDO, which catabolizes tryptophan, prevent T-cell cycle progression, enhance activation, induce T-cell death, regulate maternal T-cell immunity during pregnancy and might contribute to immunological discrimination by promoting T-cell tolerance in other circumstances [24,25].

The mechanism of IDO-mediated tolerance is not well understood, but recent findings have implicated tryptophan catabolism through the KP as one of many mechanisms involved. It has recently been found that inhibition of IDO can result in the rejection of allogenic fetuses, suggesting that tryptophan breakdown is necessary for maintaining aspects of immune tolerance. Two theories have been proposed to explain how tryptophan catabolism facilitates tolerance. One theory posits that tryptophan breakdown suppresses T-cell proliferation by dramatically reducing the supply of this critical amino acid. The other theory postulates that the downstream metabolites of tryptophan catabolism act to suppress certain immune cells, probably by pro-apoptotic mechanisms [26].

Our present study found that Kidney-replenishing herb can improve the expression of IDO mRNA and protein, improve IDO activity, modulate the balance of Th1/Th2 cytokines at the maternofetal interface to normal level, and favor maintenance of normal pregnancy when IDO activity was inhibited. The results might be related to the fact that Kidney-replenishing herb can modulate the differentiation of the trophoblast, accelerate its growth and fusion, reduce the apoptosis of trophoblasts induced by the ratio of TNF-alpha and IFN- gamma, and increase the amount of syncytiotrophoblasts. But Kidney- replenishing herb did not affect the expression of IDO mRNA and protein, IDO activity, and the Th1/Th2 cytokine balance at the maternofetal interface when IDO activity was normal and would not induce abnormality of maternal immunity during pregnancy.

In addition, we also assessed the modulating effect of Kidney- replenishing herb on maternal immunity during pregnancy with a view to studying the relationship between syncytiotrophoblasts and decidual T lymphocytes, and found that IDO was one of the key players in the maternofetal incretion-immunity network. The processes of IDO modulating the balance of Th1/Th2 cytokines at the maternofetal interface might involve modulating the proliferation, activation and apoptosis of decidual T lymphocytes. Kidney- replenishing herb modulates maternofetal immunotolerance to normal levels and maintains pregnancy by modulating the differentiation of the trophoblast, accelerating its growth and fusion, reducing the rate of apoptosis, increasing the amount of syncytiotrophoblasts, and improving the expression and activity of IDO.

The present findings provide some academic foundation for the study of the pathogenesis of abortion and the modulating effect of Kidneyreplenishing herb on the maternofetal incretionimmunity network. But further studies are still needed in order to make clear whether there are any other routes by which IDO can affect maternofetal immunity and the relationship between syncytiotrophoblasts and different classes of decidual T lymphocytes.

References

1. Erlebacher A. Why isn’t the fetus rejected? Curr Opin Immunol 2001;13:590-593.

2. Honig A, Rieger L, Kapp M, Sutterlin M, Diet J, Kammerer U. Indoleamine 2,3-dioxygenase (IDO) expression in invasive extravillous trophoblast supports role of the enzyme for materno- fetal tolerance. J Reprod Immunol 2004;61:79-86.

3. Haynes MK, Smith JB. Can Th1-like immune responses explain the immuno-pathology of recurrent spontaneous miscarriage? J Reprod Immunol 1997;35:65-71.

4. Polgar K, Yacono PW, Golan DE, Hill JA. Immune interferon gamma inhibits translarional mobility of a plasma membrane protein in preimplantation stage mouse embryos: a T-helper 1 mechanism for immunologic reproductive failure. Am J Obstet Gynecol 1996;174:282- 287.

5. Chaouat G, Assal Meliani A, Martal J, Raghupathy R, Elliott JF, Mosmann T, Wegmann TG. IL-10 prevents naturally occurring fetal loss in the CBA x DBA/2 mating combination, and local defect in IL- 10 production in this abortion-prone combination is corrected by in vivo injection of IFN-t. J Immunol 1995;154:4261-4268.

6. Clark DA, Chaouat G, Arck PC, Mittruecker HW, Levy GA. Cytokine-dependent abortion in CBA x DBA/2 mice is mediated by the procoagulant fg12 prothombinase. J Immunol 1998;160:545-549.

7. Paulsesu L, Romagnoli R, Fortinio V. Distrubution of type-1 interferon-receptors in human first trimester and term placental tissues and on isolated trophoblast cells. Am J Reprod Immunol 1997;37:443-448.

8. Prete G, Carli M, Almerigogna F, Giudizi MG, Biagiotti R, Romagnani S. Human IL-10 is produced by type 1 helper (Thl) and type 2 helper (Th2) T cell clones and inhibits their antigen-specific proliferation and cytokine production. J Immunol 1993;150:353-360.

9. Hill JA. T helper 1 immunity to trophoblast: evidence for a new immunological mechanism for recurrent abortion in woman. Hum Reprod 1995;10(Suppl 2):114-120.

10. Lihui J. The effect of herb on the Th cytokine of the maternal-fetus interface of the rat model of abortion reduced by bromocriptine (in Chinese). PhD thesis, Fudan University; 2001.

11. Tarrade A, Lai Kuen R, Malassine A, Tricottet V, Blain P, Vidaud M, Evain-Brion D. Characterization of human villous and extravillous trophoblasts isolated from first trimester placenta. Lab Invest 2001;81:1199-1211.

12. Knofler M, Saleh L, Bauer S, Vasicek R, Griesinger G, Strohmer H, Helmer H, Husslein P. Promoter elements and transcription factors involved in differentiation-dependent human chorionic gonadotrophin-alpha messenger ribonucleic acid expression of term villous trophoblasts. Endocrinology 2000; 141:3737-3748.

13. ardson BD, Cheng YH, Langland RA, Handwerger S. Differential expression of AP-2gamma and AP-2alpha during human trophoblast differentiation. Life Sci 2001;69: 2157-2165.

14. nke JT, Durning M, Johnson KJ, Bennett LK, Golos TG. SPl/SP3- binding sites and adjacent elements contribute to basal and cyclic adenosine 3′,5′-monophosphate-stimulated transcriptional activation of the rhesus growth hormonevariant gene in trophoblasts. Mol Endocrinol 1998;12: 405-417. 15. iff WT, Carlson MG, Smith SD, Levy R, Nelson DM, Sadovsky Y. Peroxisome proliferator-activated receptor- gamma modulates differentiation of human trophoblast in a ligandspecific manner. J Clin Endocrinol Metab 2000;85: 3874-3881.

16. Jacquemin P, Martial JA, Davidson I. Human TEF-5 is preferentially expressed in placenta and binds to multiple functional elements of human chorionic somatomammotropin-B gene enhancer. J Biol Chem 1997; 272:12928-12937.

17. Jacquemin P, Sapin V, Alsat E, Evain-Brion D, Dolle P, Davidson I. Differential expression of the TEF family of transcription factors in the murine placenta and during differentiation of primary human trophoblasts in vitro. Dev Dyn 1998;212:423-136.

18. Alsat E, Wyplosz P, Malassine A, Guibourdenche J, Porquet D, Nessmann C, Evain-Brion D. Hypoxia impairs cell fusion and differentiation process in human cytotrophoblast, in vitro. J Cell Physiol 1996;168:346-353.

19. Aplin JD. Hypoxia and human placental development. J Clin Invest 2000;105:559-560.

20. Sawicki G, Radomski MW, Winkler-Lowen B, Krzymien A, Guilbert LJ. Polarized release of matrix metalloproteinase-2 and -9 from cultured human placental syncytiotrophoblasts. Biol Reprod 2000;63:1390-1395.

21. Li X, Gui S, Wang H. Relationship of abortion and the expression of indoleamine 2,3-dioxygenase (IDO) in villus and syncytiotrophoblasts. J Reprod Contracept 2005;16:235-242.

22. Terness P, Bauer TM, Rose L, Dufter C, Watzlik A, Simon H, Opelz G. Inhibition of allogeneic T cell proliferation by indoleamine 2,3-dioxygenase-expressing dendritic cells: mediation of suppression by tryptophan metabolites. J Exp Med 2002;196:447-157.

23. Lee GK, Park HJ, Macleod M, Chandler P, Munn DH, Mellor AL. Tryptophan deprivation sensitizes activated T cells to apoptosis prior to cell division. Immunology 2002; 107: 452-460.

24. Mackler AM, Barber EM, Takikawa O, Pollard JW. Indoleamine 2,3-dioxygenase is regulated by IFN-y in the mouse placenta during Listeria monocytogenes infection. J Immunol 2003;170:823-830.

25. Mellor AL, Munn DH. Extinguishing maternal immune responses during pregnancy: implications for immunosuppression. Semin Immunol 2001;13:213-218.

26. Moffett JR, Namboodiri MA. Tryptophan and the immune response. Immunol Cell Biol 2003;81:247-265.

XUELIAN LI, SUIQI GUI, & HAIYAN WANG

The Hospital of Obstetrics & Gynecology, Fudan University, Shanghai, People’s Republic of China

(Received 28 December 2006; revised 28 July 2007; accepted 7 August 2007)

Correspondence: X. Li, The Hospital of Obstetrics & Gynecology, Fudan University, Shanghai 200011, People’s Republic of China. Tel: +13788965050-61320. Fax: 63455090. E-mail: xuelianlixuelian@yahoo.com.cn

Copyright Taylor & Francis Ltd. Nov 2007

(c) 2007 Gynecological Endocrinology. Provided by ProQuest Information and Learning. All rights Reserved.