Relationship of Abortion and the Expression of Indoleamine 2,3- dioxygenase (IDO) in Villus and Syncytiotrophoblasts
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Journal of Reproduction & Contraception (2005) 16 (4):235-242 Relationship of Abortion and the Expression of Indoleamine 2,3- dioxygenase (IDO) in Villus and Syncytiotrophoblasts Xue-lian LI, Sui-qi GUI, Hai-yan WANG Department of Gynecology of Integrated Traditional Chinese and Western Medicine, The Hospital of Obstetric & Gynecology, Fudan University, Shanghai 200011, China Objective To study the relationship of abortion and the expression of indoleamine 2, 3- dioxygenase (IDO) in villus and syncytiotrophoblast in vitro. Methods RT-PCR was applied to analyze the mRNA transcription of IDO in villus of normal pregnancy and inevitable abortion and JAR cells as well. Immunohistochemistry was applied to analyze the expression of IDO protein in villus. Western blot was applied to determinate the expression of IDO protein on cultured syncytiotrophoblast. High- performance liquid chromatography was applied to determinate whether there was kynurenine in cell culture medium of syncytiotrophoblast. Results The expression of IDO mRNA and protein in villus of inevitable abortion was lower than that of normal pregnancy; IDO mRNA did not express in JAR cells. IDO protein expressed on cultured syncytiotrophoblast, and there was kynurenine in cell culture medium of syncytiotrophoblast. Conclusion Appropriate expression of IDO in villus is necessary for maintenance of normal pregnancy and an active IDO protein expresses in syncytiotrophoblast. Key words: indoleamine 2,3-dioxygenase; syncytiotrophoblast; villus; abortion There is complicated pathogeny involved in spontaneous abortion, and the modulatory abnormity of incretion and immunity accounts for 80%-94%. Why is not the fetus, a homo- geneous implant, excluded by the mother? At present, the study has been to the trophoblasts, the interface of mother and fetus. How is the immunological relationship of trophoblasts and lymphocytes came from the mother? Indoleamine 2,3-dioxygenase (IDO), which mostly consists in the antigen-presenting cells of lymphoid organs and the syncytiotrophoblasts and macrophages of gestation, is the first and regulatory enzyme of the kynurenine pathway Corresponding author: Sui-qi GUI; Tel: +86-21-63455050-420; E-mail: sqgui@hotmail.com 235
(KP), the major route of L-tryptophan catabolism. The tryptophan catabolites produced through the KP induce immunosuppression of T lymphocytes[1], and may play an important role in the balance of Th cytokine at materno-fetal interface and the materno-fetal tolerance. IDO inhibitor induces the loss of fetus[2,3]. Here we mainly study the relationship of abortion and the expression of indoleamine 2,3- dioxygenase (IDO) in villus and syncytiotrophoblasts. Materials & Methods Materials Tissue collection Villus specimens were donated by healthy women undergoing elective termination of pregnancy at 6-12 weeks of gestation in Hospital of Obstetric & Gynecology, Fudan University. JAR cells were purchased from Institute of Biochemistry and Cell Biology (Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences). Chemicals Trypsin (Amresco), deoxyribonuclease I (Sigma), DMEM and bovine calf serum(Gibco), recombinant Human epidermal growth factors (rhEGF, Peprotech), Percoll(Gibco), histochemical ABC kit (Huamei), RNAex Reagent, revertAidTM first strand cDNA synthe- sis kit, Taq DNA polymerase (Fermentas), BCA-100 protein quantitative analysis kit (Shenergy), visual AP Western blotting reagent set (Ab Minus) SNBC, mouse anti- IDO monoclonal antibody (Chemoicon), mouse anti-vimentin, cytokeratin and hCG-β monoclonal antibody (Zhongshan), L-tryptophan, kynurenine (Sigma), primer (designed personally with the help of primer premier 5.00 according to gene sequence and synthesized by Bioasia). IDO forward: 5'-TCCGTGAGTTTGTCCTT-3' reverse: 5'-GCATAGTATTAGTTTGTGGC-3', product 361 bp β-actin forward: 5'-GAGCGGGAAATCGTGCGTGACATT-3' reverse: 5'-GATGGAGTTGAAGGTAGTTTCGTG-3', product 240 bp Instruments UNICOTM UV-2102C ultraviolet spectrophotometer, Perkin elmer DNA Thermal cycler, SCR-4 high-pressure electrophoretic instrument, H6-1 electrophoretic slot, Tanon GIS2010, GIS gel image disposal system, MIAS-2000 immunohistochemical measure sys- tem for colorized pathological images, Waters high-performance liquid chromatography, C- 18 column (Zorbax C18, 250 × 4.6 mm, 5 µm), Waters 2996 PDA detector. Methods RNA isolation and RT-PCR of IDO mRNA on human syncytiotrophoblasts of normal pregnancy and inevitable abortion and JAR cells Total RNA was prepared by RNAex reagent, treated with RNase-free DNase and quantified spectrophotometrically. RT products were prepared in accordance with the manu- 236
facturers’ instructions of the RevertAidTM first strand cDNA synthesis kit. The PCR reac- tion system involved 1-5 µl template DNA, 2.5 µl PCR buffer (1.5 mol/L MgCl2), 0.5 µl dNTP mix, 1 µl forward primer, 1 µl reverse primer, 0.5 µl Taq DNA polymerase and DEPC water filled in a final volume of 25 µl was incubated at 95℃ for 3 min, followed by incubation at 94℃ for 60 s, 47℃ for 60 s, and 72℃ for 60 s for a total of 30 cycles and 72℃ for 15 min. Samples were subjected to 1% agarose gel electrophoresis, stained with ethidium bromide, and photographed under UV light. As a positive control, human β-actin RNA was retrotranscribed 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 retrotranscribed. Immunohistochemical measure (ABC) The tissue specimen was embedded by paraffin, sliced up for 4 µm, and immunostained for IDO according to the guide of the histochemical ABC kit. Negative comparison was done by PBS replacing the primary antibody. The positive absolute proportion, positive rela- tive proportion and average absorbency of ten areas were measured stochastically in each slice. Isolation, culture and identify of syncytiotrophoblast The villus tissue was collected sterilely, rinsed in D-Hanks, cut into smaller pieces, and digested with 0.125% trypsin and 0.02% deoxyribonuclease I 5 min × 3 at 37℃. The supernatants were filtered over a succession of 200 µm and 60 µm pore size sieves to remove aggregates. The cells were then centrifuged at 350 × g for 10 min, resuspended in DMEM, layered over a continuous Percoll gradient (30%-70% in 5% steps of 2 ml each), then centrifuged at 37℃ and 1 200 × g for 20 min to separate different cell types. Cytotrophoblast cells between the density markers of 1.049 g/ml and 1.062 g/ml were collected, suspended in DMEM containing FCS (10%), penicillin (100 U/ml), streptomycin (100 µg/ml), plated in 24-well plates at a density of 106 cells/ml/well or in 5 ml vase at (1-5)× 10 6 cells/ml, cultured in a humidified atmosphere (95% air-5% CO 2 at 37℃) with 10 ng/ml rhEGF to promote the congregation and amalgamation, fixed and immunostained for cytokeratin, vimentin and hCG-β according to the guide of the histochemical ABC kit. Western blot analysis for expression of IDO protein in human syncytiotrophoblast cultured in vitro The protein was distilled and quantified according to the BCA-100 kit. Polyacrylamide slab gel of 10% for separation and 5% gel for concentration were used, 20 µl of sample was loaded into each well with equal SDS and protein, and subjected to electrophoresis for 1.5-2.0 h. Electrophoretic transfer of the protein onto nitrocellulose paper was done at 250 mA constant current for 1.5-2.0 h in ice. The membrane was then washed with TBS buffer and placed in 4% skim milk powder-TBS for 1.5-2.0 h to block all nonspecific binding sites on the membrane. The membrane was removed, washed, placed in a buffered solution of primary 237
monoclonal IDO antibody (1:1 000 dilution), and then left overnight at 4℃. The membrane was removed, washed, and placed in secondary antibody (alkaline phosphatased goat anti- mouse IgG, 1:2 000) for 1 h at room temperature. The membrane was washed and incu- bated for 10-30 min at room temperature with AP substrate solution(3 ml AP substrate solution+20 µl NBT solution+20 µl BCIP solution). IDO activity of syncytiotrophoblasts detected by high-performance liquid chromatography (HPLC) Samples of culture media were deproteinized with 4% trichloroacetic acid and total free tryptophan and kynurenine were assayed by HPLC on a C-18 column eluted isocratically using a solvent of 0.1 mol/L phosphate buffer (pH 4.0 with sodium hydroxide). The ratio of kynurenine/tryptophan was used to assess the IDO activity. Statistical analysis Results were analyzed statistically by t test using SPSS11.0 software package. P
1.0 *: P
Figure 3 IDO protein expression in normal villus (A) and villus of inevitable abortion (B) A: vimentin-negative; B: cytokeratin-positive; C: hCG-β-positive; D: IDO-positive Figure 4 Syncytiotrophoblasts judged by immunocytochemistry (200 ×) Figure 5 Western blot reveals IDO protein (46 kD) expressed in syncytiotrophoblasts 240
medium of syncytiotrophoblasts cultured in vitro. This fact reveals that there are IDO activ- ity in syncytiotrophoblasts cultured in vitro. We suppose that normal expression and activity of IDO in villus is an important qualification of maintaining pregnancy, and singularly lower expression or activity of IDO may be one of the reasons of abortion. Our findings could be explained by the reports that IDO expressed in trophoblasts and defends the conceptus against rejection by reducing the tryptophan level and suppressing the T cell activity[1], tryp- tophan is a necessary amino acid of cell proliferation, IDO-expressing cells could creat a local microenvironment in which levels of tryptophan are low, block the cell cycle at mid-G1, make T cells be prone to apoptosis especially apoptosis via Fas, regulate T cell proliferation and activation[4,5] IDO is inducible by cytokines such as interferon-gamma and plays a role in the balance of Th cytokine at materno-fetal interface and the inflammation and maternal tolerance of fetal allografts[6]. 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 immuno- logical discrimination by promoting T cell tolerance in other circumstances[7,8]. The mecha- nisms of IDO mediating tolerance are not well understood, but recent findings have impli- cated tryptophan catabolism through the kynurenine metabolic pathway 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[3]. It was reported that IDO reactivity could be presented by the ratio of kynurenine/ tryptophan[9]. Our research reveals that there are both kynurenine and tryptophan in the culture medium of syncytiotrophoblasts cultured in vitro, that is, there is IDO reactivity in syncytiotrophoblasts cultured in vitro. So, we speculate that the expression and reactivity in villus of early pregnancy is due to syncytiotrophoblasts. And improving the expression and activity of IDO on the materno-fetal interface may further modulate the materno-fetal im- munity to the normal level and maintain normal pregnancy. These findings provid some academic foundations for the study of pathogenesis and therapy of abortion. But further studies are still needed to make clear through which route that IDO affect materno-fetal immunity. 241
References 1. Erlebacher A. Why isn't the fetus rejected? Curr Opin Immunol, 2001, 13(5): 590-3. 2. Caucheteux SM, Kanellopoulos-Langevin C & Ojcius DM. At the innate frontiers between mother and fetus: linking abortion with complement activation. Immunity, 2003,18(2): 169-72. 3. Moffett JR & Namboodiri MA. Tryptophan and the immune response. Immunol Cell Biol, 2003, 81(4): 247- 65. 4. Terness P, Bauer TM, Rose L, et al. 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(4): 447-57. 5. Lee GK, Park HJ, Macleod M, et al. Tryptophan deprivation sensitizes activated T cells to apoptosis prior to cell division. Immunol, 2002, 107(4): 452-60. 6. Mellor AL, Chandler P, Lee GK, et al. Indoleamine 2,3-dioxygenase, immunosuppression and pregnancy. J Reprod Immunol, 2002, 57(1-2): 143-50. 7. Mackler AM, Barber EM, Takikawa O, et al. Indoleamine 2,3-dioxygenase is regulated by IFN-gamma in the mouse placenta during Listeria monocytogenes infection. J Immunol, 2003, 170(2): 823-30. 8. Mellor AL & Munn DH. Extinguishing maternal immune responses during pregnancy: implications for immunosuppression. Semin Immunol, 2001, 13(4): 213-8. 9. Schrocksnadel K, Widner B, Bergant A, et al. Longitudinal study of tryptophan degradation during and after pregnancy. Life Sci, 2003, 72(7): 785-93. (Received on June 21, 2005) 242
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