Chinese Medical Journal 2007;120(14):1247-1250
Effect of blockage of costimulatory signal on murine abortion-prone model
ZHAO Fu-xi, ZHANG Yuan-yuan, LIU Run-hua, LI Shuan-ming
ZHAO Fu-xi (Laboratory of Pathogenic Organism and Immunology, Medical College of Shanxi Datong University, Datong 037008, China)
ZHANG Yuan-yuan (Laboratory of Pathogenic Organism and Immunology, Medical College of Shanxi Datong University, Datong 037008, China)
LIU Run-hua (Laboratory of Surgery, Medical College of Shanxi Datong University, Datong 037008, China)
LI Shuan-ming (Laboratory of Pathogenic Organism and Immunology, Medical College of Shanxi Datong University, Datong 037008, China)Correspondence to:ZHAO Fu-xi,Laboratory of Pathogenic Organism and Immunology, Medical College of Shanxi Datong University, Datong 037008, China (Tel: 86-352-7158661. Fax:. E-mail:email@example.com)
Background Inhibition of the key costimulatory signals results in T cell anergy, indicating the alloantigen-specific immunologic unresponsiveness. In this study, the effect of blockage of costimulatory signal CD86 on murine abortion-prone model was studied.
Methods Thirty CBA/J female mice cohabitated with DBA/2 male or BALB/c male mice were investigated. CBA/J ×DBA/2 matings were used as the abortion-prone model, and CBA/J × BALB/c matings were used as the normal pregnant model. The abortion-prone models were divided into experimental and control groups, and the normal pregnant models were set as a normal group (10 mice in each group). The mice in the experimental group were treated with anti-mouse CD86 monoclonal antibody (mAb) (100 μg) on day 4.5 of gestation, while the controls received irrelevant-isotype matched rat IgG2b. As for the normal group, nothing was given to the mice. The mice were killed on day 13.5 of gestation, embryo resorption rate and the expression of transforming growth factor β1 (TGF-β1), plasminogen activator inhibitor 1 (PAI-1), and matrix metalloproteinase 9 (MMP-9) were detected. Then the data were analyzed by Chi-square test and Fisher’s exact test.
Results The embryo resorption rate in the experimental (8.2%) and normal groups (7.7%) was significantly lower than that of the control (23.5%, P<0.05). No significant difference was detected between the experimental and normal groups (P>0.05). The positive expression rates of TGF-β1 and PAI-1 proteins in the experimental and normal groups were significantly higher than those in the control group (P<0.05). The positive expression rate of MMP-9 protein in the experimental and normal groups was significantly lower than that in the control group (P<0.05). No significant difference in the positive expression rates of the three proteins was detected between the experimental and normal groups (P>0.05).
Conclusions Blockage of costimulatory signal CD86 at early pregnancy can treat uncertain recurrent spontaneous abortion by stimulating the expression of TGF-β1, MMP-9 and PAI-1 and reducing the embryo resorption rate.
Recurrent spontaneous abortion (RSA) is mostly caused by immunologic rejection, in which activation of antigen-specific T cell is a critical step. The activation occurs after concomitant engagement of T cell receptor (TCR) with the antigens presented on the antigen-presenting cells (APC) in association with major histocompatibility complex (MHC) class II molecules. In addition, the delivery of the costimulatory signals resulted from the interaction of several receptor-ligand complexes is also necessary under this condition.1 Inhibition of the key costimulatory signals results in T cell anergy, which indicates the alloantigen-specific immunologic unresponsiveness.2 B7 family, including B7-1 (CD80), B7-2 (CD86), and the recently discovered inducing costimulatory molecules (ICOS),3 is one of the most representative costimulatory molecules.
In this article, we treated abortion-prone murine model by injecting anti-CD86 mAb at the time of implantation, then evaluated embryo resorption rate, which indicates the state of pregnancy, and observed the changes in expressions of transforming growth factor β1 (TGF-β1), matrix metalloproteinase 9 (MMP-9), and plasminogen activator inhibitor 1 (PAI-1) proteins at the maternal-fetal interface.
Inbred strains of 8-week old female CBA/J, male DBA/2, and male BALB/c mice were obtained from Shanghai SLAC Laboratory Animal Co. Ltd (China), maintained for 2 weeks in the Laboratory Animal Facility before use, and housed 5 to 6 per cage with a 12L:12D cycle. Rat anti-mouse CD86 mAb and isotype rat IgG2b (Biolegend, USA) were purchased from Changsha Clonetimes Research Reagents Co. Ltd, (China). Rabbit anti-TGF-β1, rabbit anti-MMP-9, rabbit anti-PAI-1, and immunohisto-chemical kits were bought from Wuhan Boster Biological Technology Ltd (China).
CBA/J × DBA/2 matings were used as the abortion-prone model, and CBA/J × BALB/c matings were used as the normal pregnant model. The abortion-prone models were divided into experimental and control groups, and the normal pregnant models were set as a normal group. After overnight cohabitation of CBA/J females with DBA/2 or BALB/c males, females with vaginal plugs were arbitrarily designated as day 0.5 of gestation. Then, pregnant CBA/J mice that cohabitated with DBA/2 were randomized into experimental or control groups, pregnant CBA/J mice that cohabitated with BALB/c were designated as the normal group (10 pregnant CBA/J mice in each group).
Pregnant CBA/J mice in the experimental group received an i.p injection of 100 μg of anti-mouse CD86 mAb in 200 μl PBS on day 4.5 of gestation. Irrelevant-isotype matched rat IgG2b was administrated to the control group at the same dosage and time. As for the normal group, no antibody was given to the mice.
Preparation of placental and decidual units
Pregnant CBA/J mice were killed on day 13.5 of gestation. The uteri were removed, then the uterine horns were opened longitudinally, and placental and decidual units were separated respectively from the implantation sites of the embryo, and embedded in paraffin wax.
Embryo resorption rate
The uteri were examined macroscopically to count the number of healthy and resorbed embryos. The resorbed embryos were identified by their small size, hemorrhage and necrosis.4 The embryo resorption rate was calculated by the formula: R (%) = Re/(Re+F) × 100%, where R represents the embryo resorption rate, Re represents the number of resorbed embryos, and F represents the number of viable embryos.
To detect the TGF-β1, MMP-9, and PAI-1 proteins, immunohistochemical staining was performed according to immunohistochemical staining procedure of Wunhan Boster Ltd, China. All the positive expressions of TGF-β1, MMP-9, and PAI-1 proteins were localized in the cytoplasm of the decidual cells. Expressions of the proteins were classified into 4 grades by two methods: (1) According to the positive percentage: score 0: no positive staining; 1: positive cells < 25%; 2: positive cells 25%－50%; 3: positive cells > 50%. (2) According to the staining intensity: score 0: no staining; 1: buff; 2: buffy; 3: puce. The expression of the proteins was graded by adding up the two scores: –: score 0–1; +: 2–3; ++: 4–5; +++:＞5
Data were analyzed with SPSS13.0. The significance of difference in the embryo resorption rate among the groups was determined by Chi-square test. Fisher’s exact test was performed to determine the significance of difference in the expression of the proteins. A P＜0.05 was considered statistically significant.
Embryo resorption rate
The embryo resorption rate in the experimental and normal groups was significantly lower than that of the control. No significant difference was detected between the experimental and normal groups (Table 1).
Table 1. Embryo resorption rate of the three groups
At the maternal-fetal interface, the positive expression of TGF-β1, MMP-9, and PAI-1 proteins was located in the cytoplasm of the decidual cells and was stained buffy or puce (Fig.). The results of the immunohistochemical staining (Table 2) indicated that the positive expression rates of TGF-β1 and PAI-1 proteins in the experimental and normal groups were significantly higher than those in the control group (P<0.05). The positive expression rate of MMP-9 protein in the experimental and normal groups was significantly lower than that in the control group (P<0.05). No significant difference in the positive expression rates of the three proteins was detected between the experimental and normal groups (P>0.05).
Fig. Immunohistochemical staining showing the positive expression of TGF-β1, MMP-9, and PAI-1 proteins (original magnification ×400). A: TGF-β1; B: MMP-9; C: PAI-1.
Table 2. Expression rates of TGF-β1, MMP-9, and PAI-1 proteins in the three groups
We have known that the intervention of the costimulatory signals induced by anti-CD80/CD86 mAb could result in maternal immune tolerance to paternal antigens and inhibit maternal immune rejection to allogeneic embryo, improving the outcome of pregnancy in abortion-prone animal models.5 In addition, selective blockage of CD86 signaling could facilitate a Th2 bias at the maternal-fetal interface and expand the peripheral CD4+CD25+ regulatory T cells to rescue the abortion-prone fetus. 6 In this study, we also verified that the blockage of CD86 signaling at time of implantation could decrease the embryo resorption rate in abortion-prone murine model to a normal level. Since both the blockage of CD80/CD86 signaling and blockage of CD86 signaling improve the outcomes of pregnancy in abortion-prone mice, we proposed that CD86 signaling pathway may play a predominant role in the rejection of embryo in abortion-prone model. Previous studies have demonstrated that neither blockage of CD80 and CD86 nor that of CD86 alone could improve the outcomes of pregnancy in normal pregnant mice.6,7 The embryo resorption in normal pregnancy is most likely due to the embryo’s own chromosomal abnormality, however, the abortion resulted from genetic factor can not be improved by blockage of the costimulatory signaling pathway. Therefore, we supposed that CD86 molecule might be over-expressed in the abortion prone mice, and attempted to use anti-CD86 mAb in abortion-prone model to neutralize the over-expression of CD86 molecule.
TGF-β probably is a signal that can close immune system, exert immunosuppressive effect in normal pregnancy. 8 The expression of TGF-β mRNA of decidual cells in abortion-prone mice is significantly lower than that in normal pregnant mice, suggesting that the abnormal expression of TGF-β in decidual cells relates to abortion.9 Reduced expression of TGF-β in local decidual microenvironment would impair normal inherent immunosuppressive activity, relatively enhancing the maternal immunologic rejection to the antigens of fetus and resulting in abortion. In this study, we verified as above described that the expression of TGF-β in the control group, which had a higher embryo resorption rate, was significantly lower than that in the experimental and normal groups, showing that blockage of the CD86 signaling with anti-CD86 mAb in abortion-prone mice can increase the expression of TGF-β1 in decidual cells at maternal-fetal interface to a normal level, and improve the outcome of normal pregnancy in abortion-prone mice. MMPs, especially MMP-9, are regarded as the major proteinase degrading the extracellular matrix (ECM). 10,11 MMP-9 is expressed in the peripheral network of trophoblast cells, which were in close contact with the maternal decidua.12 Recent study has shown that positive expression rate of MMP-9 in human decidual tissues in abortion is significantly higher than that in normal pregnancy.13 MMP-9 protein was not only secreted by the trophoblast cells at maternal-fetal interface, but also detectable in the decidual tissues, suggesting that MMP-9 from both the trophoblast cells and decidual tissues play a role in degradation of the ECM. Since abundant expression of MMP-9 could excessively degrade the ECM, leading to embryo excessive invasion, we proposed that higher expression of MMP-9 probably related to the occurrence of spontaneous abortion. Our results demonstrated that the expression of MMP-9 in the experimental group that treated with anti-CD86 mAb was significantly lower than that in the control group, but was similar to that in the normal group, for which would result in fetal growth restriction.14
PAI-1, including t-PA and u-PA, is the major inhibitor of plasminogen activator (PA). Binding u-PA with its receptor (uPAR) elicits various physiologic events, which may be associated with the conversion from plasminogen to plasmin. PAI-1 can regulate the uPA/uPAR interaction by binding to the active site of its receptor.15 We thought that if the function of the uPA/uPAR interaction in abortion-prone mice was enhanced pathologically, those so-called physiologic events would not be beneficial to normal pregnancy. In the article, the expression of PAI-1 protein in the experimental group was significantly higher than that in the control group. Thus we propose that the expression of PAI-1 protein in local microenvironment after treatment with anti-CD86 mAb is increased accordingly, inhibiting the pathologic function of u-PA/u-PAR interaction.
The study of Meisser and colleagues16 has indicated that TGF-β could inhibit total gelatinolytic activity, especially the activity of MMP-9. Moreover, plasmin could activate pro-MMPs, such as pro-MMP-2 and -9, permitting PAI to indirectly regulate the remodeling of type IV collagen, the major component of the basement membrane and matrix.17 Furthermore, TGF-β can activate the transcription of the PAI-1 gene by binding smad-3 and -4 with two adjacent DNA elements in the TGF-β-responsive region of PAI-1 promotor.18 However, the relationship among TGF-β, MMP-9, and PAI-1 was uncertain yet. We would further study the problem in the following research.
In conclusion, the blockage of CD86 costimulatory signaling pathway can reduce the embryo resorption rate in the abortion-prone murine model to a normal level. In addition, the bias of expression of TGF-β, MMP-9, and PAI-1 at the maternal-fetal interface is advantageous to a successful pregnancy, indicating that blockage of CD86 can treat uncertain RSA.
1. Hancock WW, Sayegh MH, Zheng XG, Peach R, Linsley PS, Turka LA. Costimulatory function and expression of CD40 ligand, CD80, and CD86 in vascularized murine cardiac allograft rejection. Proc Natl Acad Sci USA 1996; 93: 13967-13972.
2. Kano M, Bashuda H, Yagita H, Okumura K, Morishita Y. A crucial role of host CD80 and CD86 in rat cardiac xenograft rejection in mice. Transplantation 1998; 65: 837-843.
3. Gonzalo JA, Delaney T, Corcoran J, Goodearl A, Gutierrez-Ramos JC, Coyle AJ. Cutting edge: the related molecules CD28 and inducible costimulator deliver both unique and complementary signals required for optimal T cell activation. J Immunol 2001; 166: 1-5.
4. Clark DA, McDermott MR, Szewczuk MR. Impairment of host vs graft reaction in pregnant mice. II Selective suppression of cytotoxic cell generation correlates with soluble suppressor activity and with successful allogeneic pregnancy. Cell Immunol 1980; 52: 106-118.
5. Jin LP, Zhou YH, Wang MY, Zhu XY, Li DJ. Blockade of CD80 and CD86 at the time of implantation inhibits maternal rejection to the allogeneic fetus in abortion-prone matings. J Reprod Immunol 2005; 65: 133-146.
6. Zhu XY, Zhou YH, Wang MY, Jin LP, Yuan MM, Li DJ. Blockade of CD86 signaling facilitates a Th2 bias at the maternal-fetal interface and expands peripheral CD4+CD25+ regulatory T cells to rescue abortion-prone fetuses. Biol Reprod 2005; 72: 338-345.
7. Jin LP, Li DJ, Wang MY, Zhu XY, Zhu Y, Meng Y, et al. Inducing peripheral maternal-fetal immunotolerance by blockade of costimulatory signal at the early stage of gestation of the abortion-prone murine model. Chin J Obstet Gynecol (Chin) 2004; 39: 234-237.
8. Schuster N, Krieglstein K. Mechanisms of TGF-β-mediated apoptosis. Cell Tissue Res 2002; 307: 1-14.
9. Yu YS, Lin QD, Zhao AM. Relationship between expression of TGF-β mRNA in mice decidua and spontaneous abortion. Prog Obstet Gynecol (Chin) 2002; 11: 113-115.
10. Staun-Ram E, Goldman S, Gabarin D, Shalev E. Expression and importance of matrix metalloproteinase 2 and 9 (MMP-2 and MMP-9) in human trophoblast invasion. Reprod Biol Endocrinol 2004; 2: 59.
11. Seval Y, Akkoyunlu G, Demir R, Asar M. Distribution patterns of matrix metalloproteinase (MMP)-2 and -9 and their inhibitors (TIMP-1 and TIMP-2) in the human decidua during early pregnancy. Acta Histochem 2004; 106: 353-362.
12. Harvey MB, Leco KJ, Arcellana-Panlilio MY, Zhang X, Edwards DR, Schultz GA. Proteinase expression in early mouse embryos is regulated by leukemia inhibitory factor and epidermal growth factor. Development 1995; 121: 1005-1014.
13. Liu CH, Gao YH, Guo YZ. Relationship between the matrix metalloproteinase-9/tissue inhibitor of metalloproteinase-3 in decidual tissue and spontaneous abortion. J Chin Exp Pathol (Chin) 2004; 20: 551-553.
14. Yu YH, Wang ZJ. Relationship between matrix metalloproteinase-9 expression in syncytiotrophoblast and placenta pathologic change of intrauterine growth restriction. Chin J Perinat Med (Chin) 2003; 6: 215-218.
15. Choong PF, Nadesapillai AP. Urokinase plasminogen activator system: A multifunction role in tumor progression and metastasis. Clin Orthop Relat Res 2003; 415: 46-58.
16. Meisser A, Chardonnens D, Campana A, Bischof P. Effects of tumor necrosis factor-α, interleukin-1α, macrophage colony stimulating factor and transforming growth factor-β on trophoblastic matrix metalloproteinases. Mol Hum Reprod 1999; 5: 252-260.
17. Murphy G, Stanton H, Cowell S, Butler G, Knauper V, Atkinson S, et al. Mechanisms for pro-matrix metalloproteinase activation. APMIS 1999; 107: 38-44.
18. Stroschein SL, Wang W, Luo K. Cooperative binding of Smad proteins to two adjacent DNA elements in the plasminogen activator inhibitor-1 promoter mediates transforming growth factor-β–induced smad-dependent transcriptional activation. J Biol Chem 1999; 274: 9431-9441.