1 State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China
2 To whom correspondence should be addressed. Email: pengjp{at}ioz.ac.cn
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Abstract |
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Key words:
IFN-/placenta/RT1-A/RT1-DM/uterus
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Introduction |
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Major histocompatibility complex (MHC) antigens have a central role in the development of both cellular and humoral immune responses. The regulation of their expression in the placenta, which is a semi-allogeneic organ for the maternal immune system, is a critical aspect of gestation (Hunt and Orr, 1992; Schmidt and Orr, 1993
). If strongly reduced MHC expression is of major importance for fetal survival, it is important to clarify whether MHC molecules at the maternalfetal interface can be induced in vivo, and whether this could result in inflammatory reactions eventually leading to immunological rejection (Mattsson et al., 1992
).
In mouse, rat and human haemochorial placenta, trophoblast expression of MHC class II antigens is inhibited in normal pregnancy (Yuan et al., 1994) and the enhancement of MHC class II antigen expression correlates with abortion (Athanassakis et al., 1995
). Most studies on the in vitro and in vivo effects of IFN-
have shown that both murine and human placental cells resist induction of class II surface expression (King et al., 1987
). In contrast, other reports have claimed that IFN-
induces MHC class II surface molecules on different subpopulations of murine trophoblast cells (Athanassakis-Vassiliadis et al., 1989
; Vassiliadis et al., 1994
).
Up to now, there have been no reports about the expression of HLA-DM, a class II-like heterodimer, in gestation. Variation in expression of HLA-DM has a considerable effect on antigen presentation, and regulation of these genes is likely to be a prerequisite to prevent autoimmunity (Louis-Plence et al., 2000). Mutant cells that fail to express HLA-DM are deficient in antigen processing (Green et al., 1995
). It is unclear whether the effect of IFN-
on expression of HLA-DM during gestation was one of the important factors eventually leading to immunological rejection.
IFN- treatment maximally induced class I antigen expression regardless of the resting levels of expression. Genomic imprinting of MHC class I antigens in placental tissue varies with the time of gestation and is a quantitative rather than an all or none phenomenon (Kunz et al., 1996
). No systemic studies have been reported about the effect of IFN-
on the maternalfetal interface during the whole period of pregnancy.
In the rat, the classical MHC restriction and antigen presentation function has been assigned to RT1-A-encoded class I molecules. Rat RT1-DM is the equivalent of the human HLA-DM. By studying the spatial and temporal expression of RT1-A and RT1-DM in terms of mRNA and protein levels, and the effect of IFN-, some insights may be obtained into the molecular mechanism of IFN-
effects at the maternalfetal interface during gestation.
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Materials and methods |
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According to the character of embryonic development, gestation is divided into three phases: early pregnancy (D1D9), mid gestation (D10D15) and late pregnancy (D16D19). Early gestation was also subdivided into the preimplantation period (D1D4), implantation period (D5D6) and post-implantation period (D7D9). We selected D4, D6, D9, D15 and D19 as representing the preimplantation period, implantation period, post-implantation period, mid gestation period and late gestation period, respectively. The rats allocted to different pregnancy periods were euthanized at the appropriate time. Placentas and uteri were dissected on ice. Some placentas and uteri were frozen in Eppendorf tubes and stored at 80°C until processing for RNA and protein extraction. Some placentas and uteri were fixed in 4% buffered parafomaldehyde for immunohistochemical study.
LD (low dose) rats (n = 15) in experiment 1 received vaginal muscular injections of 25 000 IU of recombinant rat IFN- (Boeringer Mannheim, Mannheim, Germany) on day 1, 3, 6, 12 and 16, three animals each time. The only difference between rats in experiment 1 and experiment 2 or experiment 3 is that 15 animals in experiment 2 and experiment 3 received vaginal muscular injection of 50 000 IU (MD, mid dose) and 10 000 IU (HD, high dose) recombinant IFN-
, respectively. Fifteen animals in group 4 received an equal volume (100 µl) of saline as sham control. Our previous work showed that vaginal muscular injection had a more apparent effect than uterine horn injection (Liu et al. 2002
)
Total RNA isolation and RTPCR
Placentas from rats were harvested aseptically from dissected uterine horns by peeling each one off carefully, leaving the maternal decidua behind. The placenta and uterus were separated from the fetal membranes, cut into small pieces and then centrifuged at 12 000 r.p.m. for 15 min at 4°C. Total RNA from placentas was isolated by using the RNAgents kit (Promega, Madison, WI). To remove contaminating nuclear DNA, total RNA was subsequently treated with DNase by incubation for 30 min at 37°C in 10 mmol/l TrisHCl (pH 7.4)/15 mmol/l MgCl2 and 10 U of RQ DNase (Promega).
All of the RTPCR reagents including M-MLV reverse transcriptase and Taq DNA polymerase were purchased from Promega. Reverse transcription reactions were performed by using 2 µg of RNA. First-strand cDNA from 100 ng of total RNA was then used for PCR with oligonucleotide primers designed to amplify the target sequences given in Table I. PCR amplification was performed with 35 cycles of 30 s denaturation at 94°C, 1 min annealing at the temperature listed in Table I, and 1 min extension at 72°C. The primers were synthesized by Biotechnology Corporation (Beijing, People's Republic of China). PCR product was detected by electrophoresis in a 2.0% agarose gel (Promega). Reactions without reverse transcriptase served as negative control. PCR signal intensities were analysed using Bio-Rad Quantity One software (Bio-Rad, Hercules, CA). The results were normalized to -actin. Three replicates were performed for all experiments.
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For reprobing, the membrane was washed in stripping buffer (100 mmol/l -mercaptoethanol, 20% SDS, 62.5 mmol/l Tris, pH 6.7) at 50°C for 30 min to strip off bound antibody after ECL detection. The membrane was reprobed with 1:300 polyclonal goat anti-actin (Santa Cruz), using the same procedure as described above. All experiments were repeated at least three times.
Immunohistochemistry
Immunohistochemical studies were performed on 4% buffered paraformalin-fixed, 10 µm thick cryosections. Endogenous peroxidase activity was blocked by a 5 min treatment with 3% hydrogen peroxide in absolute methanol. Sections were then incubated in a humid chamber with the primary antibody, either a mouse anti-rat RT1-A (Harlan) or a goat anti-RT1-DM (Santa Cruz), at a concentration of 2.5 µg/ml in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA). To minimize background staining, sections were pre-incubated with a 1:10 dilution of normal goat serum. To enhance the immunostainning, sections were digested with 10 µg/ml protease K (Sigma Chemical Company, St Louis, MO) in PBS, pH 7.6, for 15 min before incubating with the primary antibody. The sections were again washed three times in PBS and then incubated with secondary antiserum (goat anti-mouse or rabbit anti-goat IgG conjugated with HRP) at 37°C for 1 h. The antibody stains were developed by addition of diaminobenzidine (DAB). Sections were counterstained with 0.5% haematoxylin (Sigma), and then destained briefly in HClalcohol before dehydration and mounting under glass coverslips with Permount. To evaluate the specificity of the antibodies, substituting normal mouse serum or goat serum for the primary antibodies acted as negative controls.
Statistical analysis
Values are reported as the mean ± SEM. Statistical analysis was performed by one-way analysis of variance (ANOVA) and, when significant treatment effects were indicated, the StudentNewmanKeuls multirange test was employed to make pairwise comparisons of individual means.
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Results |
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Effect of IFN- on RT1-A expression in uterus
In the pre-implantation (D1D4) and post-implantation (D7D9) periods, MD IFN- treatment increased the expression of RT1-A mRNA significantly compared with the control group (P < 0.01). There was no significant difference between LD or HD IFN-
treatment groups and the control group (P > 0.05). Western blot data indicated that the RT1-A protein level in all treatment groups during the two periods increased over that of the control group (P < 0.01) (data not shown). Figures 3 and 4 showed that in the implantation period (D6), the RT1-A mRNA level and protein level of all IFN-
treatment groups increased over those of the control group, respectively (P < 0.01), and the response of RT1-A mRNA level to IFN-
is dose dependent.
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In late pregnancy (D19), LD IFN- treatment increased the expression of RT1-A significantly over that of the control group (P < 0.01). HD IFN-
treatment decreased the expression significantly over that of the control group (P < 0.01) (Figure 3). Western results are consistent with those of RTPCR (Figure 4).
Effect of IFN- on RT1-DM expression in uterus
In the implantation period (D6), MD and HD IFN- treatment increased RT1-DM mRNA expression significantly over the control group (Figure 3) and all doses increased protein levels (Figure 4) (P < 0.01)
In mid pregnancy (D15), the three doses of IFN- treatment (Figure 3) significantly decreased the level of expression of RT1-DM mRNA (P < 0.01). Figure 4 shows that MD and HD IFN-
treatment significantly decreased the level of expression of RT1-DM protein (P < 0.01), and there is no significant difference between the LD IFN-
treatment group and the control group (P > 0.05).
LD and HD IFN- treatment in late pregnancy (D19) significantly increased the RT1-DM mRNA expression (P < 0.01) (Figure 3). Western results showed increased protein levels at LD and MD IFN-
and decreased levels at HD IFN-
(Figure 4).
Effect of IFN- on RT1-A expression in placenta
In early pregnancy, all doses of IFN- treatment significantly increased the expression over that of the control group (P < 0.01) (Figure 5). In mid pregnancy, LD and MD IFN-
treatment significantly decreased the expression of RT1-A mRNA (Figure 5) and protein (Figure 6) (P < 0.01). There is no difference between control and HD in mRNA and protein levels. In late pregnancy, IFN-
treatment significantly decreased the expression of both RT1-A mRNA and protein (Figures 5 and 6) (P < 0.01).
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As shown in Figures 5 and 6, in mid pregnancy, all three doses of IFN- treatment significantly decreased the expression of RT1-DM mRNA and protein (P < 0.01). However, all three doses of IFN-
treatment significantly increased the expression of RT1-DM mRNA and protein in late pregnancy in a dose-dependent fashion. (P < 0.01).
Localization of RT1-A and RT1-DM
The results indicated that the localization of RT1-A and RT1-DM changed according to the period of pregnancy. IFN- only affected the expression levels of RT1-A and RT1-DM, and did not affect their localization at the maternalfetal interface.
During the pre-implantation and implantation periods, RT1-DM protein was mainly localized in the uterine luminal epithelium, glandular epithelium and blood vessels (Figure 7A1 and A2). During the post-implantation period, RT1-DM protein mainly localized to not only uterine luminal epithelium and glandular epithelium, but also the decidua basalis and labyrinthine zone (Figure 7B1 and B2). During mid and late gestation, RT1-DM mainly localized in the labyrinthine zone and maternal blood vessels (Figure 7C1 and C2).
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Discussion |
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Our previous work showed that IFN- did not affect MHC class II expression in trophoblasts of pregnant rabbits, but had a stimulatory effect on its expression in maternal decidua and placental lymphocyte (Liu et al., 2002
). This was consistent with the work of Mattsson et al. (1991
, 1992)
.
During the whole pregnancy period in the rat, RT1-A and RT1-DM expression in placenta was highest during early pregnancy. This suggested that these two molecules may participate in the early development of the placenta. RT1-DM expression decreased gradually in the uteri during normal pregnancy, which indicated that RT1-DM may participate in preparation for implantation.
We injected pregnant rats with three doses of IFN- in order to study the effect on expression of RT1-A and RT1-DM. It is very interesting that in early pregnancy, IFN-
treatment mainly induced RT1-A and RT1-DM expression, but inhibited their expression in mid pregnancy at the maternalfetal interface. In late pregnancy, expression of RT1-A decreased in placenta and increased in the uterus, and RT1-DM increased in both placenta and uterus with IFN-
treatment compared with control. In our experiments, some results showed a dose-dependent response, and others did not. We supposed that non-dose-dependent responses might be related to drug metabolism dynamics.
It is well known that IFN- is one of the major inducers of RT1-A and RT1-DM. It has been reported that IFN-
contributes to normal health of the mid gestational decidua (Ashkar and Croy, 1999
). Therefore, we speculated that there is a special mechanism in mid pregnancy which made IFN-
inhibit the expression of RT1-A and RT1-DM and prevented the occurrence of abortion.
In this study, we have identified that both RT1-A and RT1-DM can be expressed at the maternalfetal interface during normal pregnancy. Their localization changed during pregnancy. IFN- can modulate the expression of the two molecules at different levels throughout the whole of pregnancy.
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Acknowledgements |
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References |
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Submitted on February 20, 2005; resubmitted on March 18, 2005; accepted on April 29, 2005.
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