Mol. Hum. Reprod. Advance Access originally published online on August 17, 2007
Molecular Human Reproduction 2007 13(9):663-673; doi:10.1093/molehr/gam054
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Abortion is associated with increased expression of FasL in decidual leukocytes and apoptosis of extravillous trophoblasts: a role for CRH and urocortin
1 Laboratory of Human Reproduction, Department of Obstetrics and Gynaecology, Medical School, University of Crete, Heraklion 71003, Greece 2Department of Obstetrics and Gynaecology, Ludwig Maximilians University of Munich, Maistrasse 11, 80337 Munich, Germany 3Department of Obstetrics and Gynaecology, University of Rostock, Rostock, Germany
4 Correspondence address. Tel: +302810392131; Fax: +302810392131; E-mail: makrigia{at}med.uoc.gr
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
|---|
Human reproduction is remarkably inefficient, with more than half of spontaneous conceptions failing to complete the first trimester. However, little is known on the molecular events that take place at the implantation site during abortion. Here, we examined the hypothesis that the expression of the proapoptotic Fas/FasL system at the implantation site is impaired in abortions. We found that, in contrast to normal pregnancy, abortive deciduas contain leukocytes that are positive for FasL and extravillous trophoblasts (EVTs), which show increased expression of Fas and increased rates of apoptosis. In addition, the neuropeptides, corticotropin-releasing hormone and urocortin, were elevated in placental material obtained from abortions. In vitro, these peptides induced the expression of FasL in decidual lymphocytes (DL) obtained from elective termination of pregnancy placentas and thus potentiated the cells' ability to induce Fas-mediated apoptosis in an EVT-based hybridoma cell line. Finally, DL from abortion sites effectively induced apoptosis of EVT without prior treatment. It is possible that these events may impede successful early placentation and thus contribute to the pathophysiology of human abortion.
Key words: abortion/CRH/extravillous trophoblast/FasL/implantation
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
|---|
Human reproduction is remarkably inefficient compared with that of other mammalian species. Approximately 70% of spontaneous conceptions are lost prior to completion of the first trimester. Implantation failure and pre-clinical losses account for 85% of total pregnancy losses and clinical miscarriage for 15% (Macklon et al., 2002). Half of the cases of abortion are associated with fetal chromosomal abnormalities. Endocrine disorders, immune factors, uterine structural abnormalities, infections, chemical agents and psychological factors complete the list of known etiologies of abortion (Barnea, 1992).
The feto-maternal interface is an interweave of tissues comprised of various types of immune and non-immune cells, where complex immune phenomena take place. Starting with the formation of the adhesive endometrium and the inflammatory-like process of blastocyst implantation, an intricate network of locally acting peptides has developed to regulate these and the subsequent processes of trophoblast invasion and placentation (Anin et al., 2004; Dey et al., 2004; van den Brule et al., 2005; Makrigiannakis et al., 2006).
The molecular mechanisms that take place in the feto-maternal interface and that are associated with spontaneous abortion are largely unknown. According to early studies, spontaneous abortion is associated with particularly limited trophoblast invasion (Hustin et al., 1990; Michel et al., 1990). Currently, the most favored hypothesis on the pathophysiological events leading to abortion can be summarized as follows: reduced extravillous trophoblast (EVT) invasion results in incomplete plugging of the spiral arteries during the first weeks of pregnancy (Jauniaux and Burton, 2005). This limitation of invasion may involve both interstitial and endovascular EVT, since endovascular invasion is thought to involve a side route of interstitial invasion. Failure of vascular invasion, therefore, is possibly preceded by impaired interstitial EVT invasion (Kaufmann et al., 2003). These phenomena may lead to the early unplugging of spiral arteries and the onset of maternal blood flow into the intervillous space. Untimely oxidative stress probably occurs (Burton and Jauniaux, 2004) in a period during which the antioxidant placental defences are inadequate (Watson et al., 1997, 1998; Jauniaux et al., 2000), thus resulting in the arrest of placental development and abortion (Hempstock et al., 2003). These events are considered to be responsible for a large proportion of abortions regardless of aetiology (Jauniaux and Burton, 2005). Moreover, this hypothesis may provide mechanistic explanations for additional pregnancy complications associated with reduced trophoblast invasion, in particular pre-eclampsia and fetal growth restriction. It is suggested that spontaneous abortion, missed abortion, and early- and late-onset pre-eclampsia represent a spectrum of disorders secondary to different degrees of deficient trophoblast invasion and consequent placental oxidative stress (Burton and Jauniaux 2004).
Given its presence in immune-privileged sites such as the brain, the eye and the pregnant uterus (Niederkorn 2006), the intrauterine Fas/Fas ligand (FasL) system has been considered primarily as a mechanism utilized by trophoblasts to escape maternal immune attack. FasL-expressing trophoblasts may induce apoptosis of activated decidual lymphocytes (DL) bearing the Fas receptor (Runic et al., 1996; Uckan et al., 1997; Kauma et al., 1999; Makrigiannakis et al., 2001; Abrahams et al., 2004). However, FasL is also stored in the intracellular granules of T and NK cells and can be delivered to the cell surface upon non-specific activation of these cells (Bossi and Griffiths, 1999). In addition, trophoblasts also express low levels of Fas, thus complicating the role of the Fas/FasL system at the implantation site (Xerri et al., 1997; Huppertz et al., 1998; Runic et al., 1998; Payne et al., 1999).
It is suggested that in certain cell types, including trophoblasts, the neuropeptide corticotropin-releasing hormone (CRH) regulates FasL expression through type 1 CRH receptor (CRHR1) (Makrigiannakis et al., 2001; Dermitzaki et al., 2002). CRH is the principal mediator of the stress response in mammals and binds to CRHR1 with as much potency as its family partner, urocortin (Ucn). In the periphery, CRH and Ucn may act as important autocrine and/or paracrine regulators of immune functions (Gravanis and Margioris, 2005). The two neuropeptides are produced by the pregnant and the non-pregnant uterus, and their numerous roles in female reproductive physiology and pathophysiology are gradually being clarified (Hillhouse and Grammatopoulos 2002; Kalantaridou et al., 2004). Recently, CRH and Ucn proteins were found to be significantly elevated in cases of spontaneous abortion (Madhappan et al., 2003).
In the present study, we examine the hypothesis that spontaneous abortion in humans may be associated with aberrant expression of the Fas/FasL system at the implantation site. In addition, we explore a potential role for CRH and Ucn in spontaneous abortion.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
|---|
Reagents and drugs
All chemicals were of analytical grade and were purchased from Sigma unless otherwise stated. The CRHR1 antagonist, antalarmin, was obtained from the Laboratory of Medicinal Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases (Bethesda, MD, USA). The CRHR2 antagonist, anti-sauvagin-30, was obtained from Sigma (St. Louis, MO, USA), and the CRH and Ucn peptides were from Tocris (Bristol, UK) and Sigma, respectively.
Placental tissues
Tissues used for this study were provided from the Department of Obstetrics and Gynecology, University of Rostock. Placental tissues were obtained from Caucasian women (22–35 year old) of German ethnic background with spontaneous abortion (17 cases) or elective termination of unwanted pregnancy (ETP) (25 cases). Gestational age for both groups ranged from 7 to 12 weeks, established by menstrual history confirmed by ultrasonography. These subjects were middle class with 6 ± 2 year of post-elementary education. Samples were collected by vaginal curettage. In spontaneous abortion, curettage was performed within 24 h after diagnosis. None of the abortions was pharmacologically induced. Both groups received the same exclusionary criteria: women receiving any medication or with genetic defects, cases of chromosomal, hormonal or uterine structural abnormalities, and cases associated with infectious, autoimmune, or other systemic or local diseases were excluded. The samples had no labeling that could identify the subjects and were analysed by investigators blinded to the abortion history of the donor. Blood progesterone from patients with spontaneous abortion was quantified with a commercial enzyme immunoassay (Boehringer-Mannheim, Mannheim, Germany). This study was approved by the Ethical Committee of the Medical School, University of Rostock. Informed consent was obtained from each patient. Specimens were immediately employed in in vitro experiments, or immediately immersed in 10% formalin to later embed in paraffin, or snap frozen in liquid nitrogen for later processing, or snap frozen in a cryostat supported by tissue freezing medium. Serial cryosections (5 µm) were collected on glass slides, air dried for 2 h at room temperature (RT), fixed in acetone for 5 min at RT and stored at –40°C until required. Serial paraffin sections (5 µm) were collected on glass slides.
Cell lines
The cells used for in vitro apoptosis experiments are EVT-choriocarcinoma hybrid cells (clone AC1M88) (hereafter hyEVT), which have been previously described (Funayama et al., 1997; Gaus et al., 1997). The cells were kept in DMEM/F12 supplemented with 10% fetal calf serum (FCS), 1% L-Glutamine 200 mM, 1% Penicillin–Streptomycin (all from Invitrogen, Carlsbad, CA, USA) at 5% CO2 and 37°C.
Isolation of DL
DL were isolated as previously described (Olivares et al., 2002). Samples of decidua from different patients were not mixed in order to avoid the induction of allogeneic reaction of leukocytes. Briefly, samples from decidua of spontaneous abortion or ETP were thoroughly washed in phosphate-buffered saline, pH 7.4 (PBS). Decidual fragments were finely minced in a small volume of RPMI 1640 and then pushed through a 45 µm sieve. The resultant cell suspension was washed, layered onto Lymphoprep and centrifuged. The cells at the interface were collected in culture in complete culture medium (RPMI 1640, 10% FCS, 100 U/ml penicillin and 50 g/ml gentamicin, all from Invitrogen, Carlsbad, CA, USA) and incubated for 2 h at 37°C in an atmosphere of 5% CO2 to allow adherent cells to attach to the plastic. The supernatant containing DL was then collected and used for experiments. Purity of lymphocytes (above 96%) was confirmed by fluorescence-activated cell sorter (FACS), using either the electronically selected lymphocytic gate, or dual labeling with anti-CD56 (B&D Biosciences, Heidelberg, Germany) and anti-CD3 (Dako, Ely, Cambridge, UK) lymphocyte markers. Lymphocyte viability was microscopically determined by trypan blue exclusion. Only samples with 
90% viable lymphocytes were used.
Real-time and conventional RT–PCR
For placental RNA, total RNA was prepared using the acid guanidium thiocyanate phenol–chloroform protocol. A minimum of 30 mm3, of a mixture of decidua and villous tissue taken from macroscopically well-defined areas of the abortion and normal first trimester placentas, was used for RNA extraction.
For the detection of CRH, Ucn and actin mRNAs, 1 µg of total RNA was reversely transcribed using the Thermo-Script RT–PCR System (Invitrogen Life Technologies, Carlsbad, CA, USA). The cDNA was amplified by PCR, which was performed in a PerkineElmer DNA Thermal Cycler with the following conditions: 60 s at 95°C, 60 s at 60°C (55°C for actin) and 60 s at 72°C for 35 cycles. Aliquots of 10 µl of the amplification products (244 bp for CRH, 145 bp for Ucn and 174 bp for actin) were separated on a 2% agarose gel and visualized by ethidium bromide staining. Primers for CRH were: 5'-CAA CTT TTT CCG CGT GTT GCT-3' (forward), 5'-ATG GCA TAA GAG CAG CGC TAT-3' (reverse); for Ucn: 5'-CAG GCG AGC GGC CGC G-3' (forward), 5'-CTT GCC CAC CGA GTC GAA T-3' (reverse); for actin: 5'-CTT CCT GGG CAT GGA GTC CTG-3' (forward), 5'-CAT CCT GTC GGC AAT GCC AGG-3' (reverse). Oligonucleotides were synthesized by MWG Biotech (Ebersberg, Germany). Samples containing no reverse transcriptase (no-RT) were used to exclude contamination with genomic DNA or reagent contamination.
For Fas and FasL, the primer pairs and probes were designed using the Primer Express 1.0 program (PE Applied Biosystems, Weiterstadt, Germany). Oligonucleotide hybridization probes and primer pairs were as follows. For FasL, TaqMan probe 5'-TCC AAC TCA AGG TCC ATG CCT CTG G, forward primer 5'- AAA GTG GCC CAT TTA ACA GGC and reverse primer 5'- AAA GCA GGA CAA TTC CAT AGG TG; and for Fas, TaqMan probe 5'-AAT CAT CAA GGA ATG CAC ACT CAC CAG CA, forward primer 5'-ACT GTG ACC CTT GCA CCA AAT and reverse primer 5'-GCC ACC CCA AGT TAG ATC TGG. Primers and probes were obtained from Applied Biosystems. The primers yielded RT–PCR products of 82 (FasL) and 105 (Fas) nucleotides.
The TaqMan EZ RT–PCR kit (PE Applied Biosystems, Weiterstadt, Germany) was used for reverse transcription and amplification of both targets and standards. All real-time RT–PCR reactions were performed in duplicate with a final volume of 25 µl in the ABI PRISM 7700 SDS (PE Applied Biosystems, Weiterstadt, Germany). Reaction conditions were as follows: 2 min at 50°C, 30 min at 60°C, 5 min at 95°C, 35 cycles with 20 s at 94°C and 60 s at 60°C. Quantification of RNA standards was linear over 8 logs and the assay measures as little as 100 copies of FasL or Fas mRNA per tube. Preparation of the RNA standard was done as previously reported (Reimer et al., 2000). All results are expressed as copy numbers per 200 ng total RNA.
Immunofluorescence and immunohistochemistry
Double immunofluorescence was performed as described (Hammer et al., 2001). Briefly, the frozen sections were thawed, fixed once more in acetone for 5 min at RT and rehydrated in PBS. After blocking for 15 min with blocking solution (LSAB-Kit, Dako, Ely, Cambridge, UK), the sections were incubated either with (a) FasL specific antibody Q20 (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) diluted 1:75 for 1 h at RT followed by incubation with anti rabbit Cy-3 (1:800) (Jackson Dianova, Hamburg, Germany) or with (b) a FITC-conjugated Fas specific antibody (Dako, Ely, Cambridge, UK) diluted to 2 µg/ml, overnight at 4°C. After washing with PBS the slides were incubated either with (a) KC56-PE (anti-CD45 labeled with phycoerythrin, Coulter Instrumentation Lab, Fullerton, USA) diluted 1:50, for 1 h at RT or with (b) an anti-cytokeratin-7 antibody (Novocastra, Newcastle, UK) diluted 1:10, for 1 h at RT followed by incubation with an anti-mouse TRITC-conjugated secondary antibody (Chemicon, Temecula, USA) diluted 1:200 for 1 h at RT. The slides were finally embedded in mounting buffer containing 4',6-diamino-2-phenylindole (DAPI, Molecular Probes, Eugene, OR, USA) and examined either with (a) a confocal laser scanning microscope (Leica TCS NT, Heerbrugg, Switzerland) or with (b) a Zeiss (Jena, Germany) Axiophot photomicroscope. Percentage of FasL-positive leukocytes was estimated using the method described below for the estimation of apoptotic EVT in terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL).
For immunohistochemistry, paraffin sections were deparaffinized in xylol, rehydrated in alcohol gradient to PBS and subsequently incubated with: methanol/H2O2 (30 min), goat serum (30 min) and primary antibody (1 h at RT), which was anti-Fas diluted 1:250 (LOB3/11, Serotec), or anti-CRH, or anti-Ucn both diluted 1:250 (both from Phoenix Pharmaceuticals, Karlsruhe, Germany), or anti-cytokeratin-7 diluted 1:10 (Novocastra, Newcastle, UK). The Vectastain® Elite ABC-Kit (Vector Laboratories, Burlingame, CA, USA) was used for visualization according to manufacturer instructions. Finally, slides were counterstained with hemalaun and cover-slipped. The intensity and distribution patterns of the staining reaction were evaluated by two blinded, independent observers, including a gynaecological pathologist, using the semi-quantitative immunoreactive score (IRS), as previously described (Remmele et al., 1986).
Control experiments encompassed immunofluorescence or immunohistochemistry (i) without primary detection antibodies, (ii) with monoclonal or polyclonal non-immune antibodies as primary antibodies and (iii) with immuno-neutralization of primary antibodies by pre-incubation with the respective peptides.
In situ detection of apoptotic EVTs with TUNEL and M30
TUNEL assay in paraffin sections was performed using a commercial kit (Boehringer-Mannheim, Mannheim, Germany), according to manufacturer instructions. Thereafter, immunofluorescence for cytokeratin-7 was performed as described above. M30 assay in deparaffinized and rehydrated tissue sections was performed using the M30 Cyto-DEATH antibody (ALEXIS, San Diego, CA, USA). Tissues were incubated with M30 diluted 1:200 and then with a Cy2-labeled anti-mouse secondary antibody (Jackson ImmunoResearch, Hamburg, Germany) diluted 1:200. Trophoblasts were characterized with a Cy3-conjugated anti-cytokeratin antibody (Micromet, Munich, Germany) diluted 1:200. Tissues were incubated with all antibodies for 1 h at RT. In both TUNEL and M30 assays, five to eight sections from 16 separate tissue blocks (n = 8 for ETP, n = 8 for abortion) were examined. Apoptotic and total cytokeratin-positive interstitial EVT were counted in randomly selected microscope fields of areas of decidual tissue at a magnification of x400. The number of apoptotic nuclei of cytokeratin-positive EVT was expressed as a percentage of the total number of cytokeratin-positive EVT counted in each slide (at least 300). Each slide was independently evaluated by two investigators. Negative controls were performed by omission of TUNEL enzyme or M30 according to manufacturer instructions and omission of anti-cytokeratin antibody.
Western blot
Western Blot for FasL in DL after incubation with additives for 24 h was performed as previously described (Makrigiannakis et al., 2001). To normalize for protein content, the blots were stripped in stripping buffer (62.5 mM Tris–HCl, pH 6.7, 2% SDS, 100 mM ß-mercaptoethanol) and stained with anti-actin antibody (Chemicon, Temecula, USA). The concentration of FasL protein in each lysate was normalized versus actin. The intensity of the bands was quantified using the ImageJ imaging system. The analysis was performed four times for DL obtained from elective pregnancy terminations and, in parallel, four times for DL obtained from abortions.
FACS analysis of DL
FACS for FasL expression in DL was performed as previously described (Makrigiannakis et al., 2001). A monoclonal antibody specific for FasL (NOK-1, Santa Cruz, CA, USA) and a FITC-conjugated secondary anti-mouse antibody (Chemicon, Temecula, USA) were used both in 1:100 dilution. For a negative control, PMA+I-activated DL from ETP were analysed after omission of primary antibody. FACS was performed with FACScan (Becton Dickinson, Heidelberg, Germany), and the results were analysed with the FACScan CELLQUEST software. The cells were studied in the electrically gated lymphocyte cluster.
Apoptosis assays
The APOPercentage Apoptosis Assay (Biocolor, Newtownabbey, Northern Ireland) and FACS analysis of AC1M88 cells stained with annexin and propidium iodide (Pi) (B&D Biosciences, Heidelberg, Germany) were employed. Briefly, 2 x 104 trophoblasts were placed in 96 well plates. The cells were cultured in DMEM/F12 medium ± 2 µg of anti-Fas blocking antibody (SM1/23, ALEXIS Biotechnologies, San Diego, CA, USA) for 8 h. 2 x 105 DL were added on top of the trophoblasts resulting in target:effector cell ratios of approximately 1:10. The lymphocytes were either not pretreated with any additives or pretreated for 24 h with 10 ng/ml Phorbol 12-myristate 13-acetate (PMA) and 1 µg/ml ionomycin (I), (both from Sigma (St. Louis, MO, USA)) CRH or Ucn ± antalarmin. The cells were co-cultured for 24 h in the presence of the additives (freshly prepared), which were used for their pretreatment, or in medium alone. The wells were gently rinsed to remove the lymphocytes. Then, either APOPercentage assay was performed according to manufacturer's instructions (Johnson et al., 2003) or the wells were forcefully rinsed to recover the trophoblasts for annexin–Pi staining and FACS analysis. The latter was performed as previously described (Makrigiannakis et al., 2001) with FACScan (Becton Dickinson, Heidelberg, Germany), and the results were analysed with the FACScan CELLQUEST software. Annexin-positive cells were considered apoptotic. The experiments were performed four times.
Statistical analysis
The SPSS/PC software package version 11.01 was used for collection, processing and statistical data analysis. Statistical analysis was performed using the non-parametrical Mann–Whitney U-signed rank test for comparison of the means. P-values <0.05 were considered statistically significant. Data are presented as mean ± standard error (SEM).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
|---|
Abortion is associated with increased expression of placental FasL and Fas mRNA
Absolute copy numbers of FasL and Fas mRNAs were determined in total RNA extracts from abortion placentas (n = 11) and ETP (n = 11) by real-time RT–PCR. In abortion placentas, means of FasL and Fas mRNA copies were, respectively, 19- and 2.8-fold higher compared with placentas from ETP (P < 0.05 for both) (Fig. 1A and B).
|
Abortion is associated with expression of FasL peptide in decidual leukocytes and increased expression of Fas peptide in EVT in situ
To investigate the expression of FasL and Fas peptides in situ, double immunofluorescence and immunohistochemical stainings were performed. Prior to double labeling, serial sections from all samples were stained with the mAb MNF-116, being specific for cells of ectodermal origin, such as trophoblast cells and cells of the uterine glands, and were investigated morphologically (data not shown). Only samples containing EVT invading decidua basalis were included in this study.
In contrast to ETP (n = 8), in which mainly the decidua-invading EVT cells expressed detectable amounts of FasL (Fig. 1C), all of the investigated samples from spontaneous abortion (n = 8) showed an additional cell population being highly positive for this molecule. These cells were identified as FasL-positive decidual leukocytes by double labeling with anti-CD-45 antibody (Fig. 1D). More specifically, in the samples examined, FasL-positive decidual leukocytes varied from 0.4 to 1.1% of the total leukocyte population in normal placentas and from 30.6 to 41.1% in abortion placentas. No differences in FasL fluorescence intensity in EVT were detected between ETP and abortion. As observed by double labeling experiments for prolactin and FasL, decidual cells did not express detectable amounts of FasL in placenta from ETP or abortion (data not shown).
Immunohistochemistry analysis showed that the majority of EVT in abortion (n = 8) expressed Fas receptor (Fig. 1G), whereas few EVT stained faintly for the peptide in ETP (n = 8) (Fig. 1E). EVT were identified as such by labeling of serial sections with anti-cytokeratin-7 antibody (Fig. 1F and H). IRS scoring revealed a 16-fold increase in Fas staining intensity in EVT in abortions compared with ETP (Fig. 1I) (P < 0.05). Expression of Fas in EVT in abortion was further verified by double immunofluorescence on frozen tissue sections (Fig. 1J–M).
Abortion is associated with increased apoptosis of interstitial EVT
Apoptosis of EVT in situ was detected by double labeling of placental tissues from ETP (n = 8) and abortions (n = 8) with TUNEL/M30 assays and cytokeratin-7. Results obtained from the two assays did not differ significantly (data not shown for M30 assay). Minimal numbers (<2%) of apoptotic cells were randomly present in interstitial EVT in ETP placentas (Fig. 2). Likewise, minimal numbers (<2%) of cytokeratin-negative apoptotic cells were randomly present in decidual tissue of both groups. Increased numbers of randomly occurring apoptotic interstitial EVT (12.9 ± 4.8% in TUNEL assay and 13.7 ± 2.9% in M30 assay) were counted in decidual tissues from abortions (Fig. 2) (P < 0.05). Occasionally, EVT columns could be identified within placental tissue. We did not detect any apoptotic cells in EVT columns in any of the two groups (Fig. 2).
|
Abortion is associated with increased expression of CRH and Ucn at the maternal–fetal interface
Semi-quantitative RT–PCR was performed for CRH (244 bp) and Ucn (145 bp) in total RNAs extracted from abortion placentas (n = 11) and ETP (n = 11). Normalization of the bands' intensity versus actin (174 bp) revealed a 6.2-fold increase for CRH and a 3.4-fold increase for Ucn mRNAs in abortions compared with ETP (Fig. 3A–C) (P < 0.05). To examine whether increased levels of CRH and Ucn peptides observed in abortive material may act on EVT, decidual areas invaded by EVT were examined in serial tissue sections from ETP (n = 8) and abortions (n = 8) by immunohistochemistry. Such areas were identified by labeling of serial sections with anti-cytokeratin-7 antibody (Fig. 3E, G, I and K). IRS scoring revealed a 4.3-fold increase for CRH (Fig. 3D, F and L) and a 2.8-fold increase for Ucn (Fig. 3H, J and L) staining intensities in decidua in abortions compared with ETP (P < 0.05).
|
CRH and Ucn induce the expression of FasL in DL isolated from ETP through CRHR1
To investigate the effect of CRH and Ucn on the expression of FasL in DL, immunoblotting experiments and FACS analysis were performed. A dose of 10 ng/ml PMA together with 1 µg/ml ionomycin significantly induced the expression of lymphocytic FasL. Control cells were untreated DL from ETP which were kept for 24 h in complete culture medium and were analysed for FasL expression in parallel with treated cells and DL from abortion (Fig. 4). The intensity of the 40 kDa band, representing the membrane-bound form of the peptide, was normalized versus actin. After 24 h incubation, both CRH and Ucn at 10 nM or 100 nM significantly increased FasL in isolated DL from ETP compared to control. This effect was mediated through CRHR1 since the addition of the CRHR1 antagonist antalarmin at a 10-fold higher concentration completely reversed it, whereas the addition of the CRHR2 specific antagonist anti-sauvagine had no effect (Fig. 4A) (n = 4, P < 0.05). DL from abortion (n = 4) were analysed for FasL expression in parallel with DL from ETP, after having been kept in complete culture medium for 24 h. DL from abortion were found to express higher levels of the peptide in comparison with CRH- or Ucn-treated DL from ETP (Fig. 4A). In addition, CRH and Ucn had no effect on the expression of FasL in DL from abortion, i.e. did not alter the already high levels of FasL expression in these cells (data not shown). The immunoblot results were further validated by FACS analysis. Incubation of DL from ETP with 100 nM of CRH or Ucn for 24 h increased FasL mean fluorescent intensity (MFI) by 2.7- and 2.6-fold, respectively, (n = 4, P < 0.05) compared with control. Addition of 10-fold higher concentration of antalarmin (1 µM) completely reversed the effects of CRH and Ucn. MFI of FasL expression in DL from abortion was found to be 4.4-fold higher compared with control, thus significantly higher than CRH- or Ucn-treated DL from ETP (Fig. 4B and C).
|
CRH and Ucn potentiate the ability of dl from ETP to induce Fas-mediated apoptosis of hyEVT
All additives were initially tested for their toxic effect on hyEVT cultured alone for 32 h. APOPercentage assay revealed no toxic effect for any of the additives that were subsequently used in co-cultures of hyEVT with lymphocytes (n = 4) (Fig. 5A).
|
DL from ETP pretreated for 24 h with 100 nM CRH or Ucn and then co-cultured with hyEVT for 24 h in the presence of the respective neuropeptide showed increased cytotoxic activity against hyEVT (25.8 ± 2.2% and 25.1 ± 1.9% apoptotic hyEVT, respectively) compared with untreated DL from ETP (13.5 ± 2.1% apoptotic hyEVT). This effect was specifically mediated through CRHR1 since the addition of antalarmin completely reversed it. Eight-hour pretreatment of hyEVT with 2 µg/ml of anti-Fas blocking antibody and subsequent co-culture with CRH- or Ucn-treated DL from ETP in the presence of anti-Fas and the respective neuropeptide reduced hyEVT apoptosis to the levels observed in co-cultures with untreated DL from ETP. Similar results were obtained from both apoptosis methods applied, i.e. APOPercentage method (n= 4, P < 0.05) (Fig. 5B) and FACS analysis of Annexin–Pi stained cells (n = 4, P < 0.05) (Fig. 6).
|
DL from abortion are highly potent in inducing Fas-mediated apoptosis of hyEVT
DL from abortions cultured for 24 h in complete medium only were more potent in inducing apoptosis of hyEVT after 24 h co-culture (35.2 ± 5.2% apoptotic hyEVT) compared with CRH- or Ucn-treated DL from ETP. The apoptosis rates of hyEVT were significantly reduced (20.4 ± 3.1% apoptotic hyEVT) when the cells were pretreated with 2 µg/ml of anti-Fas blocking antibody and were subsequently co-cultured with DL from abortions in the presence of the anti-Fas ab (n = 4) (Fig. 6B).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
|---|
Here, we report that spontaneous abortion in humans is associated with expression of FasL in DL, increased expression of Fas in EVT and increased apoptosis of interstitial EVT in situ. Furthermore, we propose a functional role for the neuropeptides CRH and Ucn in the mediation of these phenomena. We have found that CRH and Ucn expression is up-regulated in abortion. The two peptides induce in vitro the expression of FasL in DL isolated from ETP and thus potentiate the cytotoxic effect of these cells against a Fas-expressing EVT-based hybridoma cell line. These effects are mediated by CRHR1, since the addition of the appropriate antagonist, antalarmin, completely reversed it. Blocking of Fas receptor in hyEVT with an anti-Fas blocking antibody also significantly reduced the levels of hyEVT apoptosis in co-cultures with lymphocytes. Importantly, isolated DL from spontaneous abortions were able to potently induce Fas-mediated apoptosis in hyEVT, without prior stimulation. In this case, blocking of hyEVT Fas receptor, partially, but significantly, inhibited the effect, thus implying that activation of additional cytotoxic mechanisms in DL occurs within an abortogenic placental environment.
Several roles have been recognized for the pro-apoptotic protein FasL. In cytotoxic T lymphocytes and NK cells, FasL stored in intracellular granules can be delivered to the cell surface to mediate cytotoxicity against virally infected or tumorigenic targets (Nagata and Golstein, 1995). Immune cell FasL has a further role in maintaining homeostasis of the immune system by deletion of autoreactive T cells (Lynch et al., 1995) as well as in triggering an inflammatory response by activating polymorphonuclear neutrophils (Seino et al., 1998). In non-immune cells, such as Sertoli cells, corneal endothelium and epithelium of the eye, EVT and several types of tumor cells, FasL is associated with promoting the immune privilege of the respective tissues. In these cases FasL induces apoptosis of infiltrating activated immune cells (O'Connell et al., 2001; Niederkorn 2006). Interestingly, an intricate system that tightly regulates sorting of FasL to different cellular compartments is suggested to be responsible for the versatility in the effects of the peptide (Bossi and Griffiths, 1999).
We and others have previously suggested that FasL synthesized by EVT promotes apoptosis of activated T cells and maintenance of early pregnancy (Runic et al., 1996; Uckan et al., 1997; Kauma et al., 1999; Makrigiannakis et al., 2001; Abrahams et al., 2004). In our present study, we could not detect dysregulation of FasL in EVT in abortions. Instead we found that decidual leukocytes and EVT became highly positive for FasL and Fas, respectively. Consequently, leukocytic FasL and EVT Fas may account, at least in part, for the increased numbers of the molecules’ transcripts that we detected in total placental RNA from abortions. Recently, Tayade et al. (2006) using a model of commercial swine, provided evidence that FasL transcripts are more abundantly expressed in lymphocytes infiltrating implantation sites which contain arresting fetuses, than in lymphocytes accumulated at healthy attachment sites. Furthermore, the authors reported high Fas transcription in trophoblasts from arresting fetuses. Although these data are consistent with our data, fundamental differences between pig epitheliochorial placentation, where the uterine epithelium is not breached by trophoblasts, and human hemochorial placentation do not permit a direct comparison of the respective results.
In support for the involvement of the Fas/FasL system in abortion-related placental apoptosis, Ejima et al. (2000) suggested that Fas-mediated apoptosis occurs in various placental cells in pregnant mice after induction of abortion by i.p. injection of LPS . A role for placental apoptosis and placental expression of apoptosis-associated proteins in induced abortion was further supported by studies in rabbit (Liu et al., 2003) and mice (Savion et al., 2002). Although these studies proposed a role for placental apoptosis in abortion, the authors did not discriminate between the different types of apoptotic cells within decidua. In addition, the particular pathophysiology of induced abortion, which involves a generalized detrimental effect of a single factor i.e. lipopolysaccharide or cyclophosphamide in placenta, limits the significance of such results for the multifactorial human spontaneous abortion. Furthermore, Kokawa et al. (1998) reported increased rates of apoptosis in syncytiotrophoblast and decidual tissue in abortions, but did not use specific markers to characterize apoptotic cells within decidua. Finally, widespread apoptosis of third trimester human EVT has been associated with the pathogenesis of pre-eclampsia (DiFederico et al., 1999).
On the basis of present and previous evidence, we hypothesized that EVT in abortion might be susceptible to Fas-mediated apoptosis induced by activated decidual leukocytes. Indeed, we found that interstitial EVT apoptosis in abortions is significantly higher than in ETP, where a very small percentage of apoptotic EVT was detected. Furthermore, we did not detect any apoptotic cells in EVT columns. A plausible explanation is that within columns, decidual leukocytes may not be able to come into intimate contact with EVT abounding these sites, but may attack trophoblasts later as they invade the decidua—the site where leukocytes reside. Another explanation could be provided by previous studies on EVT apoptosis. In accord with our study, Huppertz et al. (1998) reported that in normal placentas, proliferative EVT do not show any signs of apoptosis, whereas interstitial EVT show some apoptosis. This was discussed in relation to the abundance of the anti-apoptotic protein Bcl-2 in proliferative EVT, together with the lack of the death receptors Fas and TNF-R1 in the same cells (Huppertz et al., 2006). Having published their results only in an abstract, Huppertz et al. (1998) do not clarify the age of the examined placental samples. Nevertheless, previous (DiFederico et al., 1999; Murakoshi et al., 2003) and present observations on proliferative and interstitial EVT apoptosis largely concur, irrespective of the potentially different placental ages and source of materials (abortion, ETP, delivery of normal pregnancy).
Next, we explored in vitro a functional mechanism for our in situ findings. Activation of CRHR1 induces the expression of FasL in EVT and PC12 cells (Makrigiannakis et al., 2001; Dermitzaki et al., 2002). In addition, elevated levels of CRH and Ucn protein have recently been reported in abortions (Madhappan et al., 2003), a finding which was reproducible in our present study. We specifically localized increased expression of the peptides in EVT-invaded decidual tissue in abortive material. We, therefore, tested the hypothesis that CRH and/or Ucn act in a paracrine manner to induce the expression of FasL in decidual leukocytes and thus potentiate the ability of these cells to induce apoptosis of EVT. Our investigation was focused on DL because these cells: (i) comprise the largest population of decidual leukocytes (
80%) in first trimester placenta, including mainly a special subset of natural killer cells, the uterine natural killer cells (uNK) and a few cytotoxic T cells and (ii) are easily obtainable.
We found that CRH and Ucn induced with similar potency the expression of membrane-bound form of FasL (40 kDa) in DL from ETP, an effect mediated by CRHR1. DL from abortion spontaneously expressed high amounts of FasL, thereby confirming and further expanding our in situ data on the expression of FasL in decidual leukocytes in abortions. In contrast to DL from ETP, DL from abortions did not up-regulate FasL significantly, after treatment with CRH or Ucn (data not shown). We hypothesize that these cells do not further respond to CRH and Ucn in vitro, due to the earlier effects of a decidual environment already rich in CRH and Ucn. Using co-cultures of DL with a Fas-expressing EVT-based hybridoma cell line (data not shown), we measured higher rates of apoptosis in hyEVT when co-cultured with CRH- or Ucn-treated DL from ETP. This effect was reversed by the addition of antalarmin, or blocking of Fas receptor in hyEVT, thus suggesting that it was owed to induction of lymphocytic FasL. Notably, DL from abortions were able to potently induce apoptosis of hyEVT, without prior treatment. In this case, although hyEVT apoptosis was significantly decreased by anti-Fas blocking antibody, it did not fall to the control levels, defined by co-cultures with untreated DL from ETP. This observation suggests that in abortions there may be several abortogenic factors, apart from CRH- or Ucn-induced FasL, which act on DL to induce a number of cytotoxic mechanisms. For example, such mechanisms could involve components of cytotoxic granules, such as granzyme B and perforin which are expressed at similar levels in uNK cells and CD56dim peripheral blood NK cells (Koopman et al., 2003), and are highly expressed in decidua (Rukavina et al., 1995; Gulan et al., 1997).
Importantly, these data suggest that CRH and Ucn exert immunomodulatory effects acting directly on DL at the maternal–fetal interface. The ability of CRH and Ucn to modulate the function of immune cells, including lymphocytes, has been previously reported (Singh 1989; Singh and Leu 1990; Angioni et al., 1993; Tsatsanis et al., 2006). It is suggested that in contrast to its systemic indirect immunosuppressive effects through the HPA axis, CRH produced locally at inflammation sites acts as a potent autocrine and/or paracrine pro-inflammatory cytokine (Karalis et al., 1991; Zhao and Karalis 2002; Venihaki et al., 2003; Gravanis and Margioris 2005). In accord with these observations, we report here that CRH and Ucn potentiate the cytotoxic effect of DL through up-regulation of FasL. This effect could be either directly mediated by CRHR1 or indirectly e.g. through a CRHR1-mediated production of lymphocytic pro-inflammatory cytokines. Our study therefore supports the immunomodulatory role of peripheral CRH and the relevant neuropeptide Ucn.
DL cytotoxicity against trophoblasts has been previously investigated. uNK can be activated by interleukin-2 (IL-2) to kill trophoblasts, which are otherwise resistant to uNK cell cytotoxicity (King and Loke, 1990). Both peripheral blood and term DL may become cytotoxic against normal and tumoral cytotrophoblasts after IL-2 activation (Abadia-Molina et al., 1996). Recently, Olivares et al. (2002) reported that, unlike DL from ETP, DL from spontaneous abortions are able to potently induce apoptosis of JEG-3 cells. Although different types of trophoblasts have been employed in various studies, our data and previous data are in accord. Collectively, it appears that DL possess the armamentarium to kill trophoblasts, but their cytotoxicity is normally restrained during early pregnancy. A widely enounced theory for the quiescence of DL as well as the resistance of trophoblasts to DL cytotoxicity in normal pregnancy is the Th1/Th2 paradigm. Successful pregnancy has been associated with a shift of intrauterine and peripheral cytokine production towards the anti-inflammatory Th2 cytokines, whereas imbalance towards the pro-inflammatory Th1 cytokines due, e.g., to infection or stress might favor trophoblast rejection (Lin et al., 1993; Marzi et al., 1996; Tangri et al., 1994; Piccinni et al., 1998). Recently, the Th1 cytokines, interferon-
and tumor necrosis factor 
were shown to promote Fas expression and sensitivity in first trimester primary trophoblasts, whereas the Th2 cytokines, IL6 and IL10, increased the resistance of trophoblasts to Fas-mediated apoptosis (Aschkenazi et al., 2002).
In their study, Madhappan et al. (2003) suggested that systemic or uterine stress might induce the local release of CRH and Ucn which therefore represent a causative link between stress and abortion. Contrarily, Florio et al. (2002) propose that placenta might respond to stress by releasing CRH to protect the embryo from a hostile environment. Our present and previous studies on the effects of CRH on FasL expression at the maternal–fetal interface (Makrigiannakis et al., 2001) further foster this dispute and reveal the evident paradox. In vitro, CRH and Ucn are able to induce expression of FasL in DL and thus potentiate DL ability to kill EVT. Since both peptides are produced at the implantation site (Makrigiannakis et al., 2001; Bamberger et al., 2007) and decidual leukocytes normally do not express FasL, this mechanism appears to be functional in abortions, but is blocked in normal pregnancy. In the latter case, CRH is instead able to promote survival of EVT through the opposite mechanism (Makrigiannakis et al., 2001). To explain this contradictory role, it is tempting to speculate that within a normal hormonal/cytokine decidual environment (e.g. a Th2-cytokine-predominant environment), activation of DL is precluded and CRH acting in favor of EVT is important for their survival. However, in an abortogenic decidual environment, DL activation is permissible and elevated levels of CRH and Ucn may readily activate DL and finally be detrimental for EVT. In addition, such an environment may render EVT more susceptible to DL cytotoxicity as shown by the increase in Fas expression in these cells. Of note, in the present study, DL were used, whereas previously naïve peripheral lymphocytes of newborn children were employed (Makrigiannakis et al., 2001). The potential effect of CRH-treated EVT on DL apoptosis warrants further investigation. Nevertheless, due to the complexity of the CRH and CRH-related peptides system, investigators have often revealed dual roles for intrauterine CRH and Ucn (Hillhouse EW, Randeva H, Ladds G, Grammatopoulos D. Corticotropin-releasing hormone receptors. Biochem Soc Trans 2002;30:428-432.).
An important issue that arises from the data presented here is whether EVT apoptosis observed in situ in spontaneous abortion precedes or follows embryonic death. Of note, abortion samples were collected after diagnosis of abortion was established, meaning that the time from embryonic death to collection of material could vary in contrast to control material. EVT apoptosis could therefore be either part of the process leading to removal of unwanted decidua of a failed pregnancy or part of the process responsible for impaired placentation which may lead to pregnancy failure. Madhappan et al. (2003) suggested that elevated levels of CRH and Ucn, found in abortion placentas, activate endometrial mast cells to secrete abortogenic tryptase and IL-8. Thus, the authors associated the elevation of placental CRH and Ucn with the pathophysiology leading to abortion. The in vitro data presented here clearly indicate that CRH and Ucn are able to induce FasL expression in DL, thereby potentiating their ability to kill EVT. Accordingly, within an environment rich in CRH and/or Ucn, such as the placenta, it is possible for such a phenomenon to occur. The conditions that might prohibit DL activation in normal pregnancy can only be hypothesized at present. At the same time, apoptotic disposal of unwanted EVT after failure of pregnancy cannot be excluded.
In fairness, both events may occur in vivo and the current study cannot discriminate between the two possibilities. To do so, further studies should focus on collecting abortion samples from women who are closely monitored by ultrasound on daily basis. This material, which would be particularly difficult to find, would be collected approximately at the time point of embryonic death, thus being closer to that of elective termination. Alternatively, a large-scale study involving sufficient samples from different time points after embryonic death could be performed to monitor the progress of the apoptotic process in EVT. Such a study would be able to conclude whether apoptosis is fully up-regulated close to the time of embryonic death, thus favoring the causal hypothesis or continues to rise after embryonic death, thus favoring the decidual removal hypothesis. In addition, important evidence may be provided by studying samples from pharmacologically induced abortions as well as employing experimental animal models.
Future research should further focus on the trigger of local release of CRH and Ucn in abortions. This is also important in view of our recent data revealing a role for CRH in limiting EVT invasiveness (Bamberger et al., 2006). Increased CRH and/or Ucn could be associated with defective placentation, not only due to indirect effects through DL, but also due to direct effects on EVT invasiveness (Bamberger et al., 2006). This hypothesis is currently under investigation in our laboratory. Furthermore, investigation of the temporal and spatial regulation of death receptors in EVT, the inhibition of DL cytotoxicity in normal pregnancy and the activation of additional cytotoxic mechanisms in abortions will contribute to the understanding of abortion at the molecular level.
In summary, we provide experimental evidence which link spontaneous abortion in humans with DL-induced apoptosis of EVT. In addition, we describe a specific role for CRH and Ucn in mediating EVT apoptosis through the induction of lymphocytic FasL. Further studies are required to determine the precise role of the proposed mechanism in the process of spontaneous abortion. Elucidation of the mechanisms governing early placentation in health and disease might serve as a basis for the development of therapeutic modalities for the treatment of pregnancy complications associated with defective placentation.
|
Funding |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
|---|
Greek State Scholarships Foundation (IKYDA 2003) and Alexander Onassis Foundation (both to A.M.)
|
Acknowledgements |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
|---|
We thank Dr AM Bamberger, University Hospital Eppendorf, Hamburg, for kindly providing the hybridoma EVT cell line. Greek State Scholarships Foundation (IKYDA 2003) and Alexander Onassis Foundation to A.M.
|
Footnotes |
|---|
These two authors contributed equally. 
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
|---|
Abadia-Molina AC, Ruiz C, Montes MJ, King A, Loke YW, Olivares EG. Immune phenotype and cytotoxic activity of lymphocytes from human term decidua against trophoblast. J Reprod Immunol (1996) 31:109–123.[Medline]
Abrahams VM, Straszewski-Chavez SL, Guller S, Mor G. First trimester trophoblast cells secrete Fas ligand which induces immune cell apoptosis. Mol Hum Reprod (2004) 10:55–63.
Angioni S, Petraglia F, Gallinelli A, Cossarizza A, Franceschi C, Muscettola M, Genazzani AD, Surico N, Genazzani AR. Corticotropin-releasing hormone modulates cytokines release in cultured human peripheral blood mononuclear cells. Life Sci (1993) 53:1735–1742.[CrossRef][Web of Science][Medline]
Anin SA, Vince G, Quenby S. Trophoblast invasion. Hum Fertil (Camb) (2004) 7:169–174.[Medline]
Aschkenazi S, Straszewski S, Verwer KM, Foellmer H, Rutherford T, Mor G. Differential regulation and function of the Fas/Fas ligand system in human trophoblast cells. Biol Reprod (2002) 66:1853–1861.
Bamberger AM, Minas V, Kalantaridou SN, Radde J, Sadeghian H, Loning T, Charalampopoulos I, Brummer J, Wagener C, Bamberger CM, et al. Corticotropin-releasing hormone modulates human trophoblast invasion through carcinoembryonic antigen-related cell adhesion molecule-1 regulation. Am J Pathol (2006) 168:141–150.
Bamberger CM, Minas V, Bamberger AM, Charalampopoulos I, Fragouli Y, Schulte HM, Makrigiannakis A. Expression of urocortin in the extravillous human trophoblast at the implantation site. Placenta (2007) 28:127–132.[CrossRef][Web of Science][Medline]
Barnea E. Epidemiology, etiology of early pregnancy disorders. In: The First Twelve Weeks of Gestation.—Barnea E, Hustin J, Jauniaux E, eds. (1992) Heidelberg, Germany: Springer-Verlag. 263–279.
Bossi G, Griffiths GM. Degranulation plays an essential part in regulating cell surface expression of Fas ligand in T cells and natural killer cells. Nat Med (1999) 5:90–96.[CrossRef][Web of Science][Medline]
van den Brule F, Berndt S, Simon N, Coulon C, Le Goarant J, Munaut C, Noel A, Frankenne F, Foidart JM. Trophoblast invasion and placentation: molecular mechanisms and regulation. Chem Immunol Allergy (2005) 88:163–180.[Medline]
Burton GJ, Jauniaux E. Placental oxidative stress: from miscarriage to preeclampsia. J Soc Gynecol Investig (2004) 11:342–352.[CrossRef][Web of Science][Medline]
Dermitzaki E, Tsatsanis C, Gravanis A, Margioris AN. Corticotropin-releasing hormone induces Fas ligand production and apoptosis in PC12 cells via activation of p38 mitogen-activated protein kinase. J Biol Chem (2002) 277:12280–12287.
Dey SK, Lim H, Das SK, Reese J, Paria BC, Daikoku T, Wang H. Molecular cues to implantation. Endocr Rev (2004) 25:341–373.
DiFederico E, Genbacev O, Fisher SJ. Preeclampsia is associated with widespread apoptosis of placental cytotrophoblasts within the uterine wall. Am J Pathol (1999) 155:293–301.
Ejima K, Koji T, Tsuruta D, Nanri H, Kashimura M, Ikeda M. Induction of apoptosis in placentas of pregnant mice exposed to lipopolysaccharides: possible involvement of Fas/Fas ligand system. Biol Reprod (2000) 62:178–185.
Florio P, Severi FM, Ciarmela P, Fiore G, Calonaci G, Merola A, De Felice C, Palumbo M, Petraglia F. Placental stress factors and maternal-fetal adaptive response: the corticotropin-releasing factor family. Endocrine (2002) 19:91–102.[CrossRef][Web of Science][Medline]
Funayama H, Gaus G, Ebeling I, Takayama M, Füzesi L, Huppertz B, Kaufmann P, Frank HG. Parent cell lines for trophoblast hybridization II: AC1 and related trophoblast cell lines, a family of HGPT-negative mutants of the choriocarcinoma cell line JEG-3. Trophoblast Res (1997) 10:191–201.
Gaus G, Funayama H, Huppertz B, Kaufmann P, Frank HG. Parent cells for trophoblast hybridization I: Isolation of extravillous trophoblast cells from human chorion laeve. Trophoblast Res (1997) 10:181–190.
Gravanis A, Margioris AN. The corticotropin-releasing factor (CRF) family of neuropeptides in inflammation: potential therapeutic applications. Curr Med Chem (2005) 12:1503–1512.[CrossRef][Web of Science][Medline]
Gulan G, Podack ER, Rukavina D, Gudelj L, Rubesa G, Petrovic O, Johnson PM, Christmas SE. Perforin-expressing lymphocytes in peripheral blood and decidua of human first-trimester pathological pregnancies. Am J Reprod Immunol (1997) 38:9–18.[Medline]
Hammer A, Desoye G, Dohr G, Sattler W, Malle E. Myeloperoxidase-dependent generation of hypochlorite-modified proteins in human placental tissues during normal pregnancy. Lab Invest (2001) 81:543–554.[Web of Science][Medline]
Hempstock J, Jauniaux E, Greenwold N, Burton GJ. The contribution of placental oxidative stress to early pregnancy failure. Hum Pathol (2003) 34:1265–1275.[CrossRef][Web of Science][Medline]
Hillhouse EW, Grammatopoulos DK. Role of stress peptides during human pregnancy and labour. Reproduction (2002) 124:323–329.[Abstract]
Huppertz B, Frank HG, Kaufmann P. Apoptosis along the invasive trophoblastic pathway. Placenta (1998) 19:A35.
Huppertz B, Kadyrov M, Kingdom JC. Apoptosis and its role in the trophoblast. Am J Obstet Gynecol (2006) 195:29–39.[CrossRef][Web of Science][Medline]
Hustin J, Jauniaux E, Schaaps JP. Histological study of the materno-embryonic interface in spontaneous abortion. Placenta (1990) 11:477–486.[CrossRef][Web of Science][Medline]
Jauniaux E, Burton GJ. Pathophysiology of histological changes in early pregnancy loss. Placenta (2005) 26:114–123.[CrossRef][Web of Science][Medline]
Jauniaux E, Watson AL, Hempstock J, Bao YP, Skepper JN, Burton GJ. Onset of maternal arterial blood flow and placental oxidative stress. A possible factor in human early pregnancy failure. Am J Pathol (2000) 157:2111–2122.
Johnson JD, Ahmed NT, Luciani DS, Han Z, Tran H, Fujita J, Misler S, Edlund H, Polonsky KS. Increased islet apoptosis in Pdx1+/- mice. J Clin Invest (2003) 111:1147–1160.[CrossRef][Web of Science][Medline]
Kalantaridou SN, Makrigiannakis A, Zoumakis E, Chrousos GP. Stress and the female reproductive system. J Reprod Immunol (2004) 62:61–68.[CrossRef][Web of Science][Medline]
Karalis K, Sano H, Redwine J, Listwak S, Wilder RL, Chrousos GP. Autocrine or paracrine inflammatory actions of corticotropin-releasing hormone in vivo. Science (1991) 254:421–423.
Kaufmann P, Black S, Huppertz B. Endovascular trophoblast invasion: implications for the pathogenesis of intrauterine growth retardation and preeclampsia. Biol Reprod (2003) 69:1–7.
Kauma SW, Huff TF, Hayes N, Nilkaeo A. Placental Fas ligand expression is a mechanism for maternal immune tolerance to the fetus. J Clin Endocrinol Metab (1999) 84:2188–2194.
King A, Loke YW. Human trophoblast and JEG choriocarcinoma cells are sensitive to lysis by IL-2-stimulated decidual NK cells. Cell Immunol (1990) 129:435–448.[CrossRef][Web of Science][Medline]
Kokawa K, Shikone T, Nakano R. Apoptosis in human chorionic villi and decidua during normal embryonic development and spontaneous abortion in the first trimester. Placenta (1998) 19:21–26.[Web of Science][Medline]
Koopman LA, Kopcow HD, Rybalov B, Boyson JE, Orange JS, Schatz F, Masch R, Lockwood CJ, Schachter AD, Park PJ, et al. Human decidual natural killer cells are a unique NK cell subset with immunomodulatory potential. J Exp Med (2003) 198:1201–1212.
Lin H, Mosmann TR, Guilbert L, Tuntipopipat S, Wegmann TG. Synthesis of T helper 2-type cytokines at the maternal-fetal interface. J Immunol (1993) 151:4562–4573.[Abstract]
Liu Z, Sun QH, Yang Y, Liu JM, Peng JP. Effect of IFNgamma on caspase-3, Bcl-2 and Bax expression, and apoptosis in rabbit placenta. Cytokine (2003) 24:201–209.[CrossRef][Web of Science][Medline]
Lynch DH, Ramsdell F, Alderson MR. Fas and FasL in the homeostatic regulation of immune responses. Immunol Today (1995) 16:569–574.[CrossRef][Web of Science][Medline]
Macklon NS, Geraedts JP, Fauser BC. Conception to ongoing pregnancy: the ‘black box’ of early pregnancy loss. Hum Reprod Update (2002) 8:333–343.
Madhappan B, Kempuraj D, Christodoulou S, Tsapikidis S, Boucher W, Karagiannis V, Athanassiou A, Theoharides TC. High levels of intrauterine corticotropin-releasing hormone, urocortin, tryptase, and interleukin-8 in spontaneous abortions. Endocrinology (2003) 144:2285–2290.
Makrigiannakis A, Zoumakis E, Kalantaridou S, Coutifaris C, Margioris AN, Coukos G, Rice KC, Gravanis A, Chrousos GP. Corticotropin-releasing hormone promotes blastocyst implantation and early maternal tolerance. Nat Immunol (2001) 2:1018–1024.[CrossRef][Web of Science][Medline]
Makrigiannakis A, Minas V, Kalantaridou SN, Nikas G, Chrousos GP. Hormonal and cytokine regulation of early implantation. Trends Endocrinol Metab (2006) 17:178–185.[CrossRef][Web of Science][Medline]
Marzi M, Vigano A, Trabattoni D, Villa ML, Salvaggio A, Clerici E, Clerici M. Characterization of type 1 and type 2 cytokine production profile in physiologic and pathologic human pregnancy. Clin Exp Immunol (1996) 106:127–133.[CrossRef][Web of Science][Medline]
Michel MZ, Khong TY, Clark DA, Beard RW. A morphological and immunological study of human placental bed biopsies in miscarriage. Br J Obstet Gynaecol (1990) 97:984–988.[Web of Science][Medline]
Murakoshi H, Matsuo H, Laoag-Fernandez JB, Samoto T, Maruo T. Expression of Fas/Fas-ligand, Bcl-2 protein and apoptosis in extravillous trophoblast along invasion to the decidua in human term placenta. Endocr J (2003) 50:199–207.[CrossRef][Web of Science][Medline]
Nagata S, Golstein P. The Fas death factor. Science (1995) 267:1449–1456.
Niederkorn JY. See no evil, hear no evil, do no evil: the lessons of immune privilege. Nat Immunol (2006) 7:354–359.[CrossRef][Web of Science][Medline]
O'Connell J, Houston A, Bennett MW, O'Sullivan GC, Shanahan F. Immune privilege or inflammation? Insights into the Fas ligand enigma. Nat Med (2001) 7:271–274.[CrossRef][Web of Science][Medline]
Olivares EG, Munoz R, Tejerizo G, Montes MJ, Gomez-Molina F, Abadia-Molina AC. Decidual lymphocytes of human spontaneous abortions induce apoptosis but not necrosis in JEG-3 extravillous trophoblast cells. Biol Reprod (2002) 67:1211–1217.
Payne SG, Smith SC, Davidge ST, Baker PN, Guilbert LJ. Death receptor Fas/Apo-1/CD95 expressed by human placental cytotrophoblasts does not mediate apoptosis. Biol Reprod (1999) 60:1144–1150.
Piccinni MP, Beloni L, Livi C, Maggi E, Scarselli G, Romagnani S. Defective production of both leukemia inhibitory factor and type 2 T-helper cytokines by decidual T cells in unexplained recurrent abortions. Nat Med (1998) 4:1020–1024.[CrossRef][Web of Science][Medline]
Reimer T, Herrnring C, Koczan D, Richter D, Gerber B, Kabelitz D, Friese K, Thiesen HJ. FasL:Fas ratio—a prognostic factor in breast carcinomas. Cancer Res (2000) 60:822–828.
Remmele W, Hildebrand U, Hienz HA, Klein PJ, Vierbuchen M, Behnken LJ, Heicke B, Scheidt E. Comparative histological, histochemical, immunohistochemical and biochemical studies on oestrogen receptors, lectin receptors, and Barr bodies in human breast cancer. Virchows Arch A Pathol Anat Histopathol (1986) 409:127–147.[CrossRef][Web of Science][Medline]
Rukavina D, Rubesa G, Gudelj L, Haller H, Podack ER. Characteristics of perforin expressing lymphocytes within the first trimester decidua of human pregnancy. Am J Reprod Immunol (1995) 33:394–404.[Medline]
Runic R, Lockwood CJ, Ma Y, Dipasquale B, Guller S. Expression of Fas ligand by human cytotrophoblasts: implications in placentation and fetal survival. J Clin Endocrinol Metab (1996) 81:3119–3122.
Runic R, Lockwood CJ, LaChapelle L, Dipasquale B, Demopoulos RI, Kumar A, Guller S. Apoptosis and Fas expression in human fetal membranes. J Clin Endocrinol Metab (1998) 83:660–666.
Savion S, Lepsky E, Orenstein H, Carp H, Shepshelovich J, Torchinsky A, Fein A, Toder V. Apoptosis in the uterus of mice with pregnancy loss. Am J Reprod Immunol (2002) 47:118–127.[CrossRef][Web of Science][Medline]
Seino K, Iwabuchi K, Kayagaki N, Miyata R, Nagaoka I, Matsuzawa A, Fukao K, Yagita H, Okumura K. Chemotactic activity of soluble Fas ligand against phagocytes. J Immunol (1998) 161:4484–4488.
Singh VK. Stimulatory effect of corticotropin-releasing neurohormone on human lymphocyte proliferation and interleukin-2 receptor expression. J Neuroimmunol (1989) 23:257–262.[CrossRef][Web of Science][Medline]
Singh VK, Leu SJ. Enhancing effect of corticotropin-releasing neurohormone on the production of interleukin-1 and interleukin-2. Neurosci Lett (1990) 120:151–154.[CrossRef][Web of Science][Medline]
Tangri S, Wegmann TG, Lin H, Raghupathy R. Maternal anti-placental reactivity in natural, immunologically-mediated fetal resorptions. J Immunol (1994) 152:4903–4911.[Abstract]
Tayade C, Black GP, Fang Y, Croy BA. Differential gene expression in endometrium, endometrial lymphocytes, and trophoblasts during successful and abortive embryo implantation. J Immunol (2006) 176:148–156.
Tsatsanis C, Androulidaki A, Alissafi T, Charalampopoulos I, Dermitzaki E, Roger T, Gravanis A, Margioris AN. Corticotropin-releasing factor and the urocortins induce the expression of TLR4 in macrophages via activation of the transcription factors PU.1 and AP-1. J Immunol (2006) 176:1869–1877.
Uckan D, Steele A, Cherry, Wang BY, Chamizo W, Koutsonikolis A, Gilbert-Barness E, Good RA. Trophoblasts express Fas ligand: a proposed mechanism for immune privilege in placenta and maternal invasion. Mol Hum Reprod (1997) 3:655–662.
Venihaki M, Zhao J, Karalis KP. Corticotropin-releasing hormone deficiency results in impaired splenocyte response to lipopolysaccharide. J Neuroimmunol (2003) 141:3–9.[CrossRef][Web of Science][Medline]
Watson AL, Palmer ME, Jauniaux E, Burton GJ. Variations in expression of copper/zinc superoxide dismutase in villous trophoblast of the human placenta with gestational age. Placenta (1997) 18:295–299.[CrossRef][Web of Science][Medline]
Watson AL, Skepper JN, Jauniaux E, Burton GJ. Susceptibility of human placental syncytiotrophoblastic mitochondria to oxygen-mediated damage in relation to gestational age. J Clin Endocrinol Metab (1998) 83:1697–1705.
Xerri L, Devilard E, Hassoun J, Mawas C, Birg F. Fas ligand is not only expressed in immune privileged human organs but is also coexpressed with Fas in various epithelial tissues. Mol Pathol (1997) 50:87–91.
Zhao J, Karalis KP. Regulation of nuclear factor-kappaB by corticotropin-releasing hormone in mouse thymocytes. Mol Endocrinol (2002) 16:2561–2570.
Submitted on June 12, 2007; resubmitted on July 10, 2007; accepted on July 17, 2007.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||