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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 8 3835-3844
Copyright © 2003 by The Endocrine Society

Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL), TRAIL Receptors, and the Soluble Receptor Osteoprotegerin in Human Gestational Membranes and Amniotic Fluid during Pregnancy and Labor at Term and Preterm

M. Lonergan, D. Aponso, K. W. Marvin, R. J. A. Helliwell, T. A. Sato, M. D. Mitchell, T. Chaiwaropongsa, R. Romero and J. A. Keelan

Liggins Institute (M.L., D.A., K.W.M., R.J.A.H., T.A.S., M.D.M., J.A.K.) and Department of Pharmacology and Clinical Pharmacology (M.D.M., J.A.K.), University of Auckland Faculty of Medical and Health Sciences, Auckland, New Zealand; Perinatology Research Branch, National Institute of Child Health and Human Development, National Institutes of Health/Department of Health and Human Services (T.C., R.R.), Detroit, Michigan 48201; and Perinatology Research Branch (T.C., R.R.), National Institute of Child Health and Human Development, National Institutes of Health/Department of Health and Human Services, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Dr. Jeffrey A. Keelan, Liggins Institute, University of Auckland, Faculty of Medical and Health Sciences, 2–6 Park Avenue, Grafton, Auckland, New Zealand. E-mail: j.keelan{at}auckland.ac.nz.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have studied TNF-related apoptosis-inducing ligand (TRAIL) and its membrane-bound (R1–R4) and soluble receptors [osteoprotegerin (OPG)] in gestational membranes to assess their significance in preterm parturition and premature rupture of membranes (PROM). TRAIL was detected by ELISA in extracts of term choriodecidual (but not amnion) tissues and explant-conditioned media. Concentrations of OPG (determined using ELISA) in gestational membranes were 20- to 50-fold greater than those of TRAIL. Median OPG concentrations in amniotic fluid (AF) at 15–17 wk gestation were similar to those at term before and during labor, whereas levels in pregnancies sampled preterm were significantly elevated. OPG levels in AF from women with preterm PROM were similar to those from women in preterm labor. In contrast, in pooled AF samples (n = 23–33), TRAIL concentrations at term with and without labor were elevated compared with samples from preterm deliveries. TRAIL-R3 and -R4 decoy receptors were detected in term amnion and choriodecidual extracts by immunoblotting and were localized by immunohistochemistry to amnion epithelial cells and chorionic trophoblasts. TRAIL (100 ng/ml) had little or no effect on amnion or choriodecidual cell viability or apoptosis, although these tissues responded to TNF-{alpha} with increased prostaglandin E2 production. Our findings suggest that OPG is abundant in gestational membranes and, in concert with TRAIL decoy receptors, may protect resident cells of the fetal membranes against the proapoptotic effects of TRAIL and other related ligands during pregnancy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PLACENTA and extraplacental membranes (amnion, chorion, and decidua) are ephemeral tissues with a defined life span that ends with parturition. As apoptosis is the physiological mechanism for removal of redundant cells or tissues without eliciting an inflammatory reaction, it seems tenable that apoptosis might occur in these tissues before or during labor as part of the process of parturition. Indeed, there is mounting evidence that apoptosis occurs in the gestational membranes under normal circumstances in association with labor and in pregnancies complicated by preterm premature rupture of membranes (PPROM) as a consequence of intrauterine infection. The presence of apoptotic cells in amnion, chorion, and decidua has been documented throughout the latter half of gestation, ranging from 10–30% of all cells depending on gestational age (1, 2, 3). In the amniotic epithelium and choriodecidua, apoptotic cells increased in abundance at term (4), with higher levels of apoptosis observed in tissues at the site of membrane rupture (5) or overlaying the cervix (6). These findings in women support studies in rats showing increased apoptosis in the epithelial amnion layer accompanied by degradation of the type I collagen matrix of the amnion (7). In pregnancies complicated by intrauterine infection, there are reports of increased apoptosis of the fetal membranes (5) and elevated amniotic fluid (AF) concentrations of nucleosomal protein (8), a marker of apoptotic cell death.

The factors responsible for initiating apoptosis in fetal membranes have not been identified, although a number of candidates have been proposed, such as cytotoxic prostaglandins (PGs) (9, 10), oxidative stress (11), glucocorticoids (12), relative abundance of Bcl-related proteins (2, 13, 14), and proinflammatory cytokines (15, 16). The TNF superfamily comprises a growing number of homologous cytokine-like molecules that act via homologous type I TNF receptor-like proteins that can elicit either proapoptotic or proinflammatory signals in target tissues (17). Several investigators have suggested that Fas/CD95, a member of the TNF receptor superfamily that is expressed in the fetal membranes, might play a key role in triggering the apoptotic cascade, particularly in the presence of infection (2, 3, 15, 18). The reported presence of activated caspase-8 and -3 in membranes supports the role of activation of members of the TNF receptor superfamily in this process (15). Moreover, components of the Fas signal transduction pathway (for example, Fas-associated death domain) are expressed in these tissues (15). However, studies to date have indicated that Fas activation does not cause apoptosis in trophoblasts (1, 19).

TNF-related apoptosis-inducing ligand (TRAIL), also known as Apo2L, is a 21- to 24-kDa member of this family that has been demonstrated to induce apoptosis in T cells and a variety of tumor cells (20, 21). However, most nontransformed tissues, even those that express fully functional TRAIL receptors (TRAIL-R1 and R2), are resistant to TRAIL-induced apoptosis, and it appears that there are multiple mechanisms that protect cells from its effects. The expression of nonsignaling TRAIL decoy receptors, of which two have been characterized (DcR1/TRAIL-R3 and DcR2/TRAIL-R4), is postulated to provide TRAIL resistance by acting as a TRAIL reservoir (22). There is also some evidence that TRAIL-R4 might actively antagonize the apoptotic cascade through activation of the nuclear factor-{kappa}B (NF-{kappa}B) pathway (23), a property shared by the two functionally active TRAIL receptors (TRAIL-R1 and -R2) (24). A secreted receptor somewhat distantly related to the TNF-R superfamily, termed osteoprotegerin (OPG), is also capable of binding and neutralizing both TRAIL (25). Interestingly, it also binds TNF-{alpha}, although its most important role is probably in modulating the effects of receptor activator of NF-{kappa}B (RANK) ligand and thereby bone resorption (26).

We hypothesized that TRAIL might be a candidate proapoptotic ligand in gestational membranes with potential importance in the pathophysiology of infection-associated PROM and preterm labor. TRAIL mRNA transcripts have been identified in the placenta, and TRAIL immunoreactivity has been observed in placental Hofbaur cells and amnion epithelium at term (27). However, the relative expression of the various TRAIL receptors in the extraplacental membranes has not yet been studied, and the effects of TRAIL on gestational membranes and its relevance in pregnancies complicated by membrane inflammation and/or rupture also remain unknown. The present study was, therefore, aimed at addressing these questions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Placentas were collected at National Women’s Hospital (Auckland, New Zealand) with informed consent as approved by the Auckland ethics committee. Amnion, choriodecidual, and placental tissues from pregnancies delivered after spontaneous labor at term or after spontaneous preterm labor with and without intrauterine infection were collected and frozen in liquid nitrogen for subsequent processing for either total RNA or protein extraction. RNA was extracted and purified using the guanidinium isothiocyanate/chloroform/phenol method, and four representative samples from the four clinical groupings were selected and labeled for cDNA array analysis using a Cytokine Expression Array (R&D Systems, Inc., Minneapolis, MN) as detailed previously (28). Hybridization signals for each set of four samples were normalized to eight housekeeping genes after correcting for background and were averaged for statistical analysis (ANOVA with Bonferroni’s t test post hoc). Full details of this procedure have been recently published (28). For protein extraction, tissue aliquots were sonicated and homogenized in an aqueous hypotonic lysis buffer supplemented with protease inhibitors as previously described (29). Aliquots of homogenate extracts, stored at -80 C, were thawed and diluted in assay diluent before immunoassay analysis. Membranes from an additional two placentas were lysed and homogenized in the presence of 2% sodium dodecyl sulfate and diluted in loading buffer for SDS-PAGE and immunoblotting. Explant cultures from amnion, choriodecidual, or placental tissues from term placentas delivered before the onset of labor were prepared as previously described (30, 31), initially cultured for 24 h in 24-well culture dishes in Ham’s F-10/DMEM (membranes) or medium 199 (placenta; 1 ml/well) containing 10% fetal calf serum and antibiotics, and treated with stimuli for an additional 24 h in the same medium without serum [supplemented with 0.1% bovine {gamma}-globulin (BGG)]. Media were stored at -20 C before assay, then thawed and diluted for assay as appropriate. Explant tissue wet weights (milligrams per well) were recorded at the end of the experiments for normalization purposes. For assessment of the ability of TRAIL to induce apoptosis or inflammatory activation of gestational membranes, amnion and choriodecidual explants were treated with TRAIL or TNF-{alpha} (100 ng/ml) for 24 h in Ham’s F-10/DMEM supplemented with 0.1% BGG and antibiotics. Release of lactate dehydrogenase (LDH) as a marker of cell death was performed using a TOX-7 kit (Sigma-Aldrich Corp., St. Louis, MO) calibrated against bovine heart-derived LDH (0–2 IU/ml). Nucleosomal protein release, an index of apoptotic death, was measured using a Cell Death Detection ELISA kit (Roche Diagnostics NZ Ltd., Auckland, New Zealand) calibrated against an arbitrary positive standard included with the kit. PGE2 was measured by RIA according to previously described methods (31, 32). Amnion monolayer cultures were also performed as previously described (32) to assess the effects of TRAIL on cell viability, using trypsin/collagenase-dispersed amnion cells from term placentas plated in 96-well culture plates at a range of cell densities in Ham’s F-10 medium supplemented with antibiotics and 10% fetal calf serum. Cultures (n = 3) were allowed to grow for 3 d to establish epithelial sheets before carrying out experiments over 24 h in serum-free medium (Ham’s F-10 medium with 0.1% BGG and antibiotics). Cell viability after treatment with TRAIL or TNF-{alpha} (100 ng/ml) was assessed by measuring the mitochondrial reduction of 3-(4,5-dimethlythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) using spectrometry at 595 nm as described previously (33). DNA synthesis in response to the same conditions was measured using the bromodeoxyuridine (BrdU) method with the aid of a commercial kit (Roche Diagnostics NZ Ltd.).

AF samples were collected by transabdominal amniocentesis. All women provided informed consent before the collection of AF. The collection and use of AF were approved by the human investigation committees of participating institutions [i.e. Wayne State University, Hutzel Hospital (Detroit, MI), and Sotero del Rio Hospital (Puente Alto, Chile)] and were approved for research purposes by the institutional review board of the NICHHD. Several studies using these fluids have been published recently that include full details of their collection and clinical categorization (34, 35). In brief, samples were divided into four main groups. Group 1 consisted of women in the midtrimester (15–17 wk) of pregnancy who underwent amniocentesis for genetic indications and delivered an appropriate for gestational age infant at term (n = 13). Group 2 consisted of women with preterm labor and intact membranes. These patients were subdivided into the following categories: a) preterm labor without microbial invasion of the amniotic cavity (MIAC) who delivered at term (n = 27), b) preterm labor without MIAC who delivered preterm (<37 wk; n = 29), and c) preterm delivery with MIAC (n = 19). Group 3 consisted of women with preterm PROM, with (n = 27) and without (n = 42) MIAC. Group 4 consisted of women with term gestations (i.e. >37 wk gestation) without MIAC and was subdivided into the following categories: a) intact membranes not in labor (n = 24), and b) intact membranes in labor (n = 37). Individual samples were diluted 1:25 for OPG assay as described below. However, for the TRAIL assay, due to limited sample availability, 10-µl aliquots of samples from an individual group were pooled to generate representative pooled samples for each group.

TRAIL and OPG ELISAs

TRAIL and OPG were measured by two-site ELISAs using an enzyme-amplified fluorescence detection method. All incubations were carried out at room temperature using opaque, high protein binding LIA plates (Greiner Labortechnik, Frickenhausen, Germany). The TRAIL ELISA employed a monoclonal capture antibody (MAB687; R&D Systems) in combination with an affinity-purified biotinylated polyclonal rabbit anti-TRAIL detection antibody (PA1321BT; Cell Sciences, Inc., Norwood, MA) and was calibrated against recombinant human (rh) TRAIL, which is composed of the entire extracellular TNF-like region of the TRAIL molecule (R&D Systems). The OPG assay employed a monoclonal capture antibody (MAB8051) and an affinity-purified polyclonal goat anti-OPG detection antibody (R&D Systems) and was calibrated against an rhOPG-Fc chimera from the same supplier. Signal generation was accomplished by incubation with a biotinylated tertiary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA; OPG assay only), followed by streptavidin-biotinylated alkaline phosphatase complex (DAKO, Copenhagen, Denmark). Fluorescent substrate (4-methylumbelliferyl phosphate) was added, and fluorescence was measured (355/460 nm excitation/emission) after 1–4 h of incubation in a Wallac 1420 Multiplate Reader (PerkinElmer Life Sciences/Wallac Oy, Turku, Finland). Curve fitting and data extrapolation were performed using Workout software (Wallac). The OPG assay had intra- and interassay precisions of 2.6% (n = 18) and 12.3% (n = 5), respectively. The limit of detection was less than 5 pg/ml (<0.1 pM). The antisera for the assay exhibit less than 0.06% cross-reactivity with TNF receptor I, TNF receptor II, or CD40 (manufacturer’s information). The assay quantitatively measured OPG in explant-conditioned media (amnion, choriodecidual, and placental), aqueous tissue extracts, and AF. However, upon dilution, AF exhibited some nonlinearity with the rhOPG-Fc standard, and hence all AF samples were assayed at a single dilution (1:25). The TRAIL assay was unaffected by the presence of OPG (up to 100 ng/ml) and showed no cross-reactivity with TRANCE/receptor activator of NF-{kappa}B ligand or TNF-{alpha} (up to 100 ng/ml). The intraassay precision of the TRAIL assay was 5% (n = 11), and the limit of detection was less than 80 pg/ml. TRAIL was quantitatively recovered from AF with high efficiency. Dilution linearity studies could not be conducted due to insufficient levels of TRAIL in conditioned media and AFs.

Immunoblotting of OPG and TRAIL receptors

Immunoblotting was performed on soluble extracts of amnion, choriodecidual, and placental samples; run on a 12% SDS-PAGE gel under reducing conditions (40 µg protein/lane) on a Mini Protean II system (Bio-Rad Laboratories, Hercules, CA); and transferred to Hybond-P PVDF membrane (Amersham-Pharmacia-Biotech, Bucks, UK). Biotinylated protein markers (Bio-Rad Laboratories) and Rainbow prestained standards (Amersham Pharmacia Biotech, Little Chalfont, UK) were used to estimate protein size and confirm transfer. Membranes were incubated with affinity-purified polyclonal antisera against OPG (AF805, R&D Systems), TRAIL-R1 (no. 1139, ProSci, Inc., San Diego, CA), TRAIL-R2 (AF873, R&D Systems), and TRAIL-R3 or R4 (PX052A and PX058A, respectively, Cell Sciences, Inc.). Immunodetection was accomplished after incubation with the appropriate biotinylated secondary antisera (Jackson ImmunoResearch Laboratories), followed by streptavidin-biotinylated horseradish peroxidase complex (Amersham Pharmacia Biotech) using enhanced chemiluminescence (NEN Life Science Products, Boston, MA). Membranes were exposed to Hyperfilm ECL x-ray film (Amersham Pharmacia Biotech) for 0.5–10 min and developed using a Kodak X-OMAT processor.

Immunocytochemistry

Paraformaldehyde-fixed, paraffin-embedded placental tissue blocks (from the same placentas as those used to prepare the soluble tissue extracts) were sectioned at 7 µm and rehydrated before antigen retrieval (5 min at 100 C in a microwave in 0.5 M Tris, pH 10). Endogenous peroxidase activity was blocked with 3% H2O2 in 50% methanol, and an additional blocking step was employed using blocking reagent supplied in the tyramide signal amplification biotin system kit (PerkinElmer Life Sciences). Sections were then incubated overnight in primary polyclonal antisera (as detailed above) in the presence of 5% normal horse serum, followed by incubation (1–3 h) with biotinylated secondary antibody (Jackson ImmunoResearch Laboratories) and/or streptavidin-biotinylated horseradish peroxidase complex (30 min). Signal was amplified using tyramide amplification (TSA biotin system, PerkinElmer Life Sciences) according to the manufacturer’s instructions, and immunoperoxidase staining was developed using diaminobenzidine. Sections were not counterstained. Digital photomicrographs were taken using an Eclipse E800 microscope (Nikon, Melville, NY) fitted with a JBC (JVC, Victor Company of Japan, Ltd., Tokyo, Japan) TK-C1381 color video camera. Negatives were created from the monochrome images to enhance visualization of staining.

Statistical analysis and presentation of data

TRAIL and OPG concentrations in tissue homogenates were normalized to total protein and are expressed as the mean ± SEM. The production of OPG and TRAIL in explant culture was derived as picograms per milligram wet weight tissue and expressed as a percentage of the control to allow the results of multiple experiments to be pooled and analyzed collectively. Statistical significance was assessed by ANOVA, followed by Dunnett’s test or Bonferroni’s t test post hoc.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Our initial cDNA array studies indicated that the genes encoding TRAIL, OPG, and TRAIL-R1 to -4 were all expressed in amnion and choriodecidua from placentas delivered either at term or preterm with and without intrauterine infection (Fig. 1Go). There were no significant differences in the expression of any of these genes between any of the three gestational groups. However, in amnion there was a trend for OPG and TRAIL-R mRNA levels to be higher in the tissues delivered preterm; this trend was not apparent in the choriodecidua. Overall, the abundance of OPG, TRAIL-R2, and TRAIL-R3 mRNA appeared to be greater than that of TRAIL and the R1 and R4 receptors.



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  		FIG. 1. Expression of TRAIL and TRAIL receptors in gestational membranes as assessed by cDNA array. Total RNA was extracted from gestational tissues from placentas (n = 4/group) delivered by term spontaneous labor (TSL) and preterm labor without (PTL-) and with (PTL+) intrauterine infection (chorioamnionitis) and analyzed by cDNA array as described in Materials and Methods. Hybridization signals were corrected for background and normalized to eight housekeeping genes to correct for intermembrane variation. Data are shown as the mean ± SEM (n = 4). There were no significant differences in expression between the groups as assessed by ANOVA with Bonferroni’s t test post hoc. A, Amnion; B, choriodecidua.

 
Using a number of approaches we investigated the ability of the gestational membranes to synthesize and secrete TRAIL and OPG. Conditioned media from explant cultures of amnion, choriodecidual, and placental tissues contained concentrations of OPG (>0.5 nM) readily detectable by ELISA. Choriodecidual explants produced the greatest amount of OPG (193.3 ± 31.6 pg/mg tissue; mean ± SEM; n = 4), followed by villous placenta (11.23 ± 1.04 pg/mg tissue) and amnion (1.9 ± 0.29 pg/mg tissue). TRAIL production rates were approximately 0.5–2% those of OPG, with TRAIL being below the limits of detection in conditioned media from amnion explants. As the production of both TRAIL and OPG have been reported to be regulated by proinflammatory cytokines IL-1ß and TNF-{alpha}, factors known to act upon gestation tissues and implicated in the mechanism of preterm labor, we also investigated their effects on TRAIL and OPG production by gestational membranes. Unexpectedly, neither IL-1ß nor TNF-{alpha} at concentrations shown to exert near-maximal stimulatory effects in this model (32) elicited any change in the production of OPG or TRAIL by either amnion or choriodecidual explants. However, treatment of choriodecidual explants with LPS did increase OPG production rates by a modest, but statistically significant, amount (42.4 ± 12.3% above control; mean ± SEM; n = 4; P < 0.05).

Tissue extracts of villous placenta, amnion, and choriodecidua derived from term and preterm membranes also contained detectable levels of both OPG and TRAIL, with greatest concentrations again seen in the choriodecidual tissues and lowest levels in the amnion (Fig. 2Go). There were no trends or significant differences in OPG or TRAIL levels in tissues delivered with and without labor at term or by spontaneous preterm delivery. OPG was detected as an approximately 55-kDa protein in tissue extracts by Western blotting that was absent with antibody neutralized with rh-OPG antigen (Fig. 3Go), coinciding with the published molecular weight for native OPG. A higher molecular weight band was also detected in the tissues that was abolished by preincubation of the antiserum with OPG, possibly representing the homodimeric form.



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  		FIG. 2. Concentrations of OPG and TRAIL in gestational tissues delivered at term (with/without labor) and preterm. OPG (A) and TRAIL (B) were measured in extracts of amnion, choriodecidual, and placental homogenates obtained from pregnancies delivered at term without labor (TNL; n = 15), at term with spontaneous labor (TSL; n = 15), or at preterm labor (PTL; n = 30). Data shown are the median ± interquartile range. There were no significant differences in concentrations of immunoreactive OPG or TRAIL between any of the gestational groups (by Mann-Whitney U test).

 


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  		FIG. 3. Immunoblotting analysis of OPG in gestational membranes. Term amnion (A), choriodecidual (C), and placental (P) tissues from three placentas were lysed and sonicated in the presence of 0.5% Tween 20, and 20 µg protein (each), resolved on SDS-PAGE, then transferred to a polyvinylidene difluoride membrane for immunodetection using polyclonal anti-OPG antiserum. A parallel set of samples was subjected to immunoblotting using antisera neutralized by preincubation with rhOPG (1 µg/ml; +OPG). A dominant, approximately 55-kDa band, absent in the antigen-neutralized blots, was detectable in all tissues. A fainter, high molecular weight band was also detected that was blocked by preincubating the antiserum with rhOPG ligand.

 
OPG was abundant in AF (range, 0.059–22.6 ng/ml), with concentrations in pregnancies presenting with preterm labor significantly higher than those delivered at term (regardless of labor status) or those sampled at midtrimester (Fig. 4Go). This difference appeared to be related to gestational age at sampling, as OPG levels in AF from all groups sampled preterm (regardless of presence of preterm PROM, delivery, or MIAC) were significantly higher than those at term or midtrimester (Table 1Go). In women with preterm labor and delivery, the presence of MIAC was not associated with significantly altered AF OPG concentrations, but patients with PPROM with MIAC had a lower median AF OPG concentration than those without MIAC (Table 1Go). In contrast, mean TRAIL concentrations in AF were approximately 10-fold greater at term than at midgestation or in samples from women with preterm delivery (Fig. 5Go). TRAIL was below the limit of detection in preterm AF samples without MIAC, but was detectable (163 pg/ml) when MIAC was present. Assessment of the statistical significance of these data could not be carried out, however, because they were derived from analysis of representative pooled AF samples generated from multiple samples.



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  		FIG. 4. AF concentrations of OPG in pregnancies throughout gestation and delivery. OPG was measured by ELISA in AF samples taken from women at midpregnancy (15–17 wk gestation) for genetic screening purposes (n = 13), with preterm labor (PTL) who delivered at term (n = 27), or at term not in labor (TNL; n = 24). The horizontal line represents the mean for each group. Note the logarithmic scale of the y-axis. Statistical analysis was performed using ANOVA after log transformation of data.

 

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  		TABLE 1. Mean ± SD concentrations of OPG in amniotic fluid samples from pregnancies at various gestational ages and conditions

 


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  		FIG. 5. AF concentrations of TRAIL in samples of pooled AF from midpregnancy to term. TRAIL was measured by ELISA in pooled samples of AF from the pregnancy categories described in Fig. 4Go. Preterm samples were subdivided into those with (+) and without (-) MIAC.

 
We assessed the presence of TRAIL receptors in gestational membranes by Western blotting analysis. In two sets of homogenates of amnion, chorion, and decidua from term, nonlabored membranes, TRAIL-R1 was not visible by immunoblotting (Fig. 6AGo), although a nonspecific band at the expected position (~50 kDa) may have obscured its detection. TRAIL-R2 was also undetectable in the gestational tissue extracts, although a band of the expected size (~55 kDa) was faintly visible in the two decidual samples (Fig. 6BGo). The TRAIL-R3 (DcR1) blot was more complex, with multiple bands observed in the positive control, some of which corresponded to possible sizes expected for the R3 receptor (~55, 34, and 24 kDa; Fig. 6CGo). A 24-kDa band was present in the tissue extracts, corresponding to the reported molecular weight of the unglycosylated form of the receptor. TRAIL-R4 (DcR2) was, in contrast, clearly detectable at its expected molecular mass of about 34 kDa in all extracts and the positive control (Fig. 6DGo).



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  		FIG. 6. Immunoblotting studies of TRAIL-Rs in extracts of term amnion, chorion, and decidua. Term amnion (A), chorion (C), decidual (D), and choriodecidual (CD) tissues were lysed and sonicated in the presence of SDS, 20 µg protein (each), resolved on SDS-PAGE, and transferred to a polyvinylidene difluoride membrane for immunodetection using a polyclonal antisera for the four known TRAIL receptors (R1–R4) as described in Materials and Methods. WISH cell (HeLa-derived) extracts were run as a positive control. Molecular weight markers are shown in the left lane of each blot. The expected molecular weights of each receptor are indicated with a dotted arrow. Nonspecific bands at about 50 and 67 kDa were detected in A, C, and D.

 
TRAIL, OPG, and TRAIL receptors were localized to various cell types within the gestational membranes by immunohistochemistry (Fig. 7Go). Immunodetection was accomplished after antigen retrieval and tyramide amplification. All negative controls (primary antiserum omitted) were devoid of staining (data not shown), and each primary antiserum gave distinctly different patterns of staining. The anti-OPG antisera gave a low level of nonspecific background staining in the choriodecidua that was not localized to any particular cellular structures (Fig. 7aGo) and was not abolished by incubation of antiserum with rhOPG (not shown). In the amnion, however, specific OPG immunostaining (blocked by ligand preincubation) was localized to the basal region of the amniotic epithelium (Fig. 7gGo), although staining of the entire epithelial layer was occasionally seen in some regions. TRAIL immunostaining was minimal or undetectable throughout the amnion or choriodecidua (Fig. 7Go, b and h), although in the decidua, occasional discrete foci of TRAIL-positive cells with leukocyte-like morphology were observed (Fig. 7bGo). TRAIL-R1 staining was present sporadically throughout the chorion and decidua (Fig. 7cGo), but was absent in the amniotic epithelium (Fig. 7iGo). The mesenchymal cells of the amnion and reticular layer of the chorion (Fig. 7Go, c and i) were also positive for TRAIL-R1. Cytoplasmic TRAIL-R2 immunostaining was localized to occasional areas of decidual cells (Fig. 7dGo), but was absent in the chorion and amnio-chorion mesenchyme. However, in the amniotic epithelium, a distinctive pattern of staining was observed where the apical surfaces of the epithelial cells were intensely stained (Fig. 7jGo). TRAIL-R3/DcR1 immunostaining occurred in all cells and layers of the gestational membranes (Fig. 7Go, e and k) and was the most widespread of all the receptors. The TRAIL-R4/DcR2 antiserum stained scattered chorionic trophoblasts and the majority of mesenchymal cells at the amnio-chorion interface, whereas decidual staining was sparse and of low intensity (Fig. 7fGo). In the amnion, epithelial and mesenchymal cells were stained strongly, with a punctate staining pattern visualized in some regions of the epithelium (Fig. 7lGo).



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  		FIG. 7. Immunoperoxidase localization of OPG, TRAIL, and TRAIL-Rs in gestational membranes. Paraffin-embedded, full-thickness, term membranes were subjected to antigen retrieval and immunoperoxidase staining with TSA amplification as described in Materials and Methods. Negative controls, consisting of no primary antiserum or antigen-neutralized antiserum (OPG only), were devoid of staining (not shown). Choriodecidua is displayed in the upper panel of each set (a–f), and amnion is in the lower panel (g-l). The amnion epithelium is indicated by the white arrows. Regions of the decidua, chorion, and mesenchyme are designated D, C, and M. All photomicrographs are shown as negative images for clarity and were taken at x400 magnification.

 
To assess the capacity of TRAIL to induce cell death in amnion (the key membrane responsible for maintaining cellular integrity), we cultured trypsin-dispersed amnion epithelial monolayer cultures with TRAIL and, for comparative purposes, TNF-{alpha} in serum-free conditions at a maximally effective concentration (100 ng/ml). A range of plating densities was used, because in preliminary experiments using the amnion-like WISH cell line, cell density was found to be a key factor in determining sensitivity to apoptosis in response to TRAIL. Using the MTT assay as an index of cell viability, neither TRAIL nor TNF-{alpha} induced significant cell death over 24 h (Fig. 8AGo) regardless of cell plating density (0.125–0.5 x 106 cells/ml). Similar results were obtained over a 48-h treatment period (not shown). To rule out the possibility that TRAIL might be inhibiting cell growth without causing cell death or could even be exerting growth-promoting effects, we assessed the effects of TRAIL on DNA incorporation by amnion monolayer cultures using the BrdU method, at a seeding density of 0.25 x 106 cells/cm2. No significant effects of TRAIL or TNF-{alpha} on DNA incorporation were observed over a 24- or 48-h treatment period at concentrations ranging from 1–100 ng/ml (Fig. 8BGo). To confirm these findings we used an explant model to eliminate the possibility that losses in responsiveness to TRAIL might have occurred in culture. TNF-{alpha} was again employed as a positive control, with PGE2 production assessed to confirm responsiveness. In amnion explants, TRAIL failed to increase the release of nucleosomal protein or PGE2 production, although a small (but significant) increase in LDH release was observed (Fig. 9AGo). TNF-{alpha}, on the other hand, induced a significant increase in PGE2 production and nucleosomal protein release. In choriodecidua, TRAIL had no effect on any of the parameters measured, whereas TNF-{alpha} treatment resulted in a small increase in LDH release and a 2-fold increase in PGE2 production, which just failed to reach statistical significance (P = 0.077; Fig. 9BGo).



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  		FIG. 8. Effects of TRAIL and TNF-{alpha} on amnion cell viability and proliferation. A, Amnion epithelial cells in culture were treated in serum-free conditions with either TRAIL or TNF-{alpha} (100 ng/ml) for 24 h, and cell viability was determined by MTT assay. Data shown represent the mean ± SEM of three separate experiments performed in quadruplicate. B, Concentration-dependent effects of TRAIL and TNF-{alpha} on DNA synthesis by amnion epithelial cells in culture, assessed using the BrdU assay. Data shown represent the mean ± SEM of three or four separate experiments performed in quadruplicate.

 


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  		FIG. 9. Effects of TRAIL and TNF-{alpha} on the viability of amnion and choriodecidual explants. Explant cultures of amnion (A) and choriodecidual (B) membranes were treated over 24 h with TRAIL or TNF-{alpha} (100 ng/ml) under serum-free conditions, and the release of LDH (marker of cell death), nucleosomal protein (marker of apoptotic death), and PGE2 (marker of inflammatory activation) were measured in the media. Data were normalized to wet weight tissue per well and expressed as a percentage of the baseline control. Data shown are derived from four experiments performed in triplicate. *, P < 0.05 vs. control, by ANOVA, followed by Dunnett’s test post hoc.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This report is the first to describe the expression, production, and localization of soluble and membrane-bound TRAIL receptors in human extraplacental membranes and the role of TRAIL and its receptors in normal and complicated pregnancies. Although the proapoptotic effects of TRAIL on transformed cells have been well described (36), its role in normal tissues remains, to a large extent, unresolved. The finding that TRAIL and OPG are expressed by the gestational membranes and are present in the amniotic cavity adds further complexity to our understanding of the role of cytokines and related factors in the pathophysiology of preterm labor and PROM. Our data suggest that within the pregnant uterus, TRAIL is produced constitutively in late pregnancy by gestational membranes and accumulates in the amniotic cavity, yet the membranes themselves are protected from its proapoptotic effects by expression of decoy receptors and secretion of OPG. Hence, it appears likely that the intended target for TRAIL are cells or tissues outside of those normally found in the fetal membranes in late pregnancy. TRAIL-Rs are expressed on the surface of dendritic cells, activated T cells, and monocytes/macrophages (37). Marrow-derived cells are occasionally seen in membranes from normal pregnancies as well as those with leukocytosis as a result of intrauterine infection, and gestational maturity is accompanied by a dramatic loss of immune cells from the decidua; hence, it is possible that TRAIL may play a role in immune surveillance in the fetal membranes. This interpretation was also advanced by Phillips et al. (38) with respect to the placenta, when previously reporting that TRAIL failed to induce apoptosis in choriocarcinoma cell lines. This possibility remains speculative, however, and the hypothetical role of TRAIL in mediating leukocyte clearance in gestational tissues and placenta remains to be tested.

Evidence from our studies on tissue explants and membrane extracts indicates that the production of TRAIL by the choriodecidua is significantly greater than that of the amnion. The expression array data agree with this trend, and although TRAIL was not detectable by immunohistochemistry in any of the gestational membranes (other than some putative leukocytes), this probably reflects the sensitivity limitations of this method. Others have reported TRAIL immunoperoxidase staining in the amniotic epithelium (38). The possibility cannot be discounted that immunoreactive TRAIL measurable in amnion extracts could be derived by accumulation from adjacent tissues, whereas in the decidua, lymphocytes or other marrow-derived cells could be a major source of TRAIL, as indicated by the immunohistochemical data. Phillips et al. reported that TRAIL expression was absent in placental cytotrophoblasts (27), but detectable by immunohistochemistry in placental macrophages and syncytium (38). These findings would be consistent with the decidua, not the chorionic trophoblast, being the main source of TRAIL in the gestational membranes.

OPG production, like that of TRAIL, was far greater in the choriodecidua than in the amnion. However, OPG hybridization intensity in the cDNA array was not markedly different between amnion and choriodecidua. To confound matters further, the immunohistochemical analysis suggested that OPG abundance in the choriodecidua is low, in contrast to the strong staining observed in the amniotic epithelium. These conflicting observations remain to be resolved. The basal, rather than apical, localization of OPG in the amnion epithelium is intriguing and could be interpreted as evidence that OPG is secreted directionally by the amnion to act within the chorion and/or the decidua. Although it is possible that secreted OPG from the choriodecidua accumulates in the amnion, this is unlikely, because OPG can be found as both a membrane-bound and secreted protein and was also detectable in amnion by immunoblotting. Hence, the interesting pattern of staining in the amnion probably reflects the site of synthesis not accumulation. Alternatively, OPG may be preferentially trapped near the amniotic basement membrane; the anatomical proximity of the epithelial cells with the basement membrane might control in some way the cell’s ability to express and synthesize OPG, or substances in the AF might prevent OPG synthesis in the apical regions of the epithelium. Further studies are required to address these questions.

Interferon-{gamma} and/or TNF-{alpha} have been shown to increase TRAIL expression in immune cells and trophoblast-derived cell lines (38, 39). In contrast, our in vitro studies suggest that TRAIL production is not regulated by proinflammatory cytokines in gestational membranes. Our data suggest that TRAIL secretion into the AF may, however, be gestational age dependent, as fluids from term deliveries had markedly higher TRAIL levels than those taken at midgestation or in preterm delivery. A more thorough evaluation of AF TRAIL concentrations using multiple samples would be necessary to confirm these preliminary observations. As they stand, our data lend weight against the hypothesis that TRAIL may be involved in membrane apoptosis that is believed to occur in the presence of intrauterine infection.

Although OPG production was also unresponsive to IL-1ß and TNF-{alpha}, in the choriodecidua LPS stimulated production to a modest extent. This in itself is interesting, because many of the proinflammatory effects of LPS are mediated by the secondary release of cytokines, and it would thus appear that in these tissues LPS-stimulated OPG production is regulated in some other fashion. In contrast to TRAIL, the amounts of OPG in AF were elevated in pregnancies with preterm labor or PPROM regardless of the presence of MIAC or gestational age at delivery. The predicted association between MIAC and elevated OPG concentrations in AF was not observed: indeed, the opposite was recorded in pregnancies with PPROM. These data suggest that OPG levels in AF normally rise from midtrimester to early in the third trimester, then decline toward term, although it is not possible to rule out the preterm rise as a reflection of the pathological nature of the pregnancies sampled at this time. The increased abundance of OPG in AF of pregnancies in preterm labor could be indicative of a protective role of OPG in intrauterine tissues, perhaps to minimize the effects of TRAIL, TRANCE, or other related proteins. Indeed, a recent report has confirmed TNF-{alpha} itself as a ligand for OPG (40). Based on our quantitation, there would appear to be ample OPG to bind and neutralize the small amounts of TRAIL present in the amniotic cavity. TRANCE expression in the gestational membranes was very low by cDNA expression array, and we have been unable to detect TRANCE production by explants in vitro by ELISA (unpublished observations). The apparent decline in AF OPG concentrations at term would be consistent with a withdrawal of a factor inhibitory to inflammation/apoptosis, coinciding with increased production of inflammatory mediators and apoptosis in the gestational membranes that occurs at term. The functional importance of this possibility remains to be determined.

TRAIL-R immunoblotting studies suggested that the gestational membranes are protected from the effects of TRAIL by the expression of decoy receptors (TRAIL-R3 and -R4) in preference to the active signaling receptors (R1 and R2). Although weak R1 and R2 immunoperoxidase staining was detected in chorion and decidua, we were unable to detect these proteins in tissue extracts by Western blotting. The specificity of the antisera used in the immunohistochemical studies could not be verified absolutely due to the unavailability of receptor proteins to immunoneutralize the antisera, although each antiserum generated a specific pattern of staining that was not present in the negative controls. Hence, despite the cDNA array data showing the expression of receptor mRNA, there remains some doubt about the presence of these receptors at the protein level. In contrast, strong staining of both TRAIL-R3 and -R4 was detected in amnion, chorion, and decidual cells by immunohistochemistry, and a band corresponding to the predicted/published molecular weight of TRAIL-R4 (23) was clearly identified by Western blotting in all tissues. In the TRAIL-R3 blot, a band with a lower molecular weight (24 kDa) was detected in one amnion sample by the antisera, whereas nothing was apparent at the size previously described for TRAIL-R3 (~55 kDa). This protein has a predicted size of 24 kDa, but is highly glycosylated in most conditions described to date, causing it to migrate as a much larger protein (41). It is, therefore, possible that the band detected could be unglycosylated receptor, or it could be an unrelated molecule that cross-reacts with the antibody. Taken together with the cDNA array data, these findings suggest that R3 and R4 are present in greater abundance in the gestational membranes compared with R1 and R2, and as such we conclude that TRAIL is unlikely to be able to exert much effect on these tissues.

In this light, our finding that TRAIL causes no/minimal loss of cell viability or release of death/apoptosis markers in amnion and choriodecidua is not surprising. Under the conditions used for these experiments, TNF-{alpha} also had minimal effects on the viability parameters measured, although some evidence of cell death was observed in the explant studies. TNF-{alpha} was able to stimulate PGE2 production, however, confirming the responsiveness of the tissues. Numerous studies have described the proinflammatory effects of TNF-{alpha} in amnion and choriodecidual tissues, suggesting that in gestational membranes, activation and nuclear translocation of NF-{kappa}B would be the expected responses to this cytokine, accompanied by up-regulation of inflammation-associated genes such as PGHS-2 and matrix metalloproteases. As such, it would be unexpected for it to also be proapoptotic, since NF-{kappa}B activation is a potent inhibitor of caspase-mediated apoptosis in most systems described to date. In contrast to TNF-{alpha}, the ability of TRAIL to exert proinflammatory effects (i.e. increase PGE2 production) in explant culture was minimal, supporting the conclusion that these tissues are unresponsive to TRAIL.

In conclusion, we have identified tissues of the gestational membranes as being the source of both TRAIL and OPG. Both are present at detectable concentrations in AF, with changes observed according to gestational age or intraamniotic infection. We have found no evidence that TRAIL-mediated apoptosis might be associated with the mechanism of premature rupture of membranes or preterm labor. Indeed, to the contrary, it would appear that the expression of decoy receptors in addition to OPG protects these membranes from the actions of TRAIL. The physiological significance of TRAIL in the amniotic cavity remains to be established, as does the role of the TNF superfamily in the mechanism of membrane apoptosis and rupture.


   Acknowledgments
 
The assistance and dedication of the theater, nursing, and clinical staff at National Women’s Hospital (Auckland, New Zealand) are gratefully acknowledged.


   Footnotes
 
This work was supported by grants from the New Zealand Lottery Health Grants Board and the Health Research Council of New Zealand.

Abbreviations: AF, Amniotic fluid; BGG, bovine {gamma}-globulin; BrdU, bromodeoxyuridine; LDH, lactate dehydrogenase; MIAC, microbial invasion of the amniotic cavity; MTT, 3-(4,5-dimethlythiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NF-{kappa}B, nuclear factor-{kappa}B; OPG, osteoprotegerin; PGE2, prostaglandin E2; PPROM, preterm PROM; PROM, premature rupture of membranes; rh, recombinant human; TRAIL, TNF-related apoptosis-inducing ligand; TRAIL-R, TRAIL receptor.

Received October 31, 2002.

Accepted May 29, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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