Biol Reprod Track the topics, authors and articles important to you
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Koi, H.
Right arrow Articles by Parry, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Koi, H.
Right arrow Articles by Parry, S.
Agricola
Right arrow Articles by Koi, H.
Right arrow Articles by Parry, S.
Biology of Reproduction 64, 1001-1009 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Differential Expression of the Coxsackievirus and Adenovirus Receptor Regulates Adenovirus Infection of the Placenta1

Hideki Koia, Jian Zhanga, Antonis Makrigiannakisa, Spiro Getsiosb, Colin D. MacCalmanb, Gregory S. Kopfa, Jerome F. Strauss IIIa, and Samuel Parry2,a

a Center for Research on Reproduction and Women's Health, Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, Pennsylvania 19104 b Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, British Columbia, Canada V5Z4H4

ABSTRACT

The molecular mechanisms and pathologic significance of placental viral infections are poorly understood. We investigated factors that regulate placental infection by adenovirus, which is the most common viral pathogen identified in fetal samples from abnormal pregnancies (i.e., fetal growth restriction, oligohydramnios, and nonimmune fetal hydrops). We also determined the pathologic significance of placental adenovirus infection. Northern hybridization, flow cytometry, and immunostaining revealed that placental expression of the coxsackievirus and adenovirus receptor (CAR) varied with gestational age and trophoblast phenotype. The CAR was continuously expressed in invasive or extravillous trophoblast cells but not in villous trophoblast cells. We postulate that the villous syncytiotrophoblast, which does not express CAR and is resistant to adenovirus infection, limits the transplacental transmission of viral pathogens, including adenovirus. Conversely, extravillous trophoblast cells underwent apoptosis when infected by adenovirus in the presence of decidual lymphocytes (which simulated the maternal immune response to viral infection). Thus, adenovirus infection and/or the maternal immune response to adenovirus infection induced the death of placental cell types that expressed CAR. Consequently, we speculate that adenovirus infection of extra-villous trophoblast cells may negatively impact the process of placental invasion and predispose the mother and fetus to adverse reproductive outcomes that result from placental dysfunction.

adenovirus, apoptosis, placenta, pregnancy, trophoblast

INTRODUCTION

The placenta is a dynamic organ whose structure and function change throughout pregnancy. There is compelling evidence that the placenta plays an integral role in the vertical transmission of viruses, such as cytomegalovirus (CMV) and human immunodeficiency virus (HIV), from the mother to the fetus [1–3]. Although the sequelae of congenital viral infection (i.e., fetal anomalies, intrauterine fetal death, and persistent postnatal infection) may be devastating, very little is known about the passage of viruses through the placenta and the pathologic consequences of placental viral infection [4].

Development of the placenta is largely dependent upon the differentiation of trophoblast cells along two pathways [5, 6]. In one pathway, a subset of undifferentiated cytotrophoblast cells in anchoring placental villi invades maternal tissues and blood vessels within the decidua and myometrium. These intermediate, or extravillous, trophoblast cells engage in a dialogue with maternal cells and probably mediate suppression of the maternal immune system against the semiallogeneic conceptus [7, 8]. The inflammatory response to infection at the maternal-fetal interface has been postulated to abrogate normal placentation and predispose the mother and fetus to adverse reproductive outcomes, including miscarriage, fetal growth restriction, and pre-eclampsia [8–10]. In the other pathway of placental development, mononucleated cytotrophoblast cells within the placental villi differentiate into the multinucleated syncytiotrophoblast. As pregnancy progresses, cytotrophoblast cells become more sparse within the placental villi and the syncytiotrophoblast forms the only continuous layer separating the maternal intervillous space and the fetal capillary endothelium. Importantly, superficial breaks in this barrier do not appear to be significant factors in the vertical transmission of viruses, at least in the case of HIV [11].

We previously reported that the ability of adenovirus, herpes simplex virus, and adeno-associated virus to infect placental trophoblast cells is related to the state of trophoblast differentiation [12, 13]. Infection of villous trophoblast cells by adenovirus and herpes simplex virus is reduced secondary to decreased viral entry as the cells differentiate into syncytiotrophoblast [12, 13]. Based on these findings, we postulated that the syncytiotrophoblast is relatively resistant to viral infection. Resistance to viral entry would yield reproductive advantages, particularly because the syncytiotrophoblast has been shown to suppress other defenses against infection, including local T-cell activity [14]. Furthermore, because viral entry into host cells generally is mediated by the presence or absence of host cell receptors, we hypothesized that viral infection of trophoblast cells and resultant pathologic sequelae are determined by the differential expression of viral receptors.

Adenovirus is the most common viral pathogen identified in fetal samples obtained from abnormal pregnancies [15, 16]. In one series, adenovirus was detected in the amniotic fluid of only 2% (3 of 154) of controls [16]. However, nonimmune fetal hydrops was associated with a high incidence of viral infection (50 of 91 cases), and adenovirus was detected in the amniotic fluid from 60% of these patients [16]. Additionally, adenovirus was detected in the amniotic fluid of 30% (10 of 33) of women whose pregnancies were complicated by conditions usually attributed to abnormal placental function (i.e., fetal growth restriction, oligohydramnios) [16]. Thus, we believe that viral infection, particularly adenovirus infection, is a previously unrecognized factor in adverse obstetric outcomes.

Adenovirus entry into target cells involves attachment of the knob domain of the viral fiber protein to the recently identified coxsackievirus B and adenovirus receptor (CAR), and internalization of the virus is mediated by the binding of its penton base with {alpha}vß3 and {alpha}vß5 integrins [17–19]. Accordingly, the expression of CAR and {alpha}vß3 and {alpha}vß5 integrins is strongly correlated with host cell infectivity/resistance to adenovirus [18, 20, 21]. The integrins {alpha}vß3 and {alpha}vß5 are known to be expressed in undifferentiated and differentiated trophoblast [22, 23]. Therefore, we focused our investigation on the placental expression of CAR.

Here we present data demonstrating that the expression of CAR on trophoblast cells varies with gestational age and trophoblast phenotype. In the third trimester, the syncytiotrophoblast does not express CAR and is resistant to adenovirus entry. However, CAR appears to be a functional receptor that is continuously expressed on extravillous trophoblast cells, and treatment of these cells with adenovirus and decidual lymphocytes (to simulate the maternal immune response) induces apoptosis. Consequently, we believe that these observations suggest a mechanism by which adenoviral infection of the placenta predisposes the mother and fetus to adverse reproductive outcomes.

MATERIALS AND METHODS

Cell Preparation and Culture

Cytotrophoblast cells were isolated from third trimester human placentas using a modified protocol originally described by Kliman et al. [13, 24]. After isolation, the cells were cultured at 37°C in Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated fetal bovine serum. The cytotrophoblast cells spontaneously differentiated into syncytiotrophoblast within 48–72 h in these conditions [13, 25].

Extravillous trophoblast cells were isolated and propagated from first trimester placental tissues (8–13 wk gestation) [26]. The extravillous trophoblast cells used in these experiments were characterized by cytokeratin immunostaining (cytokeratin filaments 8 and 18) and exhibited immunoreactivity for these intermediate filament proteins [27]. The extravillous trophoblast cells were cultured in DMEM as described above.

Maternal decidual lymphocytes were isolated from term decidua using a modified protocol originally described by King et al. [28]. The purified lymphocytes were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum. Nonadherent cells were collected from the culture medium and used for experiments.

Transformed human choriocarcinoma (BeWo, JEG-3) cells and human cervical carcinoma (HeLa cells) were cultured in DMEM supplemented with 10% fetal bovine serum. Chinese hamster ovary (CHO-K1) cells were cultured in Ham F12 medium containing 10% fetal bovine serum. HeLa cells express CAR protein, while CAR is absent on the surface of CHO-K1 cells [17]. The CHO-K1 cells were transfected with CAR cDNA (CHO-CAR cells) inserted in a PCR3 expression vector that was complexed with a nonliposomal lipid reagent (FuGENE 6; Roche Molecular Biochemicals, Indianapolis, IN).

Northern Blotting

Total RNA was extracted from trophoblast cells with an RNeasy mini kit (Qiagen, Valencia, CA), separated by electrophoresis on 1% agarose gels (10 µg of RNA per lane), and transferred by standard Northern blot procedures to Hybond-N+ nylon membranes (Amersham, Piscataway, NJ). The blots were probed with a 448-bp cDNA fragment encoding a portion of human CAR (GenBank accession number Y07593) that was excised from full-length CAR cDNA in pcDNA1 plasmid (Invitrogen, Carlsbad, CA) by digestion with Pvu2. The plasmid was generously provided by J.M. Bergelson (University of Pennsylvania) [17]. Other membranes, containing 20 µg of poly(A)+ RNA isolated from villous tissue and decidual tissue at 8 wk gestation, were provided by Y. Mastumoto (Tokyo Medical and Dental University) and probed with the CAR cDNA fragment.

Indirect Immunofluorescence Assays

Trophoblast cells, HeLa cells, CHO-K1 cells, and CHO-CAR cells were treated with normal goat serum to block nonspecific binding, after which the cells were incubated with murine monoclonal antibody against human CAR (RmcB ascites provided by J.M. Bergelson, diluted 1:250 in PBS) for 1 h on ice [17]. The cells (at least 4 x 104 cells for each experiment) were then incubated with fluorescein-conjugated goat antibody to mouse immunoglobulin (diluted 1:500), and fluorescence intensity was measured using an Epics XL flow cytometer (Coulter Corporation, Hialeah, FL).

Immunohistochemistry

Permanent paraffin sections from first and third trimester placentas were deparaffinized and stained using avidin and biotinylated horseradish peroxidase complex (Vectastain ABC kit; Vector Laboratories, Burlingame, CA). After blocking nonspecific binding with normal goat serum, sections were incubated overnight at 4°C with RmcB (diluted 1:250) or monoclonal antibody against human cytokeratin peptide 18 (clone CY-90; Sigma, St. Louis, MO; diluted 1;rc400). Reaction product was developed with diaminobenzidine tetrahydrochloride, and sections were counterstained with methyl green solution. Negative controls for nonspecific binding were performed using secondary antibody without RmcB or anti-cytokeratin 18 antibody.

Binding Assays

We performed binding assays to correlate CAR expression with binding of adenovirus fiber protein to trophoblast cells. Recombinant fiber protein was produced in competent Escherichia coli that were transfected with an expression vector, pHisC5f-A3, containing a gene encoding the knob domain and a distal portion of the fiber domain of adenovirus type 5 (a gift from J. Hong, University of Alabama at Birmingham). The recombinant fiber protein (amino acids 322–581) was expressed in conjunction with an N-terminal HisX6 tag.

Radioiodination of adenovirus fiber protein was carried out by incubating 100 µg of purified fiber protein with 1 mCi 125I (Amersham; IMS30; 3000 Ci/mmol) for 5 min at room temperature in the presence of an iodination reagent (Iodobeads; Pierce, Rockford, IL). The mixture was then subjected to chromatography at 4°C on a 3-ml EconoColumn (Bio-Rad, Hercules, CA) of Bio-Gel P10 (medium pore size; Bio-Rad) that had been equilibrated with PBS. Chromatography was performed to remove the unincorporated 125I. Fractions (0.5 ml) were collected, and the void volume fractions containing the iodinated protein were collected and pooled. The specific activity of the peak fractions (4695 cpm/ng) was calculated using an LS 5000 CE (Beckman, Fullerton, CA) scintillation counter.

Primary mononuclear trophoblast cells were cultured on 24-well plates to 70% confluence and washed twice with DMEM. Cells in two wells were trypsinized before syncytialization occurred and were counted with a hemocytometer to determine the total number of cells per well. Cells in the other wells were then cultured for 0–48 h before treatment with iodinated fiber protein. The cells were incubated at room temperature for 1 h with 0.25 ml of 125I-labeled fiber solutions (0–10 nM) diluted with DMEM containing 1% fetal bovine serum. After washing three times with DMEM, the cells were solubilized in 0.25 ml of 1 M sodium hydroxide at room temperature for 20 min. Binding of the iodinated adenovirus fiber to trophoblast cells was quantified using a scintillation counter. Nonspecific binding was determined by pretreating cells with a 200-fold excess of cold ligand and was subtracted from the total binding at each concentration to determine specific binding.

Competition Assays

Competition assays were performed using adenovirus fiber protein to block CAR before infecting trophoblast cells with a recombinant adenovirus vector (Ad.CMVlacZ) [12]. Primary trophoblast cells were incubated with adenovirus fiber protein (0.1–10 µg/ml of culture medium, or 3.3–330 nM) at room temperature for 30 min. The Ad.CMVlacZ (100 PFU/cell) was added to the solution and incubated at 37°C for 2 h. After rinsing with DMEM, 1 ml of culture medium was added to each well. The cells were cultured overnight at 37°C and assessed for ß-galactosidase enzyme activity that was reported as absorbance at 420 nm ± SD. The results of cells pretreated with fiber protein were compared to untreated cells for statistical significance by two-tailed t-tests with Bonferroni corrections for multiple comparisons (one-way ANOVA). Each experiment was performed at least three times and included cells that were not pretreated with adenovirus fiber as controls.

Terminal Deoxynucleotidyl Transferase-Mediated Deoxyuridine Triphosphate Marker Nick End-Labeling Assay

Extravillous trophoblast cells were infected with wild-type adenovirus (100–1000 particles/cell) and incubated at 37°C for 2 h. The cells were washed with fresh DMEM and cultured for 24 h, at which time decidual lymphocytes were added in a 1:10 ratio to the extravillous trophoblast cells. Decidual lymphocytes were used to simulate the maternal immune response to viral infection in utero. After overnight coculture, the cells were fixed and apoptosis was detected in the extravillous trophoblast cells by 3' end-labeling of DNA fragments (Apop-Tag detection kit; Oncor, Gaithersburg, MD). Nuclei were counterstained with propidium iodide, and the number of TUNEL-positive trophoblast nuclei were counted blindly in random-selected high-power fields (x60). More than 1000 trophoblast cells were counted on each coverslip, and each experiment was performed three times. The morphology of all cells that were detected by fluorescent microscopy was reviewed using phase-contrast microscopy. The morphology of the larger extravillous trophoblast cells was easily distinguished from decidual lymphocytes. The results of the TUNEL assays were reported as percent positive cells and analyzed for statistical significance by {chi}2 tests.

RESULTS

Messenger RNA for CAR Was Expressed in Extravillous Trophoblast Cells and Choriocarcinoma Cells

Northern blot analysis was performed to determine the pattern of expression of CAR mRNA in human trophoblast cells (Fig. 1A). Different bands corresponding to CAR mRNA ranged in size from 1.2 kilobases (kb) to 6 kb, as previously reported by Tomko et al. [18]. HeLa cells were used as positive controls and CHO-K1 cells as negative controls for CAR RNA [17]. Extravillous trophoblast cells (EVT, Fig. 1A) expressed CAR transcripts at high levels, but villous trophoblast cells (VT, Fig. 1A) isolated from third trimester placentas and cultured for 12 h (cytotrophoblast cells) and 60 h (terminally differentiated syncytiotrophoblast) did not. In villous trophoblast cells, weak hybridization signals were detected at approximately 1.0 kb, which may suggest a CAR-related transcript. The CAR mRNA was also detected in transformed human choriocarcinoma cells (BeWo and JEG-3, Fig. 1A) that share properties with extravillous trophoblast cells. Analysis of poly(A)+ RNA from first trimester placentas demonstrated weak CAR expression in villous tissue (Villi, Fig. 1C), but substantial levels of CAR mRNA were observed in decidual tissue (Decidua, Fig. 1C). The villous and decidual tissue likely contained numerous cell types, including stromal cells, blood cells, and maternal decidual cells. Hence, the positive results obtained here do not verify expression of CAR in villous and extravillous trophoblast cells during the first trimester.



View larger version (98K):
[in this window]
[in a new window]
  		FIG. 1. Differential expression of CAR RNA transcripts in trophoblast cells and other cell types. A) Northern blot of total RNA from each of the indicated cell types was probed with a cDNA fragment encoding a portion of human CAR. EVT, Extravillous trophoblast cells; VT (12h), primary villous trophoblast cells isolated from term placentas and cultured for 12 h (primarily cytotrophoblast cells); VT (60h), primary villous trophoblast cells isolated from term placentas and cultured for 60 h (terminally differentiated syncytiotrophoblast). B) Hybridization of the same Northern blot with a probe for the 28S ribosomal subunit confirmed the presence of equivalent amounts of RNA in each lane. C) Northern blot containing poly(A)+ RNA from decidual tissue (Decidua) and villous tissue (Villi) at 8 wk gestation was hybridized with the CAR cDNA fragment

CAR Protein Was Expressed in Extravillous Trophoblast Cells and Choriocarcinoma Cells

To determine the levels of human CAR protein on trophoblastic cells, flow cytometric analyses of primary trophoblast cells and choriocarcinoma cells were performed (Fig. 2). HeLa cells, on which CAR was originally identified, were used as positive controls. In four separate experiments, there was a positive shift in binding of monoclonal antibody against human CAR to HeLa cells, CHO-K1 cells that were transfected with CAR cDNA (CHO-CAR cells), extravillous trophoblast cells, JEG-3 cells, and BeWo cells. However, CAR protein was not detected on CHO-K1 cells (Fig. 2B) or isolated third trimester villous trophoblast cells after 12 h and 60 h of culture (Fig. 2, E and F).



View larger version (41K):
[in this window]
[in a new window]
  		FIG. 2. Flow-cytometric analysis of human CAR expression on trophoblast cell populations. Fluorescence intensity was measured in cells that were incubated with monoclonal antibody against CAR and fluorescein-conjugated secondary antibody (shaded curves) and in cells that were incubated with fluorescein-conjugated secondary antibody alone (open curves). The Y-axis indicates cell number, and the X-axis indicates logarithm of fluorescence intensity. A) HeLa cells, B) CHO-K1 cells, C) CAR-transfected CHO-K1 cells (CHO-CAR), D) extravillous trophoblast cells (EVT) from first trimester placentas, E) villous trophoblast cells from third trimester placentas after 12 h of culture (VT [12h]), F) villous trophoblast cells from third trimester placentas after 60 h of culture (VT [60h]), G) BeWo cells, and H) JEG-3 cells

CAR Protein Was Localized to First Trimester Villous Trophoblast and Extravillous Trophoblast at All Stages of Gestation

In order to localize CAR antigen within human placental tissue, immunohistochemical staining was performed using a monoclonal antibody against human CAR protein (Fig. 3). In first trimester placentas, CAR immunostaining was detected on villous cytotrophoblast cells and at lower levels within the syncytiotrophoblast (Fig. 3A). Extravillous trophoblast cells that invade the uterine wall and maternal vasculature stained positively for CAR. In third trimester placentas, villous cytotrophoblast cells and syncytiotrophoblast were negative for CAR (Fig. 3B). However, extravillous trophoblast cells continued to express CAR protein. In consecutive sections, cells stained with trophoblast-specific anti-cytokeratin-18 antibody were identified as trophoblast cells (Fig. 3C).



View larger version (125K):
[in this window]
[in a new window]
  		FIG. 3. Immunohistochemical staining for CAR within placental tissue. A) Villous trophoblast cells (short arrow) and extravillous trophoblast cells (long arrow) stained positive for CAR at 11 wk gestation. B) In third trimester placentas, CAR antigen was not detected in villous trophoblast cells but was identified in extravillous trophoblast cells. C) Immunostaining for cytokeratin-18 was performed in serial sections to identify trophoblast cells in third trimester placental tissue. D) Negative control (secondary antibody used alone), third trimester placenta

Adenovirus Fiber Protein Mediated Adenovirus Attachment to Extravillous Trophoblast Cells but Not Term Villous Trophoblast Cells

Binding assays and competition assays using recombinant adenovirus fiber protein were conducted to determine if adenovirus infection of trophoblast cells is governed by attachment of the adenovirus fiber to its receptor (presumably CAR). Binding studies using radiolabeled adenovirus fiber protein revealed the expression of a specific receptor for fiber protein on HeLa cells, CHO-CAR cells, extravillous trophoblast cells, and JEG-3 cells similar to that previously reported for CAR protein (Fig. 4 and Table 1) [29, 30]. However, specific binding of radiolabeled fiber protein to villous cytotrophoblast cells (cultured for 12 h before binding study) was not detected (Fig. 4D). As expected, specific binding of radiolabeled fiber protein to villous syncytiotrophoblast (cultured for 60 h before binding study) was also not observed (Fig. 4E).



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 4. Analysis of adenovirus fiber binding to trophoblast cells. Data points are the averages for duplicate wells. Nonspecific binding (open squares) was subtracted from the total binding (open circles) to determine specific binding (shaded diamonds). A) HeLa cells, B) CAR-transfected CHO-K1 cells (CHO-CAR), C) extravillous trophoblast cells (EVT), and F) JEG-3 cells demonstrated specific binding to adenovirus fiber protein. D, E) Villous trophoblast cells from third trimester placentas did not bind specifically to adenovirus fiber protein


View this table:
[in this window]
[in a new window]
  		TABLE 1. The concentration of adenovirus fiber binding sites and equilibrium dissociation constants (Kd) on trophoblast and control cells

In competition experiments, the recombinant adenovirus vector, Ad.CMVlacZ, efficiently transduced extravillous and villous cytotrophoblast cells and HeLa cells (that served as positive controls), but pretreatment with recombinant adenovirus fiber protein significantly reduced transduction of HeLa cells, CHO-CAR cells, extravillous trophoblast cells, BeWo cells, and JEG-3 cells in a dose-dependent manner (Fig. 5). Maximum competition, which was characterized as minimum transduction, was observed at a fiber protein concentration of 10 µg/ml of culture medium. Mean ß-galactosidase activities in extravillous trophoblast cells were significantly reduced to 22.4% at 0.1 µg fiber protein/ml, 8.8% at 1 µg/ml, and 1.3% at 10 µg/ml, compared to activity without pretreatment (Fig. 5C). In contrast, pretreatment with adenovirus fiber protein did not inhibit adenovirus transduction of villous cytotrophoblast cells isolated from term placentas (Fig. 5D). Competition experiments using syncytialized trophoblast cells were not performed because these cells are resistant to adenovirus entry and cannot be transduced [12]. Because specific binding of adenovirus fiber protein to villous trophoblast cells was not detected, but recombinant adenovirus efficiently transduced villous cytotrophoblast cells, fiber-independent mechanisms for adenovirus attachment to villous cytotrophoblast cells must exist.



View larger version (42K):
[in this window]
[in a new window]
  		FIG. 5. Pretreatment with adenovirus fiber protein inhibited adenovirus infection of trophoblast cells. HeLa cells, CAR-transfected CHO-K1 cells (CHO-CAR), and trophoblast cells were pretreated with fiber protein (0 to 10 µg/ml of culture medium) and exposed to Ad.CMVlacZ vector. Adenovirus entry was assessed by measuring ß-galactosidase activity in the cells. Pretreatment with fiber protein significantly reduced the transduction efficiency in A) HeLa cells, B) CHO-CAR cells, C) extravillous trophoblast cells (EVT), E) BeWo cells, and F) JEG-3 cells, compared to untreated cells. Pretreatment with fiber protein did not inhibit adenovirus transduction of D) villous cytotrophoblast cells (VT [12 h]) isolated from term placentas. Data are reported as mean ± SD and the asterisk (*) indicates significant differences in ß-galactosidase activities compared to untreated cells (P < 0.05)

Adenovirus Infection Induced Apoptosis in Extravillous Trophoblast Cells in the Presence of Decidual Lymphocytes

Because extravillous trophoblast cells express CAR protein and can be efficiently transduced by recombinant adenovirus vectors at all stages of gestation, we sought to describe the cellular pathology associated with wild-type adenovirus infection and the maternal immune response. Infection with wild-type adenovirus alone or coculture of trophoblast cells with decidual lymphocytes (in the absence of adenovirus infection) did not induce cytopathic effects. However, we observed that adenovirus infection in the presence of maternal decidual lymphocytes resulted in the detachment of nonviable trophoblast cells from the culture plates. Trophoblast cells were approximately 80% confluent before treatment with adenovirus and/or decidual lymphocytes, but trophoblast cells were only 30–50% confluent after the detached cells were rinsed away. More specifically, we investigated whether adenovirus infection induced apoptosis of extravillous trophoblast cells that remained attached to the culture plates (Fig. 6, A and B). Compared to background levels of apoptosis observed in untreated extravillous trophoblast cells (<0.10% of cells observed with apoptotic nuclei), adenovirus infection of extravillous trophoblast cells (<0.10% to 0.50%) and cocultures of extravillous trophoblast cells with decidual lymphocytes (0.67%) were not associated with significantly increased levels of apoptosis (Fig. 6C). However, cocultures of extravillous trophoblast cells with decidual lymphocytes after incubation with wild-type adenovirus demonstrated significantly increased numbers of apoptotic nuclei (2.36% to 3.53%, P < 0.01).



View larger version (64K):
[in this window]
[in a new window]
  		FIG. 6. Apoptosis was induced in extravillous trophoblast cells by wild-type adenovirus in the presence of decidual lymphocytes. A) Phase-contrast image of extravillous trophoblast cells. B) Apoptotic trophoblast cells (arrow) were detected by labeling fragmented nuclear DNA (TUNEL method). C) The rate of apoptotic extravillous trophoblast cells is illustrated for each condition (± wild-type adenovirus infection [100–1000 viral particles/cell] in the presence or absence of decidual lymphocytes). The asterisk (*) indicates significant differences in the rates of apoptotic cells compared to negative controls (P < 0.01). For each condition, more than 3000 cells were counted in random high-powered fields. Original magnification x100 (A and B).

DISCUSSION

Our findings demonstrate that CAR is expressed at all stages of gestation by extravillous trophoblast cells, to which adenovirus attachment is fiber-mediated and adenovirus infection (together with the immune response to infection) induces programmed cell death (Table 2). Villous cytotrophoblast cells do not express CAR beyond the first trimester of pregnancy and adenovirus attachment to these cells is fiber independent. The villous syncytiotrophoblast also does not express CAR beyond the first trimester of pregnancy, and adenovirus does not efficiently infect these cells. Hence, the terminally differentiated syncytiotrophoblast may function as a barrier to transplacental adenovirus infection. However, earlier in gestation, the placenta, particularly invasive extravillous trophoblast cells, are susceptible to pathologic events induced by adenovirus infection.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Summary of findings that describe the presence (+) or absence (-) of CAR RNA transcripts and protein in trophoblast cells at various stages of differentiation; additionally, the ability of adenovirus to transduce trophoblast cells and to bind to these cells in a fiber-dependent fashion is listed

The CAR protein and RNA are not present in villous trophoblast cells isolated from third trimester placentas, although primary villous cytotrophoblast cells from these placentas are efficiently transduced by adenovirus vectors [12]. Additionally, preincubation with adenovirus fiber protein does not inhibit the transduction of cytotrophoblast cells by adenovirus vectors, and specific binding of adenovirus fiber protein to villous cytotrophoblast cells is not observed. Consequently, we speculate that an alternate, fiber-independent mechanism for adenovirus attachment to villous cytotrophoblastic cells exists, or that viral internalization subsequent to low-affinity binding is reduced as villous trophoblast cells syncytialize. Regarding the first possibility, alternate receptors for adenovirus have been identified. For example, the {alpha}2 domain of the heavy chain of human major histocompatibility complex (MHC) class 1 molecules has been reported to be an alternate receptor for adenovirus. However, binding to the {alpha}2 domain is a fiber-dependent process, and villous trophoblast cells do not express classic MHC 1 or 2 molecules [31–33]. Instead, trophoblast cells express a novel MHC molecule, HLA-G, the expression of which may be down-regulated by viral infection [10, 34–36]. Therefore, attachment of adenovirus to villous cytotrophoblast cells via the {alpha}2 domain is not likely. Other fiber-independent receptors for adenovirus have been proposed but have not been studied in trophoblast cells [37, 38]. Regarding the second possibility, we observed significant levels of nonspecific binding by fiber protein to villous cytotrophoblast and syncytiotrophoblast. This nonspecific binding reflects low-affinity binding to high capacity sites that are not likely to be viral receptors in the traditional sense. However, it is possible that internalization of virus subsequent to low-affinity, high-capacity binding of fiber protein is reduced as trophoblast cells syncytialize, accounting for their resistance to infection.

Adenovirus is the most common viral pathogen identified in sample materials (amniotic fluid, fetal blood, fetal ascites, pleural effusion, and tissue samples) obtained from abnormal pregnancies, but it is uncommonly found in amniotic fluid from normal pregnancies [15, 16]. Our results demonstrate that CAR is expressed on villous and extravillous trophoblast in the first trimester of pregnancy. Therefore, it is possible that maternal adenovirus infection early in gestation may precipitate trophoblast infection, transplacental passage of adenovirus, and latent fetal infection.

The cellular consequences of viral infection vary depending on the different viruses and target cell types. Many viruses, including adenovirus, have developed strategies to avoid apoptosis of target cells, because host cell death may limit the spread of viral infection [39]. In our study, however, adenovirus infection in conjunction with the maternal immune response to adenovirus infection induced cellular detachment and apoptosis of extravillous trophoblast cells in vitro. We believe that our experimental design approximated the intrauterine milieu and demonstrated convincingly that both adenovirus infection and the maternal immune response to infection (decidual lymphocytes) were necessary to induce apoptotic changes. In previous reports, increased numbers of apoptotic trophoblast cells have been associated with placental aging and fetal growth restriction [40, 41]. Quantification of apoptosis by techniques similar to ours yield similar percentages of apoptotic cells: 0.07% of normal first trimester placental cells, 0.14% of normal third trimester placental cells, and 0.24% of third trimester placental cells in gestations complicated by fetal growth restriction [40, 41]. Thus, apoptosis of extravillous trophoblast cells secondary to adenovirus infection may accelerate placental aging and limit extravillous trophoblast function (invasion of the uterine wall), predisposing to obstetric complications that result from placental dysfunction, including pre-eclampsia, fetal growth restriction, and fetal loss. In fact, a similar hypothesis was recently proposed by Fisher et al. [2], who demonstrated that cytomegalovirus infection limited extravillous trophoblast invasion into a gel matrix in their in vitro system.

The mechanisms by which human trophoblast cells influence vertical transmission of adenovirus and other viruses have not been well studied. A better understanding of the molecular basis of the interactions between viruses and trophoblast cells may yield novel strategies to prevent vertical transmission of viruses. The observations presented here confirm our initial hypothesis that differential expression of human CAR on trophoblast governs, in part, the susceptibility of the placenta to adenovirus infection. Although the syncytiotrophoblast may serve as an effective barrier to transplacental adenovirus transmission, placental infection with adenovirus early in gestation may result in fetal infection and other obstetric complications that result from placental dysfunction.

ACKNOWLEDGMENTS

We acknowledge Jeffrey M. Bergelson for his critical review of this manuscript.

FOOTNOTES

First decision: 6 June 2000.

1 S.P. was supported by a Women's Reproductive Health Research Award provided by the NICHD and by the Thomas B. McCabe and Jeannette E. Laws McCabe Fund at the University of Pennsylvania. J.F.S. was supported by the Bill and Melinda Gates Foundation. G.S.K. was supported by HD06274, HD22732, and HD33052. Back

2 Correspondence: Samuel Parry, 1352 Biomedical Research Building II/III, 421 Curie Boulevard, Philadelphia, PA 19104-6142. FAX: 215 573 5408; parry{at}mail.med.upenn.edu Back

Accepted: November 3, 2000.

Received: May 1, 2000.

REFERENCES

  1. Mühlemann K, Menegus MA, Miller RK. Cytomegalovirus in the perfused human term placenta in vitro. Placenta 1995; 16:367–373[CrossRef][Medline]
  2. Fisher S, Genbacev O, Maidji E, Pereira L. Human cytomegalovirus infection of placental cytotrophoblasts in vitro and in utero: implications for transmission and pathogenesis. J Virol 2000; 74:6808–6820[Abstract/Free Full Text]
  3. Douglas GC, King BF. Maternal-fetal transmission of human immunodeficiency virus: a review of possible routes and cellular mechanisms of infection. Clin Infect Dis 1992; 15:678–691[Medline]
  4. Kaplan C. The placenta and viral infections. Semin Diag Pathol 1993; 10:232–250
  5. Damsky CH, Fitzgerald ML, Fisher SJ. Distribution patterns of extracellular matrix components and adhesion receptors are intricately modulated during first trimester cytotrophoblast differentiation along the invasive pathway, in vivo. J Clin Invest 1992; 89:210–222
  6. Parry S, Holder J, Strauss JF III. Mechanisms of trophoblast-virus interaction. J Reprod Immunol 1997; 37:25–34[CrossRef][Medline]
  7. Pazmany L, Mandelboim O, Valés-Gómez M, Davis DM, Reyburn HT, Strominger JL. Protection from natural killer cell-mediated lysis by HLA-G expression on target cells. Science 1996; 274:792–795[Abstract/Free Full Text]
  8. King A, Loke YW. The influence of the maternal immune response on placentation in human subjects. Proc Nutr Soc 1999; 58:69–73[CrossRef][Medline]
  9. Redman CWG, Sacks GP, Sargent IL. Preeclampsia: an excessive maternal inflammatory response to pregnancy. Am J Obstet Gynecol 1999; 180:499–506[CrossRef][Medline]
  10. Schust DJ, Hill AB, Ploegh HL. Herpes simplex virus blocks intracellular transport of HLA-G in placentally derived cells. J Immunol 1996; 157:3375–3380[Abstract]
  11. Burton GJ, O'Shea S, Rostron T, Mullen JE, Aiyer S, Skepper JN, Smith R, Banatvala JE. Significance of placental damage in vertical transmission of human immunodeficiency virus. J Med Virol 1996; 50:237–243[CrossRef][Medline]
  12. MacCalman CD, Furth EE, Omigbodun A, Kozarsky KF, Coutifaris C, Strauss JF III. Transduction of human trophoblast cells by recombinant adenoviruses is differentiation dependent. Biol Reprod 1996; 54:682–691[Abstract]
  13. Parry S, Holder J, Halterman MW, Weitzman MD, Davis AR, Federoff H, Strauss JF III. Transduction of human trophoblastic cells by replication-deficient recombinant viral vectors. Am J Pathol 1998; 152:1521–1529[Abstract]
  14. Munn DH, Zhou M, Attwood JT, Bondarev I, Conway SJ, Marshall B, Brown C, Mellor AL. Prevention of allogeneic fetal rejection by tryptophan catabolism. Science 1998; 281:1191–1193[Abstract/Free Full Text]
  15. Wenstrom KD, Andrews WW, Bowles NE, Towbin JA, Hauth JC, Goldenberg RL. Intrauterine viral infection at the time of second trimester genetic amniocentesis. Obstet Gynecol 1998; 92:420–424[Abstract]
  16. Van den Veyver IB, Ni J, Bowles N, Carpenter RJ, Weiner CP, Yankowitz J, Moise KJ, Henderson J, Towbin JA. Detection of intrauterine viral infection using the polymerase chain reaction. Mol Genet Metab 1998; 63:85–95[CrossRef][Medline]
  17. Bergelson JM, Cunningham JA, Droguett G, Kurt-Jones EA, Krithivas A, Hong JS, Horwitz MS, Crowell RL, Finberg RW. Isolation of a common receptor for coxsackie B virus and adenoviruses 2 and 5. Science 1997; 275:1320–1323[Abstract/Free Full Text]
  18. Tomko RP, Xu R, Philipson L. HCAR and MCAR: the human and mouse cellular receptors for subgroup C adenoviruses and group B coxsackieviruses. Proc Natl Acad Sci U S A 1997; 94:3352–3356[Abstract/Free Full Text]
  19. Wickham TJ, Mathias P, Cheresh DA, Nemerow GR. Integrins {alpha}vß3 and {alpha}vß5 promote adenovirus internalization but not virus attachment. Cell 1993; 73:309–319[CrossRef][Medline]
  20. Huang S, Endo RI, Nemerow GR. Upregulation of integrins {alpha}vß3 and {alpha}vß5 on human monocytes and T lymphocytes facilitates adenovirus-mediated gene delivery. J Virol 1995; 69:2257–2263[Abstract]
  21. Delporte C, Redman RS, Baum BJ. Relationship between the cellular distribution of the avß3/5 integrins and adenoviral infection in salivary glands. Lab Invest 1997; 77:167–173[Medline]
  22. Vanderpuye OA, Labarrere CA, McIntyre JA. A vitronectin-receptor-related molecule in human placental brush border membranes. Biochem J 1991; 280:9–17
  23. Omigbodun A, Tessler C, Coukos G, Hoyer J, Coutifaris C. Human trophoblast adhesion to osteopontin is regulated by cell differentiation and divalent cations. J Soc Gynecol Invest 1995; 2:O126 (abstract)
  24. Kliman HJ, Nestler JE, Sermasi E, Sanger JM, Strauss JF III. Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae. Endocrinology 1986; 118:1567–1582[Abstract]
  25. Ringler GE, Strauss JF III. In vitro systems for the study of human placental endocrine function. Endocrinol Rev 1990; 11:105–123[Medline]
  26. Graham CH, Lysiak JJ, McCrae KR, Lala PK. Localization of transforming growth factor-ß at the human fetal-maternal interface: role in trophoblast growth and differentiation. Biol Reprod 1992; 46:561–572[Abstract]
  27. Neudeck H, Oei SL, Stiemer B, Hopp H, Graf R. Binding of antibodies against high and low molecular weight cytokeratin proteins in the human placenta with special reference to infarcts, proliferation and differentiation processes. Histochem J 1997; 29:419–430[CrossRef][Medline]
  28. King A, Birkby C, Loke YW. Early human decidual cells exhibit NK activity against the K562 cell line but not against first trimester trophoblast. Cell Immunol 1989; 118:337–344[CrossRef][Medline]
  29. Hsu KHL, Lonberg-Holm K, Alstein M, Crowell RL. Monoclonal antibody specific for the cellular receptor for the group B coxsackieviruses. J Virol 1998; 62:1647–1652
  30. Henry LJ, Xia D, Wilke ME, Deisenhofer J, Gerard RD. Characterization of the knob domain of the adenovirus type 5 fiber protein expressed in Escherichia coli. J Virol 1994; 68:5239–5246[Abstract/Free Full Text]
  31. Hong SS, Karayan L, Tournier J, Curiel DT, Boulanger PA. Adenovirus type 5 fiber knob binds to MHC class l {alpha}2 domain at the surface of human epithelial and B lymphoblastoid cells. EMBO J 1997; 16:2294–2306[CrossRef][Medline]
  32. Coady MA, Mandapati D, Arunachalam B, Jensen K, Maher SE, Bothwell AL, Hammond GL. Dominant negative suppression of major histocompatibility complex genes occurs in trophoblasts. Transplantation 1999; 67:1461–1467[CrossRef][Medline]
  33. Peyman JA. Repression of major histocompatibility complex genes by a human trophoblast ribonucleic acid. Biol Reprod 1999; 60:23–31[Abstract/Free Full Text]
  34. Chu W, Fant ME, Geraghty DE, Hunt JS. Soluble HLA-G in human placentas: synthesis in trophoblasts and interferon-gamma-activated macrophages but not placental fibroblasts. Hum Immunol 1998; 59:435–442[CrossRef][Medline]
  35. Rodriguez AM, Mallet V, Lenfant F, Arnaud J, Girr M, Urlinger S, Bensussan A, LeBouteiller P. Interferon-{gamma} rescues HLA class la cell surface expression in term villous trophoblast cells by inducing synthesis of TAP proteins. Eur J Immunol 1997; 27:45–54[Medline]
  36. Loke YW, King A, Burrows T, Gardner L, Bowen M, Hiby S, Howlett S, Jacobs D. Evaluation of trophoblast HLA-G antigen with a specific monoclonal antibody. Tissue Antigens 1997; 50:135–146[Medline]
  37. Huang S, Kamata T, Takada Y, Ruggeri ZM, Nemerow GR. Adenovirus interaction with distinct integrins mediates separate events in cell entry and gene delivery to hematopoietic cells. J Virol 1996; 70:4502–4508[Abstract]
  38. Hidaka C, Milano E, Leopold PL, Bergelson JM, Hackett NR, Finberg RW, Wickham TJ, Kovesdi I, Roelvink P, Crystal RG. CAR-dependent and CAR-independent pathways of adenovirus vector-mediated gene transfer and expression in human fibroblasts. J Clin Invest 1999; 103:579–587[Medline]
  39. Cuff S, Ruby J. Evasion of apoptosis by DNA viruses. Immunol Cell Biol 1996; 74:527–537[Medline]
  40. Smith SC, Baker PN, Symonds EM. Placental apoptosis in normal human pregnancy. Am J Obstet Gynecol 1997; 177:57–65[CrossRef][Medline]
  41. Smith SC, Baker PN, Symonds EM. Increased placental apoptosis in intrauterine growth restriction. Am J Obstet Gynecol 1997; 177:1395–1401[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Am. J. Pathol.Home page
F. Arechavaleta-Velasco, Y. Ma, J. Zhang, C. M. McGrath, and S. Parry
Adeno-Associated Virus-2 (AAV-2) Causes Trophoblast Dysfunction, and Placental AAV-2 Infection Is Associated with Preeclampsia
Am. J. Pathol., June 1, 2006; 168(6): 1951 - 1959.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
A. A. Dorner, F. Wegmann, S. Butz, K. Wolburg-Buchholz, H. Wolburg, A. Mack, I. Nasdala, B. August, J. Westermann, F. G. Rathjen, et al.
Coxsackievirus-adenovirus receptor (CAR) is essential for early embryonic cardiac development
J. Cell Sci., August 1, 2005; 118(15): 3509 - 3521.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
M. Terauchi, H. Koi, C. Hayano, N. Toyama-Sorimachi, H. Karasuyama, Y. Yamanashi, T. Aso, and M. Shirakata
Placental Extravillous Cytotrophoblasts Persistently Express Class I Major Histocompatibility Complex Molecules after Human Cytomegalovirus Infection
J. Virol., August 1, 2003; 77(15): 8187 - 8195.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Koi, H.
Right arrow Articles by Parry, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Koi, H.
Right arrow Articles by Parry, S.
Agricola
Right arrow Articles by Koi, H.
Right arrow Articles by Parry, S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS