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BOR - Papers in Press, published online ahead of print November 27, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.010058
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BIOLOGY OF REPRODUCTION 68, 1496–1504 (2003)
DOI: 10.1095/biolreprod.102.010058
© 2003 by the Society for the Study of Reproduction, Inc.

Immunology

B7 Family Molecules Are Favorably Positioned at the Human Maternal-Fetal Interface1

Margaret G. Petroff2,3, Lieping Chen4, Teresa A. Phillips3, Dagmar Azzola5, Peter Sedlmayr5, and Joan S. Hunt3

Department of Anatomy and Cell Biology,3 University of Kansas Medical Center, Kansas City, Kansas 66160 Department of Immunology,4 Mayo Graduate and Medical Schools, Mayo Clinic, Rochester, Minnesota 55905 Institute for Histology and Embryology,5 Karl-Franzens-University, A-8010 Graz, Austria


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 NOTE ADDED IN PROOF
 REFERENCES
 
The human placenta utilizes both active and passive mechanisms to evade rejection by the maternal immune system. We investigated the pattern of expression of the B7 family of immunomodulatory molecules B7-H1 (PD-L1), B7-2 (CD86), and B7-1 (CD80) at the term maternal-fetal interface. Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses showed that B7-H1 mRNA is abundant in term placenta and that cytotrophoblasts are sources of this message. Immunohistochemistry demonstrated that B7-H1 is constitutively expressed by the syncytiotrophoblast and by extravillous cytotrophoblasts, both of which are juxtaposed to maternal blood and tissue. By contrast, placental stromal cells, including macrophages, lacked the protein. Expression of B7-H1 protein was low in first-trimester placenta compared to second- and third-trimester tissue (P < 0.05) and was enhanced in cultured cytotrophoblasts by treatment with either interferon-{gamma} or epidermal growth factor (P < 0.05), suggesting that one or both of these mediators regulates B7-H1 expression in the placenta. RT-PCR and immunofluorescence analysis of term placental tissue revealed different patterns of expression of the immunostimulatory protein, B7-2. In contrast to B7-H1, B7-2 mRNA and protein were absent in cytotrophoblast cells but present in maternal macrophages and some fetal macrophages. The B7-1 mRNA and protein were absent at the maternal-fetal interface. These studies document expression of the B7 family proteins at the maternal-fetal interface and demonstrate that B7-H1 is positioned such that it could facilitate protection of fetal cells against activated maternal leukocytes. Conversely, B7-2 was absent on trophoblasts and was appropriately localized to fetal and maternal macrophages, which may participate in antigen presentation.

immunology, placenta, pregnancy, syncytiotrophoblast, trophoblast


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 NOTE ADDED IN PROOF
 REFERENCES
 
The semiallogeneic nature of the fetus in relation to its mother requires a defense system to ensure that the maternal immune system cannot mount an immune response against the baby. The placenta provides this protection through both active and passive means, with the principal player being the trophoblast. The trophoblast is the outermost cellular layer of the placenta, and it forms a continuous protective shell around the entire fetus. These are the only embryo-derived cells that continuously lie in direct contact with maternal blood. As such, they produce a variety of membrane-bound proteins and soluble products, such as members of the tumor necrosis factor superfamily, complement regulatory proteins, class Ib human leukocyte antigen (HLA) molecules, progesterone, and prostaglandins, which could serve to thwart potentially harmful effects of immune cells and their products [1–4].

Leukocyte activity is controlled by a balance of positive and negative signals. Whereas positive stimuli are required for initial priming and activation of the host defense mechanism, negative signals are of fundamental importance in ensuring termination of leukocyte activation. The B7 family molecules are transmembrane proteins belonging to the immunoglobulin (Ig) superfamily. Two of these molecules, B7-1 and B7-2 (also called CD80 and CD86, respectively), serve as ligands for CD28, which is constitutively expressed on most mature T cells [5]. Ligation of CD28 by B7-1 or B7-2 alone has little effect on T cells, but concomitant ligation of the T-cell receptor and CD28 provides the two requisite signals for efficient T-cell activation [6]. As a result, the mRNA for interleukin (IL)-2 and its receptor are upregulated and stabilized, antiapoptotic signals are generated, and T-cell survival and proliferation ensues. In contrast, engagement of the T-cell receptor without a costimulatory signal can lead to T-cell apoptosis or anergy [7]. A second receptor for B7-1 and B7-2 on T cells is cytotoxic lymphocyte antigen-4 (CTLA-4); engagement of this receptor provides an inhibitory signal that is imperative to the negative regulation of the immune system [8].

Recent studies have revealed several additional B7-related molecules [9–12]. The mRNA for two of these, B7-H1 (PD-L1) and B7-DC (PD-L2), are highly expressed in the human placenta [9, 12]. Both ligands were found to inhibit antigen- and anti-CD3-induced T-cell proliferation and cytokine secretion in vitro, probably through a common receptor, PD-1, which is expressed on activated T and B lymphocytes and myeloid cells [12–17]. Furthermore, mice lacking the functional PD-1 gene suffer severe autoimmune disorders targeted against organs that express the mRNA for PD-1 ligands, presumably because of the inability of these organs to sustain lymphocyte tolerance through these ligands [18–20]. Additionally, B7-H1 costimulates CD3-induced T-cell proliferation and preferentially induces secretion of several cytokines, including IL-10 [9, 21]. In addition, engagement of fully activated T-cells by B7-H1 leads to programmed cell death [22]. Therefore, B7-H1 may have diverse effects on the priming and effector phases of T cells; these effects may be mediated by a receptor other than PD-1.

We have mapped the B7-H1 protein to the first-trimester human placenta and found that its expression is restricted to villous and extravillous trophoblasts [23]. In the present study, we examined the expression of B7-H1 in first-, second-, and third-trimester placenta and extraembryonic membranes and investigated its possible means of regulation. Finally, we questioned whether the costimulatory molecules B7-1 and B7-2 are similarly distributed and whether the positioning of these molecules could give insight as to their possible functions in pregnancy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 NOTE ADDED IN PROOF
 REFERENCES
 
Tissue Specimens and Cell Lines

All tissues were obtained in accordance with protocols approved by the Human Subjects Committee at the University of Kansas Medical Center. Term placentas and fetal membranes were collected after cesarean sections, and first-trimester (5–8 wk of gestation, n = 4) and second-trimester (16–19 wk of gestation, n = 4) placentas were collected following elective pregnancy termination. Villous tissue from these samples was identified by floating of the samples in culture medium as well as by histological characterization. Tissues were either frozen in liquid nitrogen for subsequent extraction of RNA or embedded in TBS Tissue Freezing Medium (Triangle Biomedical Sciences, Durham, NC) for subsequent preparation of histological sections. All samples were stored at -80°C until further processing.

The choriocarcinoma cell lines JEG-3 and Jar were purchased from the American Type Culture Collection (Manassas, VA) and maintained at 37°C in growth medium consisting of RPMI-1640 (Sigma-Aldrich Co., St. Louis, MO) supplemented with 10% fetal bovine serum (Atlanta Biologicals, Norcross, GA), 1 mM sodium pyruvate, 50 µM 2-mercaptoethanol, and antibiotics (all from Sigma-Aldrich). For immunocytology, these cells were grown on Lab-Tek Chamber Slides (Nalge Nunc International, Naperville, IL) until confluent.

Villous Cytotrophoblast Purification and Culture

Term placentas were collected and placed on the dissecting board with the maternal side facing upward. Dissecting one cotyledon at a time, the overlying basal plate tissue was first removed and discarded to avoid contamination of the fetal tissue with maternal uterine tissue. Avoiding placental blood vessels and fibrous tissue, small pieces of placental villi were cut and placed into a sterile beaker until approximately 30 g of tissue were accumulated. After rinsing with PBS, the tissue was finely minced and dissociated as previously described [24]. The resulting enriched cytotrophoblasts were either processed for RNA extraction or subjected to immunopurification. Additionally, slides containing 50 000 cells were prepared by Cytospin centrifugation (Shandon, Inc., Pittsburgh, PA) for characterization by immunocytochemistry.

The enriched cells were subjected to a second stage of purification using magnetic microbead technology (Miltenyi Biotec, Auburn, CA) to deplete the HLA class I-positive cells from the HLA class I-negative cytotrophoblasts as previously described [25]. The purified cytotrophoblasts were then subjected to either RNA extraction or Cytospin preparation, or they were placed in culture. Immunopurified cytotrophoblasts were cultured in either eight-well chamber slides (150 000 cytotrophoblasts/well) or in 100-mm Petri dishes (9 x 106 cytotrophoblasts/plate). The cells were treated with medium alone (Iscove-modified Dulbecco modified Eagle medium containing 10% fetal bovine serum, 200 mM L-glutamine, 100 U/ml of penicillin, and 100 µg/ml of streptomycin) or medium containing either 5 ng/ml of epidermal growth factor (EGF; Peprotech, Rocky Hill, NJ) or 100 U/ml of recombinant human interferon (IFN)-{gamma} (R&D Systems, Minneapolis, MN). Medium was replenished daily for 6 days, at which time the cultures were harvested for immunocytochemistry or RNA or protein extraction.

Decidual Macrophage Purification

Decidual tissue from normal term pregnancies was collected after vaginal deliveries at Karl-Franzens-University in accordance with approved human subjects protocols. The CD14-positive decidual macrophages were isolated by flow cytometric sorting as previously described [26], shipped to the Kansas laboratory, and stored at -20°C until RNA was extracted.

RNA Extraction and Analysis

Reagents for the preparation and analysis of RNA were purchased from Life Technologies (Grand Island, NY) unless otherwise noted. The RNA was extracted from villous tissue and cell preparations using Trizol reagent according to the manufacturer's instructions. For extraction of RNA from decidual macrophages, 250 µg/ml of glycogen were added to the Trizol reagent to maximize yield.

The RNA (300 ng to 1 µg) was reverse transcribed using Murine Moloney Leukemia Virus reverse transcriptase and oligo-dT12–18 primers in 20-µl reaction volumes. Four microliters of the reverse transcription (RT) reaction were then subjected to polymerase chain reaction (PCR) using Taq DNA polymerase. Primers for B7-2 and B7-H1 were designed using PrimerSelect software (DNASTAR, Madison, WI) and purchased from Gemini Biotech (Alachua, FL). Primer sequences were as follows: B7-1 forward, 5'-AAT TGT TGG CTT TCA CTT T-3'; B7-1 reverse, 5'-AGC GTC TTT TTC ATA CTT CA-3'; B7-2 forward, 5'-GGC AGG ACC AGG AAA AC-3'; B7-2 reverse, 5'-CAG CCA ATC AAA CAG ACA AG-3'; B7-H1 forward, 5'-ACG CAT TTA CTG TCA CGG TTC C-3'; and B7-H1 reverse, 5'-GAC TTC GGC CTT GGG GTA GC-3'. Primers for ß-actin were chosen from previously published sequences [27]. Polymerase chain reaction was carried out for 35 cycles at 53°C annealing temperature for B7-2, 45 cycles at 56°C annealing temperature for B7-H1, and 30 cycles at 58°C annealing temperature for ß-actin. Identities of amplicons were confirmed by dye terminator sequence analysis at the University of Kansas Medical Center Biotechnology Support Facility.

For Northern blot analysis, 10 µg of total RNA were electrophoresed on a 1.2% (w/v) agarose/6.12% (v/v) formaldehyde/20 mM 4-morpholinepropanesulfonic acid (MOPS) gel and transferred to a nylon membrane (Schleicher and Schuell, Keene, NH). Complementary DNA probes for B7-H1 [9] or ß-actin, prepared as a PCR product, were labeled with [{alpha}-32P]dCTP (ICN Pharmaceuticals, Costa Mesa, CA) using the RTS RadPrime DNA Labeling System (Life Technologies) to a specific activity of approximately 1 x 109 cpm/µg. Northern blots were prehybridized for at least 20 min in QuickHyb hybridization buffer (Stratagene, La Jolla, CA) and hybridized with 2 x 106 cpm of labeled B7-H1 or ß-actin cDNA per 1 ml hybridization buffer for 1 h at 68°C. This was followed by two washes in 2x SSC/0.1% SDS at room temperature and two washes in 0.1x SSC/0.1% SDS at 60°C (1x SSC: 0.15 M sodium chloride and 0.015 M sodium citrate). Blots were exposed to Hyperfilm (Amersham-Pharmacia, Piscataway, NJ) at -80°C.

Western Blot Analysis

Protein from placental tissue and cultured cells was collected by lysis in PBS containing detergents (1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) and protease inhibitors (100 µg/ml of phenylmethylsulfonic acid, 10 µg/ml of aprotinin, and 10 µg/ml of leupeptin). Protein concentration was determined using the DC Assay (Bio-Rad, Hercules, CA), and 25 µg of protein were electrophoresed under denaturing conditions on a vertical mini-gel apparatus (Bio-Rad). The protein was transferred to a supported nitrocellulose membrane (Schleicher & Schuell), blocked in 3% nonfat milk in Tris-buffered saline, and probed with either goat anti-human B7-H1 (1 µg/ml; R&D Systems) or rabbit anti-actin (1:5000; Sigma-Aldrich). In parallel, identical blots were incubated with goat or rabbit IgG to document the absence of nonspecific immunoglobulin binding. Incubations with primary antibodies were performed overnight at 4°C. After stringent washing and probing with the corresponding anti-goat or anti-rabbit horseradish peroxidase-labeled secondary antibody (Jackson ImmunoResearch, West Grove, PA), bound antibodies were detected using the enhanced chemiluminescent detection system (Pierce Biotechnology, Rockford, IL) and autoradiography. Densitometric intensities were obtained using Gel-Pro image analysis software (Media Cybernetics, Silver Spring, MD).

Immunohistology

For immunoperoxidase staining, 10-µm tissue sections were taken from frozen term placentas using a cryostat. Cells grown on chamber slides were fixed in acetone for 7 min at -20°C (cell lines) or 1% paraformaldehyde in PBS for 20 min at room temperature (primary cultured cytotrophoblasts). Nonspecific immunoglobulin binding was blocked in 10% normal horse serum. Primary antibody (anti-B7-H1, clone 2H1 [22]) or its isotype-specific control (IgG1, 6.7 µg/ml; Pharmingen, San Diego, CA) was incubated with the tissue sections or cells for 1 h at room temperature. After the addition of secondary antibody (biotinylated horse anti-mouse IgG; Vector Laboratories, Burlingame, CA), the samples were depleted of endogenous peroxidases by incubation in 0.5% H202/methanol. Reactivity was detected using the streptavidin-peroxidase and AEC reagent sets (Zymed, San Francisco, CA). The tissues and cells were then lightly counterstained with hematoxylin, coverslipped, and viewed by light microscopy. Positive staining was detected as a red-brown coloration of the tissues and cells.

For immunofluorescence, frozen placental sections were fixed in acetone, and nonspecific binding was blocked in 10% human serum and 10% mouse serum for 1 h at room temperature. After removal of the blocking solution, phycoerythrin-conjugated anti-human B7-1, B7-2, CD14, or their isotype-specific controls (Pharmingen) were applied to the tissue sections at 1:5 dilutions and incubated at 37°C for 1 h. After rinsing the sections in PBS/0.3% Tween 20, the sections were coverslipped and viewed by fluorescence microscopy.

Statistical Analysis

Semiquantitative data from Western blots were obtained by taking ratios of densitometric intensities of B7-H1 and actin. Statistical analysis was performed using one-way (placental samples) or two-way (cultured cytotrophoblasts) analysis of variance with Student-Newman-Keuls multiple comparisons tests to detect differences between means. Cell culture experiments were blocked on sample preparation to account for sample-to-sample variation, and differences were considered to be statistically significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 NOTE ADDED IN PROOF
 REFERENCES
 
Trophoblast Cell Expression of B7-H1 in Second-Trimester and Term Placenta

We previously established that B7-H1 mRNA is present in the placenta of first-trimester pregnancy [23]. Figure 1A shows that B7-H1 mRNA is also present in total cellular RNA of three different term placentas. Using a full-length cDNA probe for B7-H1, we identified two transcripts with sizes of approximately 4.0 and 1.8 kilobases (kb).



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  		FIG. 1. Identification of B7-H1 mRNA in term placentas and cytotrophoblasts. A) Northern blot analysis of B7-H1 (top panel) and ß-actin (bottom panel) using total RNA of three different human term placentas. B) RT-PCR of RNA from freshly isolated human villous cytotrophoblasts using primers for B7-H1 (left panel) and Northern blot of total RNA from cultured cytotrophoblasts (cTB) for B7-H1 (right panel)

To determine whether term placental trophoblast cells are a source of B7-H1 mRNA, we purified villous cytotrophoblasts from term placenta and employed RT-PCR. As shown in Figure 1B, B7-H1 mRNA was detected in total RNA derived from these cells. Sequence analysis of the cloned RT-PCR product confirmed the identity of this transcript. Furthermore, Northern blot analysis of cultured term villous cytotrophoblast cells confirmed that these cells contain the 4.0-kb transcript of B7-H1 mRNA (Fig. 1B).

Having found that trophoblast cells are sources of B7-H1 mRNA within the placenta, we employed Western blot analysis and immunohistochemistry to determine whether the message is translated into protein. Using a polyclonal goat anti-human antibody generated against the extracellular domain of B7-H1, we detected a major band at approximately Mr 50 000 in protein extracts of first-, second-, and third-trimester placentas (Fig. 2A). These results confirm previous findings of B7-H1 protein in the first-trimester placenta, and they extend those previous findings to show that placental B7-H1 protein is expressed throughout pregnancy. Statistical analysis revealed that B7-H1 expression is higher in second- and third-trimester placentas in comparison to first-trimester tissue (P < 0.05) (Fig. 2B).



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  		FIG. 2. Immunodetection of B7-H1 protein in total protein extracts from first- and second-trimester and term placenta. A) Immunoblot analysis of B7-H1 (top panel) and actin (bottom panel) from placental tissue. B) Semiquantitative analysis of B7-H1 protein across gestation. Data are expressed as the mean of densitometric ratios ± SEM of four samples from each stage. Asterisks (*) denote significant differences (P < 0.05)

To map the expression of B7-H1 in the second-trimester and term placenta, we employed immunohistochemistry. Analysis of frozen term villous placenta and fetal membranes revealed that the B7-H1 protein is highly expressed by several populations of trophoblast cells. Similar to first-trimester placenta, the villous syncytiotrophoblast of second-trimester and term placenta, which are bathed in maternal blood throughout pregnancy, prominently expressed B7-H1 (Fig. 3, A and B). Whereas some cytoplasmic staining of the syncytiotrophoblast could be observed, B7-H1 immunoreactivity was often strongest at the microvillous membrane, which interfaces with the maternal blood. Additionally, B7-H1 immunoreactivity was detected on villous cytotrophoblasts and cell islands of second-trimester placenta (Fig. 3A). In contrast, positive staining could not be demonstrated in Cytospin preparations of term villous cytotrophoblasts (data not shown). Extravillous cytotrophoblasts within the basal plate (Fig. 3C) and chorion membrane (Fig. 3D) at term also bound the B7-H1 antibody. The B7-H1 antibody did not bind to other mesenchymal cells of the fetal villi (Fig. 3, A and B), whereas a low degree of staining was observed within some cells of the maternal decidua (Fig. 3D, arrows) and fetal connective tissue between the amnion epithelium and chorion membrane (Fig. 3D, arrowheads). The isotype-specific control antibody at the same concentration failed to stain any cells (Fig. 3, E–H).



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  		FIG. 3. Immunohistochemical localization of B7-H1 in placental villi, basal plate placenta, and fetal membranes. A) Second-trimester placental villi. Arrows and arrowheads demarcate location of villous cytotrophoblasts and syncytiotrophoblast, respectively; asterisks show locations of cytotrophoblast cell islands. B) Term placental villi. Arrowheads show location of syncytiotrophoblast. C) Basal plate placenta showing extravillous cytotrophoblasts. D) Fetal amnion and chorion membranes with associated maternal decidua. Arrows show positively staining maternal cells; arrowheads show positive fetal cells within the connective tissue (CT). E–H) Isotype-matched antibody controls for the B7-H1 antibody. cTB, Cytotrophoblast; MBS, maternal blood space. Original magnification x200 (A, E, B, F, D, H) and x400 (C, G)

Trophoblastic tumor cell lines can be utilized as models for normal trophoblasts, and our previous observations indicated that the choriocarcinoma cell line JEG-3 expresses B7-H1 mRNA [23]. We therefore wished to determine whether B7-H1 protein is expressed in JEG-3 and Jar cells. In agreement with the results of mRNA analyses of these lines [23], immunocytochemical analysis showed that JEG-3 cells expressed B7-H1, whereas Jar cells did not (data not shown).

Induction of B7-H1 Protein in Cultured Term Cytotrophoblasts by EGF and IFN-{gamma}

The low level of protein expression in term villous cytotrophoblasts suggested that B7-H1 expression may be regulated by exogenous factors in trophoblasts. We therefore used Western blot analysis and immunocytochemistry to determine whether B7-H1 expression could be modified by factors known to be associated with placental development. Both enriched (before HLA class I-positive cell depletion) or immunopurified cytotrophoblasts were cultured for 6 days in the presence of EGF or IFN-{gamma}. As expected [25], treatment of the purified trophoblasts with EGF resulted in the development of extensive areas of flat, multinucleated cells, suggesting that the cells had syncytialized in vitro. In contrast, control and IFN-{gamma}-treated purified trophoblasts, though clustered in small colonies, appeared to remain as single cells (data not shown). Cytotrophoblasts treated with either EGF or IFN-{gamma} exhibited enhanced expression of B7-H1 as detected by immunocytochemistry (Fig. 4, A–C). Western blot analysis revealed a 3- to 4-fold increase in B7-H1 expression as compared to untreated controls (P < 0.05) (Fig. 4, D and E). These results therefore establish a link between known modulators of placental function and expression of B7-H1.



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  		FIG. 4. Immunocytochemistry (A–C) and Western blot analysis (D) of B7-H1 in cytotrophoblasts cultured for 6 days. A–C represent an experiment in which enriched cytotrophoblasts were cultured in the presence or absence of treatments, and D represents an experiment in which purified cytotrophoblasts were cultured. A) Untreated cytotrophoblasts. B) EGF-treated cytotrophoblasts. C) IFN-{gamma}-treated cytotrophoblasts. D) B7-H1 (top panel) and ß-actin (bottom panel). E) Semiquantitative analysis of B7-H1 protein in cultured cytotrophoblasts. Data are expressed as the mean of densitometric ratios ± SEM of four experiments from different cytotrophoblast preparations. Asterisks (*) denote significant differences from untreated cytotrophoblasts (P < 0.05)

Identification of B7-2 mRNA in Term Placental mRNA

Because of the prominent expression of the immunoinhibitory B7 family protein B7-H1 in the placenta, we next asked whether the immunostimulatory molecules, B7-1 and B7-2, are also expressed at the maternal-fetal interface. B7-2 mRNA was not detectable in villous placental total cellular RNA by Northern blot analysis using a PCR-generated cDNA probe (results not shown). However, by using primers specific for B7-2, a product of the expected size (445 base pairs) was amplified from term villous RNA by RT-PCR (Fig. 5A). Sequence analysis confirmed that this amplicon was derived from B7-2 mRNA, and omission of the reverse transcriptase enzyme from the RT reaction demonstrated that the product was not the result of contaminating genomic DNA. In contrast, B7-1 mRNA (not shown) was undetectable in placental tissue by either Northern blot analysis or RT-PCR, even though it was readily identifiable in the positive control cell line, Raji.



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  		FIG. 5. RT-PCR analysis of B7-2 in term placenta (A), freshly isolated enriched and purified cytotrophoblasts (cTB) (B), and cell lines (C). Top panels, Ethidium bromide-stained B7-2 RT-PCR products; bottom panels, ß-actin RT-PCR products

To determine whether the B7-2 message was derived from cytotrophoblast cells or cells of mesenchymal origin, further analyses were performed on cytotrophoblasts at two different stages of the purification procedure, as described in Materials and Methods. Figure 5B shows that the first population of cytotrophoblasts, termed enriched cytotrophoblasts, contained B7-2 mRNA detectable by RT-PCR. This population is derived from cytotrophoblasts before the depletion of HLA class I-positive, non-cytotrophoblastic cells. However, highly purified cytotrophoblasts, examined after depletion of the HLA class I-positive cells, did not contain the message for B7-2 (Fig. 5B). Additionally, the trophoblast-derived cell lines JEG-3 and Jar both lacked detectable mRNA for B7-1 and B7-2 (Fig. 5C).

Expression of B7-2 Protein by Fetal Macrophages

Further studies using immunofluorescence histochemistry were performed to map the expression of the B7-2 protein. Figure 6A shows that, in agreement with the results of the mRNA analyses (Fig. 5B), the villous trophoblast layers lacked expression of the B7-2 protein. Additionally, only a few mesenchymal cells bound the phycoerythrin-conjugated anti-B7-2 antibody. The intensity of staining exhibited by these cells was low, and most cells showed no B7-2 immunoreactivity. Staining of an adjacent villous section with a similarly labeled antibody against the macrophage marker CD14 (Fig. 6B) established that the B7-2-positive cells were macrophages. It was also readily apparent that whereas a few fetal macrophages in the placenta expressed B7-2, most macrophages within the same placental tissue samples were B7-2 negative. Phycoerythrin-conjugated, isotype-matched control antibodies failed to stain any cells.



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  		FIG. 6. Immunofluorescence analysis of B7-2 and CD14 in adjacent sections of term placental villi (A–C), amnion (D–F), and amniochorion (G–I). A, D, and G) Staining for B7-2, showing a low degree of staining (arrow) and absence of staining (large arrowheads) in corresponding CD14-positive cells. B, E, and H) Small paired arrowheads mark the location of syncytiotrophoblast. C, F, and I) Phase-contrast images of corresponding tissue sections. CT, Fetal connective tissue. Original magnification x 100 (A–C and G–I) and x200 (D–F)

To determine whether fetal macrophages associated with the extraplacental membranes and extravillous cytotrophoblasts expressed B7-2, amnion and chorion membranes were examined for the presence of this protein in parallel with CD14. B7-2 was detectable on cells associated with the connective tissue between the amnion epithelium and chorion membranes (Fig. 6, D and G). Staining of an adjacent section with anti-CD14 antibody confirmed the identity of these cells as macrophages (Fig. 6, E and H). Analysis of chorion membrane demonstrated that chorion cytotrophoblast cells lack B7-2 protein (Fig. 6G).

Expression of B7-2 mRNA and Protein by Maternal Decidual Macrophages

Previous studies have demonstrated the expression of both immunostimulatory and immunoinhibitory B7 family molecules on human macrophages. We therefore mapped the expression of these molecules on maternal macrophages associated with the uterine decidua. Figures 3D and 6, G and H, show maternal decidua associated with chorion membrane. Within this tissue, high numbers of macrophages are identifiable by CD14 immunoreactivity in histological sections (Fig. 6H). Both B7-H1 and B7-2 could be identified in these cells (Figs. 3D and 6G). Consistent with these observations, RT-PCR analysis of mRNA from highly purified (>95%) preparations of decidual macrophages revealed that both B7-2 and B7-H1 mRNA are present in these cells (data not shown). In contrast, B7-1 message was not amplified from these samples by RT-PCR. Together, these results reveal that both B7-H1 and B7-2 are expressed by maternal macrophages at the maternal-fetal interface.

Figure 7 summarizes the results of protein expression of B7-H1 and B7-2 at the maternal-fetal interface, emphasizing the reciprocal nature of their localization. Whereas B7-2 expression was found only in maternal and fetal macrophages and not in trophoblasts, B7-H1 expression was especially prominent only in the fetal trophoblast subpopulations that are in direct contact with maternal blood and tissue.



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  		FIG. 7. Summary of B7-H1 and B7-2 protein expression at the maternal-fetal interface. "+" indicates the presence of the specified protein as determined by immunohistochemistry, whereas "-" indicates the protein's absence. "+/-" indicates low or absent expression of the protein. cTB, cytotrophoblasts; MBS, maternal blood space; M{phi}, macrophages; sTB, syncytiotrophoblast


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
NOTE ADDED IN PROOF
 REFERENCES
 
Recently, it has become increasingly apparent that maternal immune tolerance to the semiallogeneic fetus is achieved and maintained not only through a lack of expression of the highly polymorphic HLA molecules by fetal cytotrophoblasts but also by an abundance of immunoinhibitory signals. However, maintaining the potential for a beneficial immune response in case of infection of fetal tissue is also required. In the present study, we have demonstrated that two members of the B7 family, B7-2 and B7-H1, are differentially expressed at the maternal-fetal interface and are positioned therein as to retain this beneficial balance.

These studies definitively show that fetal cells in direct contact with maternal blood (villous syncytiotrophoblast) and tissue (extravillous chorionic cytotrophoblasts and basal plate cytotrophoblasts) are the major source of B7-H1 protein at the maternal-fetal interface throughout pregnancy. Because these are the only fetal cells that are exposed to maternal blood and tissues, the positioning of B7-H1 is consistent with a role for B7-H1 in the immunological protection of the fetus against the maternal immune system. Furthermore, placental B7-H1 is more abundant during the second and third trimesters as compared to the first trimester. Significantly, during the second trimester (13 wk of gestation and thereafter), the chorionic villi are first exposed to maternal blood [28], raising the possibility that exposure to maternal blood induces B7-H1 expression in the placenta. Additionally, these results suggest that the acquisition of placental B7-H1 correlates with the onset of exposure of fetal tissues to the potentially harmful maternal immune cells.

While investigating possible regulators of B7-H1 expression, we found that cytotrophoblast B7-H1 can be increased by EGF. The EGF stimulates term villous cytotrophoblasts to form a syncytium in vitro [25], and it may have a similar role in syncytialization in vivo [29]. It is possible that B7-H1 induction by EGF is secondary to differentiation of the cells and results only after induction and action of another regulator(s) of its expression. However, our preliminary data suggest that B7-H1 protein increases as early as 24 h after initiation of EGF treatment, suggesting that EGF upregulates B7-H1 expression either before or concomitantly with differentiation of the cells. Still, additional studies are required to dissect the mechanism of EGF in bringing about B7-H1 expression and to establish whether a direct link exists between EGF and B7-H1 gene expression.

We also found that prolonged IFN-{gamma} treatment increases B7-H1 expression in cultured cytotrophoblasts. Previously, we and others have found that IFN-{gamma} increases B7-H1 mRNA in JEG-3 cells and monocytes after as little as 4 h [23,12], suggesting a direct effect of IFN-{gamma} on B7-H1 gene expression. Despite its proinflammatory properties, this cytokine is present in placenta [30, 31] and may contribute to activation of maternal macrophages [30, 32]. Even in low concentrations, IFN-{gamma} can be immunosuppressive by inducing expression of immunoinhibitory molecules on cells [33, 34]. Induction of B7-H1 by IFN-{gamma} treatment in cytotrophoblasts therefore raises the possibility that one function of placental IFN-{gamma} may be to induce, by either direct or indirect mechanisms, immunomodulatory molecules such as B7-H1.

The function of B7-H1 expression in the placenta is as yet uncertain. While costimulating growth of primarily CD4+ T cells, B7-H1 may selectively promote differentiation of these cells to produce certain cytokines such as IL-10, suggesting a role in differentiation of Th2-type lymphocytes [9, 21]. Additionally, B7-H1 can inhibit proliferation of lymphocytes through cell cycle arrest, induction of apoptosis, or both [12, 22], and it has recently been implicated as playing a role in evasion of the immune system by tumors [22, 35]. A sister ligand of B7-H1 that also binds PD-1 [12, 36], B7-DC has also been identified in the placenta [12]. Mice with a deficiency in PD-1 and, thus, an inability to respond to either ligand undergo a breakdown in peripheral immunotolerance and, consequently, suffer autoimmune attack of organs that express these ligands [18, 19]. These data, together with our findings of the distribution of B7-H1 in the placenta, strongly suggest that placental B7-H1 promotes maternal immunotolerance of the fetus or maintains a favorable Th1/Th2 balance that is thought to be important in maintaining pregnancy. Development of gene-targeted mice that lack functional genes encoding B7-H1 and/or B7-DC will be key in determining the role of these ligands in the placenta.

In the present study, we found a lack of B7-1 expression at the maternal-fetal interface. However, B7-2 was expressed by both maternal macrophages and some fetal macrophages. This is consistent with known patterns of B7 expression in other tissues; B7-2 is constitutively expressed on macrophages of many tissues as well as their blood monocyte precursors [37–41]. Consistent with this pattern of expression is B7-2's proposed role in the initiation of a T-cell response [6]. Another source of B7-2 may be the decidual cells themselves; recent studies suggest that these cells express B7-2 and are immunologically competent [42]. On the other hand, B7-1 expression is usually induced; whether placental and decidual macrophages would express this molecule on induction by activating cytokines during infection is not known.

Interestingly, we found that B7-2 protein is differentially expressed by different subsets of fetal macrophages. Macrophages associated with the connective tissue between the amnion epithelium and chorion membranes clearly expressed B7-2, whereas those within the placental villi expressed little or no B7-2. A possible explanation is a difference in the state of differentiation or maturity of these cells. Fetal and newborn leukocytes have reduced immunocompetence as compared to adult cells, and the acquisition of HLA expression by fetal macrophages is gradual [43–47].

Our finding that villous and membrane-associated fetal macrophages express B7-2 is consistent with the idea that these cells can serve in an immunological capacity. Both populations express HLA class I and class II, and villous macrophages can support a mixed lymphocyte reaction in vitro [44, 48, 49]. Furthermore, the number of activated macrophages and CD4+ T cells increases in cases of villitis of unknown cause, implying that a T cell-mediated immune response, in which B7-mediated costimulation would be required, can occur within the villous placental mesenchyme [50]. Therefore, it is possible that fetal and/or maternal macrophages in the placenta and its associated membranes present antigen to maternal T cells to ward off infection.

Cytotrophoblasts, including extravillous cytotrophoblasts that express class Ia and class Ib HLA molecules [51, 52], lacked B7-1 and B7-2 mRNA and protein. When combined with an HLA class Ia or class II signal, B7-1 and B7-2 costimulate cytotoxic and helper T-cell responses in vivo and in vitro. The absence of costimulatory molecules on trophoblast cells may ensure that these cells do not elicit a "danger" signal to the maternal immune system, perhaps instead contributing to the establishment of immunological tolerance in vivo [53–59].

In summary, these results are the first, to our knowledge, to map B7 family molecules in macrophages and trophoblasts at the maternal-fetal interface. Both molecules have the potential to profoundly influence leukocyte activation and, therefore, likely are key players in trophoblast regulation of the maternal immune system. The distribution of these molecules could indicate a mechanism for active suppression of the maternal immune system while ensuring that the ability to protect against foreign pathogens is not compromised.


   NOTE ADDED IN PROOF
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 NOTE ADDED IN PROOF
REFERENCES
 
After acceptance of this paper, two relevant reports were published by others. First, signaling through B7-2 was shown to induce indoleamine 2,3-dioxygenase (IDO) synthesis in dendritic cells upon ligation by CTLA-4, which is present in placental fibroblasts [60, 61]. As IDO promotes tolerance in murine pregnancy [62], this reveals a possible function of B7-2 in the placenta. Second, Brown et al. [63] reported similar distribution of B7-H1 as was found by us [current study; 23], and that B7-DC is present in placental endothelial cells. These findings further stress the importance of placental B7 molecules.


   ACKNOWLEDGMENTS
 
The authors wish to thank Christian Caluba for expertise in flow cytometric cell sorting and Kimberly Morgan for technical assistance.


   FOOTNOTES
 
1 Supported by NIH grants HD26429 (J.S.H.), HD33994 (Subproject, J.S.H.) from the Kansas Reproductive Sciences U54 Center, CA97085 (L.C.), and grants from the Franz-Lanyar-Stiftung (P.S.) and the Lied Endowed Basic Science Fund (M.G.P.). Salary support for M.G.P. was provided by NIH National Research Service Award HD08660. Back

2 Correspondence: Margaret G. Petroff, Department of Anatomy and Cell Biology, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160-7400. FAX: 913 588 7180; mpetroff{at}kumc.edu Back

Received: 7 August 2002.

First decision: 3 September 2002.

Accepted: 5 November 2002.


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 DISCUSSION
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