ARTICLE |
Correspondence to: Renate Fuchs, Dept. Pathophysiology, University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria. E-mail: renate.fuchs@akh-wien.ac.at
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Summary |
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Human placental alkaline phosphatase (PLAP) was localized at the apical and basal plasma membrane of syncytiotrophoblasts and at the surface of cytotrophoblasts in term chorionic villi using immunoelectron microscopy. Similarly, apical and basolateral PLAP expression was found in polarized trophoblast-derived BeWo cells. Trophoblasts isolated from term placentas exhibited mainly vesicular PLAP immunofluorescence staining immediately after isolation. After in vitro differentiation into syncytia, PLAP plasma membrane expression was upregulated and exceeded that observed in mononuclear trophoblasts. These data call for caution in using PLAP as a morphological marker to differentiate syncytiotrophoblasts from cytotrophoblasts or as a marker enzyme for placental brush-border membranes. (J Histochem Cytochem 49:11551164, 2001)
Key Words: human placental syncytiotrophoblasts, placental alkaline phosphatase, BeWo cells
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Introduction |
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Human heat-stable placental alkaline phosphatase (PLAP) is a glycosyl phosphatidylinositol (GPI)-anchored sialoglycoprotein composed of two identical 66-kD subunits (
Using cryo-immunoelectron microscopy, we here demonstrate that PLAP is highly expressed in STBs and, to a lesser extent, in CTBs in term placental villi. In agreement with these data, PLAP was found in freshly isolated as well as in cultured mononuclear trophoblast cells from term placentas. On syncytium formation in vitro, PLAP expression was highly upregulated. In situ, the enzyme was located at both plasma membrane domains of the syncytium, although at relative higher density at the apical compared to the basal side. An apical and basolateral PLAP distribution was also found in the polarized choriocarcinoma cell line BeWo.
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Materials and Methods |
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Chemicals and Cell Lines
Unless stated otherwise, all chemicals used were purchased from Sigma Chemical (St Louis, MO) and were of highest purity available. Reagents for immunoelectron microscopy were from Merck (Darmstadt, Germany). BeWo cells clone b24, kindly provided by Alan Schwartz (School of Medicine, Washington University; St Louis, MO) and Arie van der Ende (Laboratory of Cell Biology; Medical School, University of Utrecht, The Netherlands), were cultured in DMEM high glucose with 10% (w/v) FCS, 100 U/ml penicillin, and 0.1 mg/ml streptomycin and maintained in plastic culture flasks. Cells were seeded onto 12-mm permeable filters (0.45 µm, high density; Becton Dickinson Labware, Franklin Lakes, NJ) to obtain polarized cell monolayers as described (
SDS-PAGE and Western Blotting
PLAP purified from human placentas (Sigma) was used as positive control. Total microsomes were prepared from polarized BeWo cells and term placental tissue by centrifugation of the respective cell homogenate (in PBS, pH 7.4, containing 3 mM EDTA) at 100,000 x g for 1 hr. The microsomal pellets were solubilized in 150 mM phosphate buffer, pH 7.4, containing 60 mM N-octyl ß-D-glucopyranoside, 10 mM D-gluconic acid lactone, 1 mM EDTA. Proteins were separated on 10% reducing SDS-PAGE gels and transferred to nitrocellulose membranes. Membranes were blocked with 5% (w/v) dried milk powder in PBS. hPLAP was detected by incubation with rabbit anti-hPLAP antibody (1:200; Signet, Dedham, MA), followed by incubation with a secondary horseradish peroxidase-conjugated, goat anti-rabbit antibody (1:14,000). The blots were developed with an enhanced chemiluminescence kit (Pierce; Rockford, IL).
Immunoelectron Microscopy
Blocks of placental villous tissue (2 mm in diameter) from 13 term placentas were fixed in 5% (w/v) paraformaldehyde (PFA) in 0.2 M piperazine-N,N'-bis[2-ethanesulfonic acid], pH 7.0, cryoprotected with polyvinylpyrollidine/sucrose, and frozen in liquid nitrogen. Ultrathin frozen sections (60 nm) were prepared (
Trophoblast Isolation and Culture
Human placentas were obtained from non-complicated full-term pregnancies. Trophoblasts were isolated by trypsin/DNAse digestion of dissected tissue using a modification of the procedure of
Immunofluorescence
At the times indicated in the figure legends, trophoblasts were washed with ice-cold PBS+ (PBS containing 1 mM CaCl2 and 1 mM MgCl2), fixed, and permeabilized with -20C methanol for 10 min. To determine PLAP expression at the plasma membrane only, cells were fixed with 3% PFA (w/v) in PBS. Unspecific binding sites were blocked with 1% (w/v) BSA in PBS for 30 min and PLAP was detected by indirect immunofluorescence using a mouse anti-hPLAP antibody (clone 8B6, 1:10; DAKO) or a rabbit anti-hPLAP antibody (1:20; Signet), with identical results. After incubation with a TRITC-conjugated goat anti-mouse IgG or a DTAF-conjugated goat anti-rabbit IgG (both 1:100; Accurate, Westbury, NY) the nuclei were labeled with Hoechst dye (0.5 µg/ml in PBS for 10 min). Cells were mounted in Immuno Fluore mounting medium (ICN Biochemicals; Aurora, OH) and viewed with a Zeiss Axioscope 2MOT. Digital images were processed with the Zeiss KS400 imaging program.
Polarized BeWo cells were washed with PBS+ and preincubated in Leibovitz 15 medium (L-15; Life Technologies) containing 0.5% (w/v) BSA and 1 g/liter glucose for 30 min at 37C. Cells were cooled to 4C and mouse anti-hPLAP antibody (clone 8B6, 1:10 in L-15 containing BSA and glucose; DAKO) or rabbit anti-hPLAP antibody (1:20 in L-15 containing BSA and glucose; Signet) was added to either the apical or basolateral compartment for 1 hr at 4C. Cells were washed with PBS+, fixed with 3% PFA in PBS, and quenched with 50 mM NH4Cl in PBS. Unspecific binding was blocked with 1% (w/v) BSA in PBS and PLAP immunoreactivity was detected with TRITC-conjugated goat anti-mouse IgG (1:100; Accurate) or DTAF-labeled secondary antibody (1:50 in L-15 containing BSA and glucose; Accurate). The staining pattern obtained was identical for the monoclonal and polyclonal anti-PLAP antibodies.
Quantification of Plasma Membrane PLAP Expression and Transferrin Binding in Polarized BeWo Cells
To quantitate PLAP expression at the apical and basolateral plasma membrane, cells were washed with ice-cold PBS+ and IgG-binding sites present at the plasma membrane were blocked with hIgG (1 mg/ml, Endobulin; Baxter AG, Vienna, Austria) in L-15 containing 0.5% (w/v) BSA and 1 g/liter glucose for 30 min at 4C. Anti-hPLAP antibody (1:20 in L-15; Signet) was bound to the apical (100 µl) or basolateral plasma membrane (30 µl) at 4C for 60 min. Unbound antibody was removed by washing with PBS+ and cells were incubated (60 min at 4C) with DTAF-labeled secondary antibody (1:50 in L-15; Accurate). After washing the cells with PBS+, filters were removed from the filter holders, cells were lysed in 0.6 ml PBS, pH 7.4, containing 0.5 % (w/v) Triton X-100, and centrifuged at 12,000 x g for 10 min to pellet insoluble material. DTAF-fluorescence in 250 µl supernatant was determined in a Cytofluor 2300 (Millipore; Astroscan, Isle of Man, British Isles) using a standard filter set [excitation 485 nm (slit 20 nm); emission: 530 nm (slit 25 nm)]. Background fluorescence was determined under identical conditions but omitting the first antibody. One experiment of three individual experiments is shown. Data given (mean ± SD) are fluorescence in arbitrary units after subtraction of background fluorescence from 10 parallel samples. Transferrin binding to the apical and basolateral side was determined after preincubating the cells for 30 min at 37C in L-15 medium to deplete endogenous transferrin. Cells were then cooled to 4C and FITCtransferrin (100 µg/ml) in L-15 was allowed to bind to either the apical or the basolateral plasma membrane for 1 hr at 4C. Cells were washed with PBS and processed as described above to determine the amount of cell-associated FITCtransferrin after subtraction of background fluorescence. Data shown are the mean ± SD from one typical experiment out of three carried out sixfold. Statistical significant differences were calculated using Student's t-test.
Morphometrical Analysis to Determine the Apical: Basolateral Ratio of the Surface Area of Polarized BeWo Cells
BeWo cells grown as a polarized monolayer on permeable filters were fixed with 2.5% (w/v) glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, postfixed with 2% (w/v) osmium tetroxide, dehydrated in ascending ethanol series, and embedded in Epon resin. Ultrathin sections cut perpendicular to the filters were contrasted with aqueous 2% uranyl acetate and 2% alkaline lead citrate (
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Results |
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In Situ Localization of PLAP in Term Chorionic Villi by Immunoelectron Microscopy: Non-polarized Expression in STBs and CTBs
PLAP was immunolocalized on ultrathin cryosections of human term placentas using the immunogold technique (
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PLAP Is Expressed in Mononuclear and Multinuclear Freshly Isolated and Cultured Term Trophoblasts
The expression pattern observed in situ in term placentas indicated that PLAP is expressed at a low level in the CTBs and is upregulated upon differentiation into STBs. To investigate whether a similar expression pattern of PLAP is also found in isolated CTBs, trophoblasts were isolated from term placentas, cultured, and examined for PLAP expression after 0 hr, 24 hr, and 48 hr in culture by indirect immunofluorescence. Immediately after isolation, two main populations of cells were observed: single round mononuclear cells and cell aggregates (Fig 2A and Fig 2B). Both populations revealed a patchy, presumably vesicular PLAP staining (Fig 2A) that was clearly distinct from the typical uniform labeling of PLAP at the plasma membrane (compare to Fig 2G). The localization of PLAP in intracellular vesicles was confirmed by the almost complete absence of PLAP immunoreactivity in trophoblasts fixed with PFA. Under this condition, only PLAP at the plasma membrane is accessible to anti-PLAP antibodies (Fig 2C). An intracellular localization of the enzyme could be attributed to its internalization during trypsin digestion of term placental tissue, similar to the effect of collagenase treatment of rat liver on endocytosis of hepatocyte apical plasma membrane proteins (
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Non-polarized PLAP Expression in Polarized BeWo Choriocarcinoma Cells
Because we were unable to obtain a continuous polarized syncytium during in vitro cultivation to quantitate PLAP expression at the apical and basolateral side of STBs, trophoblast-derived BeWo cells were used instead. We and others have shown that BeWo cells, which can be grown as a polarized tight monolayer on permeable filters, represent a useful in vitro system for study of STB functions (
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Second, expression of PLAP at either the apical or basolateral plasma membrane was investigated by immunofluorescence microscopy. As shown in Fig 4A and Fig 4B, PLAP was detected at the apical (Fig 4A) and the basolateral plasma membrane domains (Fig 4B). This expression pattern is clearly in contrast to the almost entirely apical localization of PLAP in MDCK cells (0.001) was obtained for apical to basolateral transferrin receptor expression in BeWo cells. Similarly, higher amounts of anti-PLAP antibody bound to the basolateral compared to the apical side, yielding an apical:basolateral ratio of 1:1.52 (statistically significant difference 0.01
p
0.001). Thus, at steady state about 1.5 times more PLAP and transferrin receptors are located at the basolateral than at the apical membrane of BeWo cells.
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To investigate whether these differences in apical/basolateral PLAP and transferrin receptor distribution can be explained by different surface areas, we examined ultrathin Epon sections of polarized BeWo cells that were cut perpendicular to the filters (not shown). In contrast to STBs in situ, microvilli were irregular and less abundant (see also p
0.001; Fig 4E). Thus, when normalized to the apical and basolateral surface areas, respectively, equal concentrations of PLAP and transferrin receptors are found at either plasma membrane domain.
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Discussion |
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In situ localization of PLAP in term placental villi by immunoelectron microscopy revealed PLAP expression in both STBs and CTBs. Based on the high level of resolution of the methodology applied, our data clearly demonstrate that PLAP cannot serve as a morphological differentiation marker for STBs during isolation and in vitro culture. It is generally assumed that CTBs isolated from term placental tissue aggregate, fuse, and differentiate into multinuclear syncytia in vitro (
PLAP expression in villous STB in situ was not restricted to the apical (brush-border) plasma membrane (Fig 1B1D). This is in analogy to Na+/K+-ATPase, which has an exclusive basolateral localization in small intestinal epithelial cells and which has recently been found to be present to a higher extent at the apical than at the basal membrane of term STBs (
Although BeWo cells exhibit some properties of villous STBs (e.g., placental hormone secretion), they differ with respect to syncytium formation, differentiation, and protein expression pattern (
The discrepancy of apical/basal PLAP expression in STBs in situ and in BeWo cells might also be due to the nature of PLAP itself. Two closely related genes encode heat-stable alkaline phosphatases: the PLAP-1 gene (term placental enzyme) and the PLAP-2 gene (germ-cell or PLAP-like enzyme). Whereas primarily PLAP-1 and only trace amounts of PLAP-2 transcripts are expressed in term placentas, the opposite expression pattern is found in BeWo cells (
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Acknowledgments |
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Supported by grants from the Austrian Science Foundation P-12084-MED and P-14079-MED to R.F.
We are grateful to Cordula Westermann and Dorothea Budde for excellent technical assistance.
Received for publication September 29, 2000; accepted March 21, 2001.
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