Journal of Histochemistry and Cytochemistry, Vol. 49, 1155-1164, September 2001, Copyright © 2001, The Histochemical Society, Inc.


ARTICLE

Placental Alkaline Phosphatase Expression at the Apical and Basal Plasma Membrane in Term Villous Trophoblasts

Karl Leitnera, Roman Szlauera, Isabella Ellingera, Adolf Ellingerb, Klaus-Peter Zimmerc, and Renate Fuchsa
a Departments of Pathophysiology, Medical School at Münster University, Münster, Germany
b Histology and Embryology II, Medical School at Münster University, Münster, Germany
c University of Vienna, Vienna, Austria, and Department of Pediatrics, Medical School at Münster University, Münster, Germany

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


  Summary
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 49:1155–1164, 2001)

Key Words: human placental syncytiotrophoblasts, placental alkaline phosphatase, BeWo cells


  Introduction
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Human heat-stable placental alkaline phosphatase (PLAP) is a glycosyl phosphatidylinositol (GPI)-anchored sialoglycoprotein composed of two identical 66-kD subunits (Holmgren and Stigbrand 1976 ) present at high levels in placental trophoblasts and at trace amounts in the lung, endocervix, and Fallopian tube (Goldstein et al. 1982 ; Hamilton-Dutoit et al. 1990 ). In the placenta, expression of PLAP mRNA (Okamoto et al. 1990 ) and protein (Jones and Fox 1976 ) starts at around Week 7 of gestation and steadily increases with duration of gestation. At term, villous and, to a lesser extent, extravillous trophoblasts exhibit PLAP immunoreactivity (Bulmer and Johnson 1985 ; Caulfield et al. 1992 ). Although expression of PLAP in villous syncytiotrophoblasts (STBs) has been demonstrated unequivocally (Jones and Fox 1976 ), its localization in villous cytotrophoblasts (CTBs) is controversial. Weak immunoreactivity of anti-PLAP monoclonal antibodies was observed in CTBs in situ (Bulmer and Johnson 1985 ) and in CTB aggregates cultured in vitro (Cervar et al. 1999 ). In contrast, others found no reactivity in CTBs in chorionic villi (Hartmann et al. 1989 ; Ribitsch et al. 1990 ). A precise localization is, however, of major importance for the use of PLAP as morphological differentiation marker to study STBs functions in vitro. This in vitro system is based on the ability of isolated villous trophoblasts to differentiate into STBs during culture (Bloxam et al. 1997a , Bloxam et al. 1997b ). The differentiation is accompanied by morphological changes, such as formation of multinuclear syncytia, and biochemical alterations, such as upregulation of membrane and secretory proteins. Because of the lack of a bona fide and generally accepted STB marker, PLAP expression has been used to demonstrate the formation of multinuclear STBs during in vitro culture (Bax et al. 1989 ; Bullen et al. 1990 ).In all polarized cells examined thus far, PLAP is found at the apical plasma membrane with its enzymatic portion facing the extracellular space. When human PLAP is expressed in MDCK cells, a polarized dog kidney cell line, 90% of the enzyme is targeted to the apical surface (Lisanti et al. 1988 ). Although it was initially assumed that this is due to its GPI anchor, it has recently been shown that PLAP contains an apical sorting signal in its protein ectodomain (Lisanti et al. 1990 ; Lipardi et al. 2000 ). In villous STBs, high levels of PLAP have been localized at the apical brush-border membrane (Jones and Fox 1976 ). It is unclear, however, whether the enzyme is restricted to the apical membrane domain, because alkaline phosphatase activity has also been detected at the basal plasma membrane by enzyme histochemistry (Jones and Fox 1976 ). Inherent to this procedure, the presence of other phosphatases complicates the interpretation of these data. Nevertheless, PLAP is used as an apical marker enzyme to follow the isolation and purification of placental brush-border membranes (Truman and Ford 1984 ; Illsley et al. 1990 ; Eaton and Oakey 1994 ). The precise localization of PLAP at the villous CTBs and at the apical and basal membrane domain of STBs is still missing.

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.


  Materials and Methods
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 (Ellinger et al. 1999 ). Fluorescein isothiocyanate (FITC) was coupled to human diferric transferrin as described (van Renswoude et al. 1982 ).

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 (Tokuyasu et al. 1985 ) using a Leica cryoultramicrotome (block temperature -110C, knife-temperature -100C). Grids with the attached thawed sections were blocked with 5% (w/v) fetal calf serum (FCS; Life Technologies, Rockville, MD) in PBS, pH 7.4, and then incubated with mouse monoclonal anti-hPLAP antibody (clone 8B6, undiluted; DAKO, Glostrup, Denmark) for 45 min at room temperature (RT). After a washing step with PBS, the sections were incubated with 12-nm gold-conjugated goat anti-mouse antibody (dilution 1:10; DIANOVA, Hamburg, Germany), washed again with PBS, washed with A.bidest., and subsequently contrasted and embedded by incubation with methylcellulose/uranyl acetate on ice [9:1 mixture of 2% (w/v) methylcellulose and 4% (w/v) uranyl acetate, freshly prepared]. Samples were examined in a Philips 400 electron microscope. Under these incubation conditions no background labeling was noted on nuclei and mitochondria of trophoblasts. Sections incubated with gold-conjugated goat anti-mouse IgG without first antibody revealed no detectable staining. The specificity of MAb 8B6 (raised against PLAP expressing Hep-2 cells) has been well characterized by ELISA, immunohistochemistry, and competitive binding studies (Durbin et al. 1988 ).

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 Kliman et al. 1986 . About 80 g villous tissue was minced, washed in PBS, pH 7.4, and digested three times (20 min at 37C) in Hanks' balanced salt solution containing 25 mM Hepes, 0.125% (w/v) trypsin (Life Technologies; Rockville, MD), and 0.5 µg/ml DNase DN-25. The supernatants were pooled, cells were pelleted at 500 x g (10 min at 4C), and resuspended in keratinocyte growth medium (KGM); Life Technologies) containing 5 ng/ml EGF, 50 µg/ml bovine pituitary extract, and 0.65 mM Ca++. These cells were layered on top of a pre-formed, discontinuous 70% to 5% Percoll density gradient (Pharmacia; Uppsala, Sweden) and centrifuged at 1300 x g for 20 min at 4C. Material banding at densities between 1.048 and 1.062 g/ml was collected, washed, and resuspended in KGM. Contaminating, HLA class I-positive cells were removed (Douglas and King 1990 ) using a mouse anti-human HLA-I (W6/32) antibody (Sigma) coupled to magnetic beads (Dynabeads M-450, sheep anti-mouse IgG-coated; Dynal, Oslo, Norway). The resulting purified trophoblast preparation consisted of about 99% cytokeratin-positive cells determined by immunofluorescence with a mouse anti-cytokeratin antibody (AE-1/AE-3 pan-cytokeratin; Signet) that recognizes cytokeratin 1–8, 10, 14–16, and 19 (Sun et al. 1983 ). Trophoblasts were seeded onto 8-well plastic chamber slides at a density of 5 x 105 cells/cm2 and cultured for up to 48 hr in KGM [containing 10% (w/v) fetal calf serum, penicillin (100 IU/ml) and streptomycin (100 µg/ml)]. The culture medium was changed daily.

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 FITC–transferrin (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 FITC–transferrin 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 (Reynolds 1963 ), examined in a Philips 400 electron microscope, and representative micrographs were taken randomly at a magnification of x8000. The micrographs (final enlargement x24,000) were covered with a square grid (7.5 mm, equivalent to 0.31 µm of the photograph) that was aligned at an angle of 15° with the apical surface, and intersections of the grid with the apical and basolateral membrane, respectively, were counted. The numbers of the membrane–grid intersections of the apical and basolateral side, respectively, in 15 micrographs were determined according to Griffiths 1993 . This ratio is related to the respective surface area. The mean intersections ± SD are depicted. Statistical significant differences were calculated using Student's t-test.


  Results
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 (Griffiths and Hoppeler 1986 ). An overview of a villous area examined is shown in Fig 1A. PLAP was found in villous STB (Fig 1B–1D) as well as in CTB (Fig 1D and Fig 1E). Many gold particles, indicating PLAP, marked the apical plasma membrane of STBs (Fig 1B). The labeling intensities at the apical cell surface were more pronounced in areas rich than in areas low in microvilli. Apical PLAP labeling was comparable in different villi from one placenta but varied among the 13 individual term placentas examined. In contrast to the PLAP labeling density at the apical plasma membrane, weaker staining was observed at the basal side of STBs (Fig 1C and Fig 1D). At term, placental villi are devoid of a continuous CTB layer underneath the STBs, and CTBs are only rarely found (Jones and Fox 1991 ). These villous CTBs exhibited a homogeneous distribution of PLAP around their entire surface (Fig 1D and Fig 1E). No background labeling could be observed in nuclei (Fig 1F) and mitochondria of STB. Furthermore, negative controls carried out by incubating the cryosections with gold-conjugated goat anti-mouse IgG but omitting the first antibody revealed no detectable staining in placental microvilli (Fig 1G).




View larger version (258K):
[in this window]
[in a new window]
 
Figure 1. In situ localization of PLAP on ultrathin cryosections of a human term placental villus by immunoelectron microscopy. Cryosections were incubated with mouse anti-hPLAP antibody (clone 8B6) followed by 12-nm gold-conjugated goat anti-mouse antibody. (A) Low-magnification micrograph of an ultrathin cryosection through a term placental villus. Bar = 1 µm. The squares drawn in the overview indicate the areas presented in B–F. (B) Strong labeling of PLAP was observed at the microvillous surface of the STB. (C) In contrast to the apical side, the basal plasma membrane of the STB showed lesser staining with the anti-PLAP antibody. (D,E) PLAP immunoreactivity was observed along the entire plasma membrane of the CTB. (F) No background labeling could be observed on nuclei. (G) Cryosections incubated with gold-conjugated goat anti-mouse IgG without first antibody revealed no detectable staining at the apical membrane. STB, syncytiotrophoblast; CTB, cytotrophoblast; BM, basement membrane. Bars = 0.1 µm.

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 (Graf et al. 1984 ). Because we were unable to detect significant PLAP labeling in the endoplasmic reticulum and Golgi apparatus in STBs and CTBs in term placentas in situ (data not shown), PLAP-positive vesicles in freshly isolated trophoblasts are most likely not derived from the secretory pathway. After 24 hr in culture, 60–80% of the isolated trophoblasts had formed clusters in which PLAP was primarily localized at the cell surface (Fig 2D). After 48 hr in culture, syncytial elements, identified by their closely accumulated nuclei (arrows in Fig 2H) exhibited a high level of plasma membrane fluorescence at the microvillous surface (Fig 2G and Fig 2I). In agreement with the in situ localization of PLAP in CTBs and STBs shown above, the enzyme is expressed by isolated and cultured mononuclear (CTB) and multinuclear (STB) trophoblasts.



View larger version (89K):
[in this window]
[in a new window]
 
Figure 2. Immunofluorescence localization of PLAP in freshly isolated trophoblasts (A,C) and after 24 hr (D–F) and 48 hr (G–I) in culture. Trophoblasts were fixed and permeabilzed with methanol (A–I, except C) or fixed with 3% (w/v) PFA in PBS (C). PLAP was detected with mouse anti-PLAP antibody 8B6 followed by TRITC-conjugated goat anti-mouse IgG. Nuclei were stained with Hoechst dye (B,E,H). (F) Negative control, secondary antibody only. After 48 hr in culture, PLAP was highly expressed at the microvillous cell surface of syncytia (G) with closely accumulated nuclei (arrows, H). Objectives used: A–F 63-fold; G–H 40-fold; I 100-fold.

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 (Cerneus and van der Ende 1991 ; Ellinger et al. 1999 ). First, the specificity of the rabbit anti-hPLAP antibody used was verified by Western blotting analysis of total microsomes prepared from confluent polarized BeWo cells and from term placental tissue using purified human PLAP as positive control. As shown in Fig 3, the antibody reacts with a single band in BeWo cells of the same electrophoretic mobility (about 66 kD) as purified PLAP. The faint additional band of about 58 kD seen in placental microsomes is due to proteolytic degradation of PLAP (Abu-Hasan and Sutcliffe 1985 ). Densitometry of these bands revealed that 10 times more PLAP/µg microsomal protein is expressed in term placenta compared to BeWo cells, indicative of the lower extent of differentiation of BeWo cells.



View larger version (57K):
[in this window]
[in a new window]
 
Figure 3. Western blotting analysis of PLAP expression in polarized BeWo cells and term placenta. Confluent BeWo cells and term placental tissue were homogenized in PBS, pH 7.4, containing 10 mM EDTA. Total microsomes were prepared by 60-min centrifugation at 100,000 x g. Proteins (3 µg purified PLAP, 25 µg placental and 50 µg BeWo microsomes) were separated on 10% reducing SDS-PAGE, blotted onto nitrocellulose membranes, and incubated with rabbit anti-PLAP antibody (1:200; Signet) followed by horseradish peroxidase-conjugated goat anti-rabbit IgG (1:14,000). Blots were developed with an enhanced chemiluminescence kit. The polyclonal antibody recognizes a band of about 66 kD in term placenta and BeWo cells. The lower molecular weight band in placental microsomes is due to proteolysis of PLAP.

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 (Arreaza and Brown 1995 ) and is indicative of its non-polarized distribution in trophoblasts. Next, the amount of anti-PLAP antibody bound to either the apical or basolateral cell aspect was quantitated by fluorescence spectroscopy (Fig 4C). For comparison, apical and basolateral FITC–transferrin binding to polarized BeWo cells was included in this experiment (Fig 4D). In agreement with Cerneus et al. 1993a , a ratio of 1:1.57 (statistically significant difference, p<=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.



View larger version (27K):
[in this window]
[in a new window]
 
Figure 4. Apical and basolateral expression of PLAP and transferrin receptors in polarized BeWo cells. BeWo cells were cooled to 4C and mouse anti-PLAP antibody 8B6 was added to either the apical (A) or the basolateral (B) compartment for 1 hr at 4C. After fixation with 3% (w/v) PFA, PLAP was detected with TRITC-conjugated goat anti-mouse IgG. Immunofluorescent images of the apical (A) and basolateral side (B) are depicted. Objectives used: A,B 100-fold. (C) Rabbit anti-PLAP antibody was bound to the apical and basolateral side, followed by DTAF-conjugated secondary antibody. Cells were lyzed and DTAF fluorescence intensities in cell lysates were determined in a Cytofluor. Data given are the mean ± SD from one experiment carried out tenfold. (D) FITC–transferrin was bound to the apical or basolateral plasma membrane for 60 min at 4C and cell-associated fluorescence was determined thereafter as described in Materials and Methods. Means ± SD from one experiment carried out sixfold are depicted. (E) Electron micrographs of Epon sections of polarized BeWo cells (final enlargement x24,000) were covered with a square grid and intersections of the grid with the apical and basolateral membrane, respectively, were counted. The intersections (mean ± SD) from 15 micrographs are shown. Statistical significant differences (C–E) were calculated using Student's t-test. **0.01>=p>=0.001; ***p<=0.001.

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 Cerneus and van der Ende 1991 ). Determination of the surface area by counting the intersections between the respective plasma membrane domain and a square grid according to Griffiths 1993 revealed an apical to basolateral surface area of 1:1.51 (statistically significant difference 0.01>=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.


  Discussion
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 (Bloxam et al. 1997a , Bloxam et al. 1997b ). Although villous STBs are isolated along with CTBs (Bloxam et al. 1997b ), they either do not adhere or tend to disappear during culture. Thus far, PLAP served as a presumptive marker for STBs for the morphological characterization of these cultures. In line with this hypothesis, freshly isolated trophoblasts were found to be cytokeratin-positive (Bax et al. 1989 ; Bullen et al. 1990 ), but only 10% of this population were PLAP-positive cells (i.e., CTBs); after 48 hr in culture all cells expressed PLAP (Bullen et al. 1990 ). However, this is in contrast to the results presented in this investigation, in which almost all isolated cytokeratin-positive cells (= trophoblasts) were also PLAP-positive immediately after isolation (compare Fig 2A and Fig 2B). The low vesicular staining of PLAP in isolated cells might have escaped detection in previous investigations (Bax et al. 1989 ). The lack of detection of freshly isolated PLAP-positive cells in previous investigations is, however, not due to the release of PLAP by trypsin digestion during trophoblast isolation. Trypsin treatment of isolated brush-border membranes has been shown to result in cleavage of only a 9-kD fragment from the N-terminal domain of the subunit (Abu-Hasan and Sutcliffe 1985 ). Although PLAP was expressed at the plasma membrane in mononuclear cells (CTB) cultured for 24–48 hr (Fig 2D), PLAP expression was strongly upregulated on fusion of mononuclear cells into syncytia (Fig 2G). Therefore, PLAP is found in CTBs and STBs in situ as well as in isolated and cultured mononuclear trophoblast cells and syncytial elements. Given that different isolation procedures and culture conditions drastically influence the degree of differentiation of trophoblasts (Bloxam et al. 1997a ), we conclude that PLAP expression in isolated cultured trophoblasts does not necessarily indicate the presence of fully differentiated syncytiotrophoblasts.

PLAP expression in villous STB in situ was not restricted to the apical (brush-border) plasma membrane (Fig 1B–1D). 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 (Johansson et al. 2000 ). The apical and basal localization of PLAP in STBs is in analogy to its steady-state distribution between the apical and basolateral plasma membranes of trophoblast-derived BeWo cells, cultured as a polarized monolayer (Fig 4). In these cells, about 1.5 times more PLAP and transferrin receptors, respectively, were found basolaterally than apically. Because morphometrical analysis revealed that the basolateral surface area is 1.5 times larger than the apical surface, the surface densities of PLAP and transferrin receptors are identical at both plasma membrane aspects of BeWo cells. For the transferrin receptor, it has been shown that 80% of newly synthesized receptor is initially inserted into the basolateral domain. The steady-state distribution is then achieved by different rates of transcytosis of apical and basolateral receptors (Cerneus et al. 1993a ). It remains to be demonstrated whether newly synthesized PLAP is initially sorted to the apical domain and subsequently transcytosed to the basolateral side, with less transcytosis taking place in the opposite direction in BeWo cells. This assumption is supported by the observation that PLAP together with maternal IgG is found in clathrin-coated vesicles isolated from placental tissue (Makiya et al. 1992 ), by histochemistry of alkaline phosphatase activity in vesicles (endosomes?) in the vicinity of the brush-border membrane (Jones and Fox 1976 ), and by endocytosis of PLAP in BeWo cells (Cerneus et al. 1993b ). Alternatively, different sorting mechanisms might operate in MDCK and BeWo cells that direct PLAP to the apical and basolateral membrane after synthesis in BeWo cells. Such differences were also found between MDCK cells and neurons, the latter exhibiting non-polarized PLAP distribution after transfection (Kollins et al. 1999 ). However, the non-polarized distribution of PLAP in BeWo cells does not indicate a general loss of surface polarity: (a) the kinetics of transferrin transcytosis and endocytosis differ between the apical and basolateral side (Cerneus et al. 1993a ); (b) specific low pH-dependent IgG binding (indicative of plasma membrane expression of hFcRn) is observed at the apical but not at the basolateral side; and (c) four times as much IgG is transcytosed from the apical to the basolateral side as in the opposite direction (Ellinger et al. 1999 ).

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 (King et al. 2000 ). Our data also indicate a lesser degree of differentiation when PLAP expression is compared in placental tissue and BeWo cells by Western blotting. Considering that term placental tissue contains other cell types in addition to villous STBs and CTBs, the amount of PLAP/µg microsomal protein found in placental tissue is nevertheless 10 times higher than in BeWo cells. The low level of expression and the non-polarized distribution of PLAP in BeWo cells are reminiscent of the situation observed in CTBs in situ (Fig 1). Nevertheless, BeWo cells are the only immortalized trophoblast-derived cell line that forms polarized tight monolayers on permeable filters to study transepithelial transport processes.

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 (Povinelli and Knoll 1991 ). Whether PLAP-1 transcripts in term placental tissue arise from a different cell type than PLAP-2 transcripts has not been shown. Because PLAP-1 and PLAP-2 differ by only seven amino acids (Watanabe et al. 1991 ), most antibodies (such as the ones used in this study) detect both PLAP isoforms. It is unknown thus far whether PLAP-1 and PLAP-2 are sorted differently in polarized cells (STB, BeWo), giving rise to different extents of expression at the apical and basal membranes, respectively. Although the actual amount of PLAP expressed at the entire apical and basal surface of villous STB in situ is unknown, the data presented in this investigation call for caution in using PLAP as a marker enzyme for the brush-border membrane.


  Acknowledgments

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.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Abu-Hasan NS, Sutcliffe RG (1985) Placental alkaline phosphatase integrates via its carboxy-terminus into the microvillous membrane: its allotypes differ in conformation. Placenta 6:391-404[Medline]

Arreaza G, Brown DA (1995) Sorting and intracellular trafficking of a glycosylphosphatidylinositol-anchored protein and two hybrid transmembrane proteins with the same ectodomain in Madin-Darby canine kidney epithelial cells. J Biol Chem 270:23641-23647[Abstract/Free Full Text]

Bax CMR, Ryder TA, Mobberley MA, Tyms AS, Taylor DL, Bloxam DL (1989) Ultrastructural changes and immunocytochemical analysis of human placental trophoblast during short-term culture. Placenta 10:179-194[Medline]

Bloxam DL, Bax BE, Bax CMR (1997a) Culture of syncytiotrophoblast for the study of human placental transfer. Part II: production, culture and use of syncytiotrophoblast. Placenta 18:99-108[Medline]

Bloxam DL, Bax CMR, Bax BE (1997b) Culture of syncytiotrophoblast for the study of human placental transfer. Part I: isolation and purification of cytotrophoblast. Placenta 18:93-98[Medline]

Bullen BE, Bloxam DL, Ryder TA, Mobberley MA, Bax CM (1990) Two-sided culture of human placental trophoblast. Morphology, immunocytochemistry and permeability properties. Placenta 11:431-450[Medline]

Bulmer JN, Johnson PM (1985) Antigen expression by trophoblast populations in the human placenta and their possible immunobiological relevance. Placenta 6:127-140[Medline]

Caulfield JJ, Sargent IL, Ferry BL, Starkey PM, Redman CW (1992) Isolation and characterisation of a subpopulation of human chorionic cytotrophoblast using a monoclonal anti-trophoblast antibody (NDOG2) in flow cytometry. J Reprod Immunol 21:71-85[Medline]

Cerneus DP, Strous GJ, van der Ende A (1993a) Bidirectional transcytosis determines the steady state distribution of the transferrin receptor at opposite plasma membrane domains of BeWo cells. J Cell Biol 122:1223-1230[Abstract]

Cerneus DP, Ueffing E, Posthuma G, Strous GJ, van der Ende A (1993b) Detergent insolubility of alkaline phosphatase during biosynthetic transport and endocytosis—role of cholesterol. J Biol Chem 268:3150-3155[Abstract/Free Full Text]

Cerneus DP, van der Ende A (1991) Apical and basolateral transferrin receptors in polarized BeWo cells recycle through separate endosomes. J Cell Biol 114:1149-1158[Abstract]

Cervar M, Blaschitz A, Dohr G, Desoye G (1999) Paracrine regulation of distinct trophoblast functions in vitro by placental macrophages. Cell Tissue Res 295:297-305[Medline]

Douglas GC, King F (1990) Differentiation of human trophoblast cells in vitro as revealed by immunocytochemical staining of desmoplakin and nuclei. J Cell Sci 96:131-141[Abstract]

Durbin H, Milligan EM, Mather SJ, Tucker DF, Raymond R, Bodmer WF (1988) Monoclonal antibodies to placental alkaline phosphatase: preclinical evaluation in a human xenograft tumour model of F(ab')2 and Fab fragments. Int J Cancer Suppl 2:59-66[Medline]

Eaton BM, Oakey MP (1994) Sequential preparation of highly purified microvillous and basal syncytiotrophoblast membranes in substantial yield from a single term human placenta: inhibition of microvillous alkaline phosphatase activity by EDTA. Biochim Biophys Acta 1193:85-92[Medline]

Ellinger I, Schwab M, Stefanescu A, Hunziker W, Fuchs R (1999) IgG transport across trophoblast-derived BeWo cells: a model system to study IgG transport in the placenta. Eur J Immunol 29:733-744[Medline]

Goldstein DJ, Rogers C, Harris H (1982) A search for trace expression of placental-like alkaline phosphatase in non-malignant human tissues: demonstration of its occurrence in lung, cervix, testis and thymus. Clin Chim Acta 125:63-75[Medline]

Graf J, Gautam A, Boyer JL (1984) Isolated rat hepatocyte couplets: a primary secretory unit for electrophysiologic studies of bile secretory function. Proc Natl Acad Sci USA 81:6516-6520[Abstract]

Griffiths G (1993) Fine Structure Immunocytochemistry. Heidelberg, Springer-Verlag

Griffiths G, Hoppeler H (1986) Quantitation in immunocytochemistry: correlation of immunogold labeling to absolute number of membrane antigens. J Histochem Cytochem 34:1389-1398[Abstract]

Hamilton–Dutoit SJ, Lou H, Pallesen G (1990) The expression of placental alkaline phosphatase (PLAP) and PLAP-like enzymes in normal and neoplastic human tissues. An immunohistological survey using monoclonal antibodies. APMIS 98:797-811[Medline]

Hartmann M, Dohr G, Ribitsch D, Siwetz G, Desoye G, Kessler H (1989) GZ116, a trophoblast specific monoclonal antibody. Placenta 10:489

Holmgren PA, Stigbrand T (1976) Purification and partial characterization of two genetic variants of placental alkaline phosphatase. Biochem Genet 14:777-789[Medline]

Illsley NP, Wang ZQ, Gray A, Sellers MC, Jacobs MM (1990) Simultaneous preparation of paired, syncytial, microvillous and basal membranes from human placenta. Biochim Biophys Acta 1029:218-226[Medline]

Johansson M, Jansson T, Powell TL (2000) Na+-K+-ATPase is distributed to microvillous and basal membrane of the syncytiotrophoblast in human placenta. Am J Physiol 279:R287-294[Abstract/Free Full Text]

Jones CJ, Fox H (1976) An ultrahistochemical study of the distribution of acid and alkaline phosphatases in placentae from normal and complicated pregnancies. J Pathol 118:143-151[Medline]

Jones CJ, Fox H (1991) Ultrastructure of the normal human placenta. Electron Microsc Rev 4:129-178[Medline]

King A, Thomas L, Bischof P (2000) Cell culture models of trophoblast II: trophoblast cell lines—a workshop report. Placenta 21:S113-119[Medline]

Kliman HJ, Nestler JE, Sermasi E, Sanger JM, Strauss JF, III (1986) Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae. Endocrinology 118:1567-1582[Abstract]

Kollins KM, Powell SK, Rivas RJ (1999) GPI-anchored human placental alkaline phosphatase has a nonpolarized distribution on the cell surface of mouse cerebellar granule neurons in vitro. J Neurobiol 39:119-141[Medline]

Lipardi C, Nitsch L, Zurzolo C (2000) Detergent-insoluble GPI-anchored proteins are apically sorted in Fischer rat thyroid cells, but interference with cholesterol or sphingolipids differentially affects detergent insolubility and apical sorting. Mol Biol Cell 11:531-542[Abstract/Free Full Text]

Lisanti MP, Le Bivic A, Saltiel AR, Rodriguez–Boulan E (1990) Preferred apical distribution of glycosyl-phosphatidylinositol (GPI) anchored proteins: a highly conserved feature of the polarized epithelial cell phenotype. J Membr Biol 113:155-167[Medline]

Lisanti MP, Sargiacomo M, Graeve L, Saltiel AR, Rodriguez–Boulan E (1988) Polarized apical distribution of glycosyl-phosphatidylinositol-anchored proteins in a renal epithelial cell line. Proc Natl Acad Sci USA 85:9557-9561[Abstract]

Makiya R, Thornell L-E, Stigbrand T (1992) Placental alkaline phosphatase, a GPI-anchored protein, is clustered in clathrin-coated vesicles. Biochem Biophys Res Commun 183:803-808[Medline]

Okamoto T, Seo H, Mano H, Furuhashi M, Goto S, Tomoda Y, Matsui N (1990) Expression of human placenta alkaline phosphatase in placenta during pregnancy. Placenta 11:319-327[Medline]

Povinelli CM, Knoll BJ (1991) Trace expression of the germ-cell alkaline phosphatase gene in human placenta. Placenta 12:663-668[Medline]

Reynolds ES (1963) The use of lead citrate at high pH as an electron-opaque stain in electron miscroscopy. J Cell Biol 17:208-213[Free Full Text]

Ribitsch D, Dohr G, Hartmann M, Pilz G, Salmhofer H, Desoye G (1990) GZ 100, 101, 106, 107, 111, 112, 116, 121: a series of monoclonal antibodies against human trophoblast antigens. Acta Histochem Suppl XXXVIII: 227–231

Sun TT, Eichner R, Nelson WG, Tseng SC, Weiss RA, Jarvinen M, Woodcock–Mitchell J (1983) Keratin classes: molecular markers for different types of epithelial differentiation. J Invest Dermatol 81:109s-115s[Abstract]

Tokuyasu KT, Maher PA, Singer SJ (1985) Distributions of vimentin and desmin in developing chick myotubes in vivo. II. Immunoelectron microscopic study. J Cell Biol 100:1157-1166[Abstract]

Truman P, Ford HC (1984) The brush border of the human term placenta. Biochim Biophys Acta 779:139-160[Medline]

van Renswoude J, Bridges KR, Harford JB, Klausner RD (1982) Receptor-mediated endocytosis of transferrin and the uptake of Fe in K562 cells: identification of a nonlysosomal acidic compartment. Proc Natl Acad Sci USA 79:6186-6190[Abstract]

Watanabe T, Wada N, Kim EE, Wyckoff HW, Chou JY (1991) Mutation of a single amino acid converts germ cell alkaline phosphatase to placental alkaline phosphatase. J Biol Chem 266:21174-21178[Abstract/Free Full Text]