The appearance of transferrin receptors on cultured human cytotrophoblast and in vitro-formed syncytiotrophoblast

Placenta (1988), 9,387-396

The Appearance of Transferrin Receptors on Cultured Human Cytotrophoblast and in Vitro-formed Syncytiotrophoblast

M. B. BIERINGS, J. P. VAN DIJK

H. J. ADRIAANSEN”

&

Departments of Chemical Pathology and Cell Biology, Immunology and Genetics, Erasmus University Rotterdam, PO Box 17.38,3000 DR Rotterdam, The Netherlands Paper accepted I0.3.I988

INTRODUCTION Maternal-fetal iron transport starts with the binding of maternal transferrin to transferrin receptors at the apical side of syncytiotrophoblast in human placenta (Fletcher and Suter, 1969; Galbraith et al, 1980). The presence of transferrin receptors at this villous membrane has been shown using immunofluorescence on cryostat sections (Faulk and Galbraith, 1979; Galbraith et al, 1980; Galbraith, Galbraith and Faulk, 1980). Ultrastructural studies showed transferrin (King, 1976; Parmley, Barton and Conrad, 1985) and its receptor (Parmley, Barton and Conrad, 1985), as did binding studies on a biochemical preparation of this membrane (Wada, Hass and Sussman, 1979; Brown, Molloy and Johnson, 1982). Placental transferrin receptor has also been isolated from placental homogenate (Seligman, Schleicher and Allen, 1979) and microvillous membrane preparations (Wada, Hass and Sussman, 1979; Loh et al, 1980; Brown and Johnson, 1981; Tsunoo and Sussman, 1983). Recently the presence of transferrin receptors was also reported on the basal plasma membrane of human placental syncytiotrophoblast by Vanderpuye, Kelley and Smith (1986) using a purified biochemical preparation of this membrane (Kelley, Smith and King, 1983). Using cryostat tissue sections and a monoclonal antibody against the transferrin receptor, transferrin receptors were shown to be absent from villous cytotrophoblast (Hsi, Yeh and Faulk, 1982; Bulmer and Johnson, 1985). Cytotrophoblast is the proliferative cell type in human placenta, even at term. This cell type is thought to give rise to syncytiotrophoblast through cellular fusion (Kliman et al, 1986; Kliman, Feinman and Strauss, 1987). Kliman et al (1986), using a standard trypsinization procedure (Hall et al, 1977) and a Percoll gradient centrifugation addition, isolated a mononuclear cell population from human term placentae. These cells are thought to be cytotrophoblast, since they tend to fuse in vitro and start producing Schwangerschaftsprotein (SP,), human chorionic gonadotrophin (hCG) and human placental lactogen (hPL) (Kliman et al, 1986; Kliman, Feinman and Strauss, 1987). Being a proliferative cell type, it is surprising that cytotrophoblast lacks transferrin receptors. It is well known that the presence of transferrin receptors is associated with cellular proliferation and activation (Trowbridge and Omary, 1981; May and Cuatrecasas, 1985). or43-4004/88/040387

+ IO $03.00/o

17 1988 Baillike Tindall Ltd

Plarentu (19X8), Vol. 9

388

We isolated mononuclear cell populations from human term placentae, characterized them and studied their behaviour in vitro. A well-characterized cell culture method for trophoblast can become an important tool in the study of placental iron uptake.

MATERIALS

AND METHODS

Mononuclear cell preparation Normal term human placentae were obtained within half an hour of spontaneous vaginal delivery. Villous structures were cut out from the maternal side, washed extensively, minced, and subjected to the trypsinization procedure described by Hall et al (1977) with the Percoll gradient addition of Kliman et al (1986). Essentially, the villous tissue was subjected to three 3o-min trypsin-DNase digestions using, respectively, 5, 3.3 and 2.5 ml/g tissue of a calcium- and magnesium-free solution of Earle’s balanced salts (CMFS, Gibco) containing 1250 BAEE units trypsin (type III, Sigma Chemical Co., or I : 250, Sigma) and 475 Kunitz units DNase I, grade II (Boehringer Mannheim) per millilitre. The resultant cell suspensions were centrifuged through fetal calf serum (FCS, Flow Labs). The resultant cells were pooled, resuspended in MI99 (Flow Labs) and carefully layered on a preformed Percoll gradient (Percoll from Pharmacia), 5 to 70 per cent Percoll with CMFS in 5 per cent steps of 3 ml each, and centrifuged for 20 min at I 200 g at room temperature. The gradient was then fractionated by inserting a glass capillary from the top to the bottom of the tube, collecting I ml fractions. These fractions were washed once, using Earle’s balanced salts solution (EBSS, Gibco), and checked using phasecontrast microscopy. Mononuclear cell fractions were used for cell culture and characterization, using immunofluorescence and immunoperoxidase staining procedures. Cell culture Cells were counted using a Burker couniing chamber. They were diluted and plated out at a density of 6 x 10~ cells/ml in j-ml Petri dishes, containing thin glass coverslips, coated with fetal calf serum. Culture medium was hi199 (Flow Labs) supplemented with 4 mM L-glutamine, 20 per cent FCS, 50 pg/ml gentamicin or penicillin IOO U/ml/streptomycin ( IOO pg/ml) and fungizone (2.5 pg/ml, Gibco). All cultures were incubated in humified 5 per cent CO,/95 per cent air at 37°C for designated times. After 24 h non-adherent cells were gently washed off and the medium was replaced. Immunofluorescence Immunological marker analysis. Mononuclear cells were incubated with monoclonal antibodies directed against a B cell antigen, T cell antigens, and myeloid/monocytic antigens. Detailed information about the monoclonal antibodies used is summarized in Table I. For immunological marker analysis of cultured adherent cells non-fixed slides were incubated with antibodies against the monocytic antigen CD14 (My4) and the transferrin receptor (661G10). As a second-step reagent we used a fluorescein isothiocyanate- (FITC-)conjugated goat anti-mouse immunoglobulin antiserum (Central Laboratory of the Blood Transfusion Service, Amsterdam, The Netherlands). The fluorescence stainings were evaluated using Zeiss (Carl Zeiss, Oberkochen, FR Germany) and Leitz (Ernst Leitz, Wetzlan, FR Germany) microscopes. The microscopes were equipped with phase-contrast facilities. Zmmunoperoxidase. All reagents were from DAK0 corporation (Santa Barbara, USA). Immunoperoxidase stainings were done on paraffin sections of term villous tissue, methanolfixed smears of Percoll-purified mononuclear cell populations and fixed cells cultured on glass

Brrrzn~s, ,4druransen, mn Dik: 7‘ransferrin Receptors in Cultured C.ytu- and S.yncyt~otrophoblast Table I Immunological marker analysis on mononuclear gation using trypsin type III

cells from Percoll gradient centrifu-

Percentage of positive cells

Antigen recognized

CD (antibody)

I I

B cell antigen TI antigen T3 antigen Monocytic antigen Myeloid antigen HLA-DR, non-polymorphic antigen Transferrin receptor (Tg antigen)

CD20 (Bib) CD5 (Leu-1,) CD3(I.cu-4,) CD14 (My 4h) Cl>15 (VIM-D5”) anti-HI.A-DR’ 66rG10

389

iv;

iv;1

4o z

0.5 16 o

o 17 o

39 3’

‘3 I*

16

z

I

N) = not determined.

” Cluster of differentiation as proposed by the Workshops on Human Leukocyte Differentiation Antigen (Paris, France, 1982; Boston, MA, USA, 1984; Oxford, UK, 1986).

j, Coulter Clone, Hialeah, FL, USA. Becton Dickinson, Sunnyvale, C.4, USA.

/ Dr W. Knapp, Vienna, Austria. Dr J. ,I1 van de Rijn, Amsterdam,

coverslips.

Fixation

per cent)

at room

placing

them

The Netherlands.

with 20 per cent (v/v) hydrogen

was for 20 min in methanol temperature.

Paraffin

was removed

for 30 min at 56°C before

slides were placed in absolute

transferring

methanol

for 5 min with 3 per cent hydrogen

from them

the sections

to a toluene

for 3 min and rehydrated.

peroxide

to remove

before

staining

by

bath for 3 min. Then

Sections

any remaining

peroxide (3

were then covered

endogenous

peroxidase

activity. All incubations Specimens

were followed

were first covered

immunoglobulins. bodies against

by rinsing

with normal

hPL, hCG and SP, were applied. After 20 min specimens

immunoglobulins

for 20 min.

The

This was followed

by a 4o-min

incubation

Specimens

Brdll

mounting

incubations,

had been cultured

were washed

were incubated coverslips

this was replaced

by nor-

with swine anti-rabbit

substrate

hydrogen

and covered

solution

(PAP). of amino-

peroxide.

with

Mayer’s

in 2 per cent (v/v) NH,OH,

direct wnmunoJuorescence. for approximately

These

40 h. Coverslips

(BrdU,

twice.

fixed in 70 per cent ethanol 0.07 M NaOH

water

of

anti-

haematoxylin

they were mounted,

for using

medium.

ing IO PM bromodeoxyuridine coverslips

pH 5.2, containing

binding

(rabbit-raised)

of peroxidase/antiperoxidase

with a freshly prepared

with distilled and dipped

controls

and incubated

step consisted

pH 7.6, for 30 min.

non-specific

off and primary

In negative

were washed

third

in o. I M acetate buffer, were rinsed

8 min. After being washed glycergel

in 0.05 M Tris, to suppress

serum

After 20 min excess serum was tapped

mal swine serum.

ethylcarbazole

and washing swine

Sigma

for 2 min followed

After

by immersion

with a FITC-conjugated

were done with cells which

were washed

Co.) was applied.

For the visualization for 30 min.

experiments

of BrdU

air-drying,

mouse anti-BrdU

After

2 h in the

incorporation

the coverslips

in o. I M Na,B,O,,

were washed twice before being mounted

twice and medium

antibody

for microscopic

containincubator

coverslips

pH 8.5. Subsequently (Becton

were

were immersed Dickinson).

in they The

examination.

RESULTS Mononuclear cell preparation and characterization The pellets from three consecutive trypsinizations were loaded on a Percoll gradient.

Centrifu-

Placenta (198X), Vol. ()

390 Table 2. Immunological

marker analysis on Percoll-purified mononuclear cells: results from six different experiments, using trypsin type III Percentage Number

1

ofpositivecells

CD14 (My41

66IGIO

2

40 9

3’ 8

3 4

3 I

4 z

z

‘7 16

I2 16

gation resulted in three distinct layers: a bottom layer, consisting primarily of erythrocytes; a top layer, consisting of cellular debris and tissue fragments; a middle layer, with a density in the range 1.048 to 1.062 g/ml as determined by parallel-run gradients loaded with density marker beads (Pharmacia), which consisted of mononuclear cells. The middle layer consisted of one or several closely related bands. These bands were always difficult to separate, using phase-contrast microscopy. Immunological marker analysis revealed that the cell preparations contain only a few B and T lymphocytes, but a variable amount of monocytes. An example of such an analysis is given in Table I. The variability in the percentages of monocytes found in four different experiments is given in Table 2. It appeared that fractions from Percoll gradients contain somewhat fewer monocytes towards lower density (results not shown), but no fraction was entirely free of monocytes. Table 2 also shows that the amount of cells staining positively for monocyte markers correlates highly with the percentage of cells containing transferrin receptors in their outer membrane. All these suspensions gave rise to syncytiotrophoblast formation in vitro (see next section). All these experiments were done using trypsin type III. The use of trypsin I : 250 resulted in higher cell yields, making cell culture easier. Immunoperoxidase staining on fixed smears of Percoll-purified mononuclear cells was negative for all three antigens examined (SP,, hCG and hPL). Cell culture and characterization of cultured cells In all experiments discussed in this section trypsin 1:250 was used to improve cell yield. When cultured, cells readily adhered and after 24 h approximately 70 per cent of adherent cells were mononucleated, round, or with villus-like cytoplasmic protrusions; 20 per cent were binucleated. Approximately IO per cent of all cells contained 3, 4 or 5 nuclei. Using phasecontrast microscopy it is practically impossible to judge whether cells are adjacent or truly syncytial. Immunoperoxidase-processed coverslips are clearer in this respect. Probably only ultrastructural evidence can truly reveal a syncytial nature. During prolonged culture periods, the percentage of mononucleated cells slowly decreased (60 per cent at 40 h, 20 per cent at 72 h) and the relative amount of multinucleated groups or syncytia increased. After 72 h in culture 25 to 35 per cent of cells contained more than ten nuclei, sometimes up to 50. Immunoperoxidase staining for hCG, hPL and SP, on paraffin sections gave a pronounced red colour of similar intensity for all three antigens, strictly limited to syncytiotrophoblast. A negative control, replacing the first antibody with normal swine serum, was entirely negative. Immunoperoxidase staining for SP,, hCG and hPL on methanol-fixed coverslips gave the

Bwrings, Ad&amen,

van Dtjk: Transfer&

Receptors in Cultured cyto- and Syncytrotrophobkst

391

Figure 1. Morphology of cultured cells after q h in culture: syncytial structure, containing four nuclei and manonucleated cells. Note the villus-like cytoplasmic protrusions. In the original colour photograph, cytoplasm is coloured red, produced by immunoperoxidase staining directed against SP,. Nuclei were counterstained with haematox!lin. x 1250.

following results. For SP,, after 24 h, the red colour reaction was strongly positive. Both mononucleated and multinucleated cells were positive. The reaction occurred mainly in the perinuclear region, and only slightly in cytoplasmic protrusions. An example is shown in Figure I. In occasional mononucleated cells the colour reaction was only very slightly positive. After 40 h the colour was even more intense, and more pronouncedly perinuclear. At this time no negative-staining cells were seen on the coverslips studied. Expanding syncytial structures coloured less intensely. In some cases mononucleated cells apparently in the process of fusion with syncytial structures can be seen. These mononucleated cells react extraordinarily strongly. After three days in culture, very large syncytia were seen with a reaction which varied within syncytia. The strongest reaction was again perinuclear. Mononucleated cells appear to react less intensely at this time. hCG peroxidase staining on coverslips resulted in a markedly less intense colour reaction, with a stronger progressive time-course. Mainly binucleated cells and multinucleated syncytia gave a positive reaction. The same applies to apparently-fusing mononucleated cells. This colour intensified over the first 40 h. After 72 h expanding syncytia reacted less strongly. At this time point, mononucleated isolated cells reacted only weakly. The peroxidase reaction for hPL was less strong than that for hCG. After 24 h most cells

392 Table 3. Immunological marker analysis on cultured cells: percentage of monocytic cells and transferrin receptor-positive cells in different experiments. Trypsin 1:250 was used to isolate cells Culture period (hours)

Percentage of positive cells CD14 (My41

661GIO

‘9

2

16

36 36 40 40

0

9 9

55 45 96

0

82

ii:

0 7

iy

reacted slightly positively. A stronger reaction was seen from small groups with two or three nuclei. Few mononucleated cells were entirely negative. After 72 h the intensity of the colour reaction was not markedly increased for this antigen. Syncytial structures were apparently homogeneously weakly positive. Negative control slides were always included and were always entirely negative. The results of the immunological marker analysis on cultured cells at different time-points for the monocytic marker CD14 and the transferrin receptor are summarized in Table 3. It is clear that the appearance of transferrin receptors on cultured cells is time-dependent. Both mononucleated and multinucleated cells reacted positively. Since it is nearly impossible to distinguish with certainty between these forms, using uncoloured specimens, we have not tried to quantify this. It is noteworthy that no clearly syncytial structures were seen that did not stain positively for transferrin receptor. Figure 2 shows an example of immunofluorescence studies. Incubation for 2 h with BrdU of cells cultured for 40 h revealed that less than 2 per cent of these cells were active in DNA synthesis.

DISCUSSION We isolated a mononuclear cell suspension from term human placental villous tissue. Our results indicate that this cell suspension consists primarily of cytotrophoblast and monocytes. Kliman and co-workers, using the same isolation procedure, studied contamination by endothelium, macrophages and fibroblasts. They did not find any of these three cell types (Kliman et al, 1986). Recently Main, Strizki and Schochet (1987), also using this isolation method, described the presence of I .2 to 8.2 per cent (depending on the marker used) of monocytes in mononuclear cell preparations. This was in the Percoll gradient zone with a density of 1.048 to 1.055 g/ml. The relative proportion of monocytes increased with increasing density (density 1.055 to 1.075 g/ml; 13 to 23 per cent monocytes). This appears to be similar to our results. Immunological marker analysis on freshly prepared mononuclear cell suspensions from a number of experiments clearly shows that there is a good correlation between the percentage of cells positive for the monocytic marker CD14 and the transferrin receptor respectively. This suggests that monocytes are the main, if not only, cell type having transferrin receptors. (Only a double-staining procedure, which we have not yet attempted, could prove this.) This would

Bwings, .4drraansen, wn Dijk: Transj~rrrn Receptors in Cultured Cyto- and Synrytiotrophoblast

393

F’qure 2. Phase-contrast (a) and fluorescence (b) microscopy of cells cultured for 40 h, using indirect immunofluorescencc for transferrin receptor using a monoclonal antibody as described. Apparently two groups of cells are making contact and may be fusing. Positive fluorescence can be seen on the whole outer cell membrane. x 630.

394

Phcenra (ry&Y), Vol. !,

imply that cytotrophoblast lacks transferrin receptors. That would be in accordance with findings from cryostat sections (Faulk and Galbraith, 1979; Galbraith et al, 1980; Galbraith, Galbraith and Faulk, 1980). In vitro, as is shown by us, the transferrin receptor appears on both cytotrophoblast and syncytiotrophoblast. The latter is a highly differentiated, not mitotically active terminal cell stage. Apparently cytotrophoblast is an exception to the rule that mainly undifferentiated, proliferative cell types contain many transferrin receptors (Trowbridge and Omary, 1981; Huebers and Finch, 1987). It also indicates that normal cytotrophoblast differs considerably from BeWo chorion carcinoma cells in this respect (van der Ende et al, 1987). All marker analyses on Percoll cells were done using trypsin type III. Trypsin 1:250 results in higher yields and is therefore more suitable for culture purposes. Initial cell populations can differ, however, depending on the type, and moreover batch, of trypsin used (Morrish and Siy, 1986). The undifferentiated state of cytotrophoblast in vivo results in a lack of markers. So far, only research applications of monoclonal antibodies directed against first-trimester cytotrophoblast have been reported (Butterworth et al, 1985; Loke et al, 1986; Contractor and Sooranna, 1986). Some of these monoclonals have been shown to react specifically with villous cytotrophoblast, preterm or term. It would be of great interest to use these monoclonals to characterize Percoll gradient-purified mononuclear cells. In vitro, in our system, cytotrophoblast lacks many physiological environment factors: basement membrane and cellular contacts with cyto- and syncytiotrophoblast. In vitro, cytotrophoblast is exposed to calf transferrin in the culture medium. This could also influence the expression of the transferrin receptor. Our results show that under these in vitro conditions cytotrophoblast does not remain in its undifferentiated state. The loss of cell contact could very well induce the expression of transferrin receptors. On the other hand, expression of the transferrin receptor can be part of an integrated gene program, gradually expressed in the transition of cyto- to syncytiotrophoblast. Ulloa-Aguirre et al (1987) showed that bromo-CAMP regulates the expression of both hCG and fibronectin in cytotrophoblast cultures. This could also be the case for the transferrin receptor. Transferrin receptor, hCG, SP, and hPL possibly follow distinct and complex time-courses as if responding to several independent regulatory signals. Our immunoperoxidase results show that freshly prepared cytotrophoblasts lack SP,, hCG and hPL. In vitro, SP, is rapidly and strongly expressed. hCG follows somewhat later, and hPL is thought to be only weakly positive. This is in accordance with Kliman’s findings (Kliman et al, 1986). In particular, the expression of SP, and to a somewhat lesser extent hCG and hPL is, in vitro, not limited to syncytiotrophoblast. The same applies to the transferrin receptor. After 60 to 72 hours the vast majority of cells and cellular complexes express transferrin receptors, whether mononucleated or multi,nucleated. This is not related to DNA synthesis in preparation for cell division, as in mitogen-stimulated T lymphocytes (Neckers and Cossman, 1983). After two to three days in culture our culture system could serve as a model to study receptor-mediated transferrin uptake in trophoblast.

SUMMARY

Using trypsinization and Percoll gradient centrifugation, mononuclear cell populations were prepared from human term placentae. These populations consist mainly of cytotrophoblast, which probably lacks transferrin receptors. In vitro, transferrin receptors appear on these

Bwrrngs, 4drwansm, an DiJk: Trunsferrm Receptors in Cultured C’yto- and .~,~nc)~t~otrophohlast

mononuclear cells and on syncytiotrophoblast fusion and proliferative state.

formed in vitro, independently,

395

of cellular

ACKNOWLEDGEMENTS The authors wish to thank Mr M. J, Kroos and Mrs J. M. Wijkhuijs for their excellent technical assistance. We would also like to thank the I~epartment of Obstetrics (Professor I-I. C. S Wallenburg) for their kind cooperation in supplying us writh placentae.

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is required

for DNA synthesis and cell division and is regulated

by interleukin

2. Proceedmgs of the Nattonal

Academy of Sciences of the USA, 80.3494-3498.

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van der Ende, A., Du Maine,

A., Simmons,

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Vanderpuye, 0. A., Kelley, L. K. & Smith, C. H. (1986) Transferrin receptors in the basai plasma membrane of the human placental syncytiotrophoblast. Placenta, 7,405-4x5. Wada, H. G., Hass, P. E. & Sussman, H. H. (1979) Transferrin receptor in human placental brush border membranes. 3oournal of Biological Chemistry, 254, IZ 62912 635.