Expression of the proliferation markers Ki67 and transferrin receptor by human trophoblast populations

Journal of Reproductive immunology, 14 (1988) 291--302

291

Elsevier Scientific Publishers Ireland Ltd.

JR100577

Expression of the proliferation markers Ki67 and transferrin receptor by human trophoblast populations J u d i t h N . Bulmer% L y n n M o r r i s o n a a n d P e t e r M . J o h n s o n b "Department of Pathology, University of leeds, Leeds LS2 9JT and ~Department of Immunology, University of Liverpool, Liverpool L69 3BX (U.K.) (Accepted for publication 7 November 1988)

Summary lmmunohistochemical techniques were used to investigate the expression of proliferation markers (Ki67 and transferrin receptor) by fetal trophoblast in normal human pregnancy. In placental villous tissue, transferrin receptor was detected not only on the apical syncytiotrophoblastic membrane but also on the proximal portion of cytotrophoblast columns, an area of high cellular proliferative activity. The majority of cells in cytotrophoblast columns and shell showed nuclear reactivity with Ki67. Villous syncytiotrophoblast was uniformly unreactive with Ki67 but a proportion of the underlying cytotrophoblast was Ki67-positive throughout pregnancy. Occasional Ki67-positive trophoblast cells were identified within chorion laeve at term. In contrast, interstitial and endovascular extravillous trophoblast in maternal uterine decidual tissue failed to label with either proliferation marker. Thus, chorionlc villous cytotrophoblast and extravillous trophoblast in the chorion laeve appear to retain their proliferative capacity into late pregnancy. Cytotrophoblast columns represent a zone of cellular proliferation which may be dependent on transferrin. Key words:

human placenta; trophoblast; proliferation;

transferrin-

receptor; Ki6Z Introduction The chorionic villi in early h u m a n placentae are covered by inner cytotrophoblastic and outer syncytiotrophoblastic layers. In early pregCorrespondence to: Dr. J.N. Bulmer. 0165-0378/88/$03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

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nancy, cytotrophoblast proliferates from the tips of tertiary villi, forming columns of cytotrophoblast which mushroom outwards to form a cytotrophoblastic shell. Trophoblast from this shell invades maternal uterine tissue, replacing the endothelium of spiral arteries and migrating through decidua and inner myometrium (Boyd and Hamilton, 1970; Pijnenborg et al., 1980). Villous cytotrophoblast persists throughout gestation and is generally accepted to be the mitotic tissue of the placenta (Boyd and Hamilton, 1967, 1970). Mitotic figures are not generally observed in syncytiotrophoblast. Although amitotic division of syncytiotrophoblast was originally suggested as mechanism of rapid placental growth in early pregnancy (Wislocki and Bennett, 1943; Hertig et al., 1956), uptake of tritiated thymidine in autoradiographic studies was observed only in cytotrophoblast (Richart, 1961; Tao and Hertig, 1965; Hou et al., 1968). The near diploid distribution of syncytiotrophoblastic DNA in early placentae also suggests little or no replicative capacity (Galton, 1962). Trophoblast possesses proliferative capacity at term, as evidenced by successful culture of normal term placentae (Tominaga and Page, 1966; Hou and Swanson-Beck, 1967; Fox, 1970). In the present study the expression of two cellular proliferation markers by trophoblast in early and late pregnancy has been compared. Ki67 is a monoclonal antibody reactive with proliferating cells at all phases of the cell cycle except Go (Gerdes et al., 1983, 1984). Iron-laden transferrin can act as a mitogenic growth factor for several cell types and cell surface expression of the transferrin receptor correlates with proliferative activity in many normal and neoplastic human tissues (Sutherland et al., 1981; Trowbridge and Omary, 1981). Transferrin receptor has been detected on villous syncytiotrophoblast in normal human placentae (Galbraith et al., 1980; Johnson, 1984; Yeh et al., 1987), but has been reported to be absent from cytotrophoblast in term placental bed and amniochorion tissues (Johnson and Molloy, 1983). Materials and methods

T/ssues Fresh fragments (5--10 mm size) of chorionic villi and maternal uterine decidua, identified by macroscopic appearance, were obtained from 5 elective aspiration pregnancy terminations performed at 7--11 weeks' gestational age. Normal first trimester uteroplacental tissues were also obtained from 5 pregnancy hysterectomies ranging from 7--13 weeks' gestation: specimens were dissected from the placental bed to include chorionic villi, decidua and myometrium. Ten normal term placentae were obtained immediately following vaginal delivery and specimens were dissected from the maternal-facing surface to include both chorionic villi and basal plate.

293

Fragments (5--10 mm) of reflected amniochorion were mounted in a pile supported by OCT embedding medium (Raymond Lamb, London). All first trimester and term placental tissues were quenched in liquid nitrogen-cooled isopentane and stored sealed in liquid nitrogen until use. Cryostat sections (5/am) were mounted on clean glass slides, air dried overnight and f'Lxed for 10 min at room temperature in acetone.

Antibodies Three murine monoclonal antibodies (mAbs) were employed. DAKO-PC (clone Ki67) (Dakopatts A/S, Denmark) labels proliferating cells in all human tissues, reacting with cells at all stages of the cell cycle except the Go phase (Gerdes et al., 1983, 1984). DAKO-anti-transferrin receptor (clone BerT9) (Dakopatts A/S) is directed against the human transferrin receptor. Selected specimens were also labelled with CAM 5.2 (Becton Dickinson, CA, U.S.A.), a marker of low molecular weight cytokeratins, which reacts with both villous and extravillous trophoblast throughout pregnancy (Makin et al., 1984; Bulmer et al., 1986). Selected specimens were also labelled with four further murine mAbs raised against human placental microvilli (GB16, GB18, GB19 and GB22) which have been shown to react with the transferrin receptor (Yeh et al., 1987).

Immunohistochemistry Tissue sections were labelled by an indirect immunoperoxidase technique as described in detail previously (Bulmer and Sunderland, 1984). Briefly, frozen sections were rehydrated in Tris buffered saline (TBS) (pH 7.6) and incubated with primary mAb for 45 min at room temperature. The dilutions used were; Ki67:1 in 5; anti-transferrin receptor: 1 in 20; CAM 5.2:1 in 10. After two brief washes in TBS, sections were incubated for 30 min with peroxidase-conjugated rabbit anti-mouse immunoglobulin (Dakopatts A/S, Denmark) diluted 1:50 in TBS. After two further TBS washes, the reaction was developed with 3,3'-diaminobenzidine (DAB). After washing with excess water, sections were lightly counterstained with Mayer's haematoxylin, dehydrated, cleared and mounted in synthetic resin. In some sections the brown nuclear DAB reaction product obtained with DAKO-PC was partly obscured by the haematoxylin counterstain. An alternative methyl green counterstain failed to give satisfactory staining of uteroplacental tissues (J.N. Bulmer, unpublished observation). Hence, sections were labelled with DAKO-PC using a streptavidin-biotin-peroxidase kit (Zymed, CA, U.S.A.) as well as by the indirect immunoperoxidase technique. The sections were incubated sequentially with the primary rnAb (30 min), biotinylated anti-mouse immunoglobulin (10 min) and streptavidin-peroxidase (5 min) as per the supplier's instructions. The reaction was

294

developed with aminoethylcarbazole (AEC) to give a bright red reaction product. Sections were lightly counterstained with haematoxylin and mounted in aqueous mountant. The reaction patterns obtained with DAKOPC by the streptavidin-biotin technique were comparable with those seen by indirect immunoperoxidase but the bright red AEC reaction product was not obscured by the nuclear counterstain. Tissue sections of a gliohlastoma multiforme were used as a positive control for DAKO-PC. Negative controls were performed at all levels of the immunohistochemical reaction. Results The results are summarised in Table 1.

First trimester placentae Sections were initially labelled with DAKO-anti-transferrin receptor; four further anti-transferrin receptor mAbs (GB16, GBIS, GB19 and GB22) produced comparable results. The transferrin receptor was localised to the apical membrane of villous syncytiotrophoblast (Fig. la). Villous cytotrophoblast did not label, as noted by others (Yeh et al., 1987), but reactivity

TABLE

I

Reactivity of trophoblast with D A K O - P C

(Ki67)

and anti-transferrin receptor.

First trimester placenta

Term placenta

Ki67

Ki67

Transferrin

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-

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-

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-

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NA

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-

NA

NA

Chorion laeve c T E n d o m e t r i a l gland epithelium

NA

NA

+

-

-

+ + +

-

syncytiotrophoblast; cytotrophoblast; NA, not applicable; - , no positive cells; + , 0 - - - 2 5 % cells positive; + + , 2 5 - - 5 0 % cells positive; 4. + 4- 5 0 - - - 7 5 % cells positive; 4- 4- 4- 4 - , 7 5 - - 1 0 0 % cells positive.

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Fig. 1. C r y o s t a t sections o f first t r i m e s t e r u t e r o p l a c e n t a l tissues labelled w i t h (a) D a k o - a n t i - t r a n s f e r r i n receptor, Co, c) Ki67 u s i n g a n i n d i r e c t i m m u n o p e r o x i d a s ¢ t e c h n i q u e . (a) S h o w s I o c a l i s a t i o n o f t r a n s f e r r i n receptor t o the a p i c a l m e m b r a n e o f villous s y n c y t i o t r o p h o b l a s t . Co) S h o w s reactivity o f Ki67 w i t h villous c y t o t r o p h o b l a s t (am'rowed) a n d c y t o t r o p h o b l a s t c o l u m n s (C). (c) S h o w s lack o f reactivity o f Ki67 w i t h e x t r a v i l l o u s t r o p h o b l a s t in m a t e r n a l d e c i d u a . M a g n i f i c a t i o n : (a) x l 0 0 ; Co) x l 0 0 ; (c) ><250.

was observed in the proximal portion of the cytotrophoblast columns close to the villi and in the outer aspect of the cytotrophoblast shell. There was no reactivity with any interstitial or endovascular trophoblast in maternal uterine decidual and myometrial tissues. Endometrial gland epithelial cells were transferrin receptor-positive with most intense reactivity on their basal aspect, as described previously (Johnson and Bulmer, 1984). Many maternal cells in decidua also reacted for the transferrin receptor: these included large decidualised stromal cells and smaller lymphocyte-like cells. Occasional intensely positive cells were identified close to endometrial glands, as described earlier (Johnson and Bulmer, 1984). Villous syncytiotrophoblast was uniformly unreactive with Ki67. In contrast, villous cytotrophoblast showed nuclear staining of up to 50o70 of cells (Fig. lb), the proportion of positive cells varying both within and between specimens. Ki67 consistently reacted with at least 75o70 and often almost all cells within cytotrophoblast columns (Fig. lb). There was also labelling of the majority of trophoblastic cells on the outer aspect of the cytotrophoblast shell. Interstitial and endovascular trophoblast within maternal uterine tissues were uniformly unreactive with Ki67 (Fig. l c) and comparison of

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Fig. 2. Cryostat sections of (a) term villous palcenta, (b) term basal plate, (c) term amniochorion labelled with Ki67 by streptavidin-biotin-peroxidas¢. (a) Shows labelling of scattered cytotrophoblast cells in chorionic villi (arrowed). (b) Shows small groups of Ki67-positive trophoblast in the basal plate. (c) Shows reactivity of single cells in chorion laeve (arrowed). Magnification: (a) 160; (b) 400; (c) 160.

adjacent sections labelled respectively with CAM 5.2 and Ki67 confirmed this impression. Endometrial gland epithelial cells in early pregnancy were generally unreactive with Ki67. Several small scattered lymphocyte-like cells within decidual tissue showed nuclear reactivity with Ki67; these were predominantly in the zona spongiosa and most probably represented the endometrial stromal granulocytes (K6rnchenzellen), which have recently been shown to be granulated lymphocytes (Bulmer et al., 1987; Pace et al., 1988).

Term placenta Villous syncytiotrophoblast showed expression of transferrin receptor on its apical membrane whereas villous cytotrophoblast was uniformly negative, as was extravillous trophoblast in the basal plate. Decidual cells in basal plate showed weak reactivity for transferrin receptor. Cytotrophoblast in the chorion laeve was also uniformly unreactive with the five anti-transferrin receptor mAbs, although there was weak labelling of the attached decidua parietalis. Villous syncytiotrophoblast did not label with Ki67. Ki67-positive cytotrophoblastic cells were identified in most chorionic villi (Fig. 2a),

299

although these were less numerous than in first trimester placental tissues. Rare extravillous trophoblast cells in the basal plate reacted with Ki67, often situated in small groups (Fig. 2b). In contrast with early pregnancy decidua, rare Ki67-positive lymphoid cells were identified in basal plate tissue. The chorion laeve consistently contained individual Ki67-positive trophoblast (Fig. 2c): cells showing Ki67-nuclear reactivity were randomly distributed in both maternal and fetal aspects. Discussion

The tissue distribution of two cell proliferation-associated markers has been examined in human uteroplacental tissues. The detection of transferrin receptor on villous syncytiotrophoblast in pre-term and term pregnancy is in accord with other studies (Galbraith et al., 1980; Johnson, 1984; Yeh et al., 1987). The role of the syncytiotrophoblastic transferrin receptor has been a source of speculation. Although it is most likely to be associated with iron uptake to fetal tissues and has evolved for this reason, an immunological role has also been suggested whereby transferrin-binding restricts available iron in the intervillous spaces and hence impairs proliferative responses of maternal blood leucocytes at this site (Johnson, i984). Syncytiotrophoblast is a terminally differentiated non-dividing multinucleated cell and expression of the transferrin receptor is not in keeping with its proposed role as a proliferation marker. Villous syncytiotrophoblast did not show any labelling with Ki67, and hence the expression of transferrin receptor at this site clearly cannot be correlated with cellular proliferative activity. Moe (1971) observed metaphase spreads in both syncytial sprouts and villous syncytiotrophoblast in placentae removed from women given colchicine before elective pregnancy termination. He argued that the mitotic rate within syncytiotrophoblast was low and hence only apparent after metaphase arrest: this explained the deficiency of mitoses in other studies. The lack of reactivity with Ki67, which labels proliferating cells at all stages of the cell cycle excluding Go, does not lend support to this suggestion. Cytotrophoblast has been established as the proliferating cell within normal chorionic villous tissue. Although the transferrin receptor was not detected on villous cytotrophoblast even in early pregnancy, Ki67 reacted with villous cytotrophoblast in both early pregnancy and at term. The presence of proliferating cells in late pregnancy is not surprising since the term placenta is capable of in vitro proliferation (Loke, 1983). Columns of cytotrophoblast proliferate from the tips of chorionic villi in early pregnancy (Boyd and Hamilton, 1970). Maximal mitotic activity has been reported in these cytotrophoblast columns, particularly in the proximal portion (Wislocki and Bennett, 1943). In the present study, the majority of cells in

300

cytotrophoblast columns and shell labelled with Ki67 in early pregnancies. Cytotrophoblast columns and shell were also transferrin receptor-positive, the proximal part labelling more intensely than the distal parts. Recently, c-myc and c-sis oncogenes have been detected in the cytotrophoblast columns and shell of normal early human placentae (Pfeiffer-Ohlsson et al., 1984; Goustin et al., 1985). Both these proto-oncogenes are considered to be concerned with cell growth and proliferation, the c-s/s product showing structural homology with the/1-chain of PDGF (Waterfield et al., 1983). Thus, there is supporting evidence that cytotrophoblast columns are a zone of maximal proliferative activity. The chorion laeve is formed by retrogression of chorionic villi at the abembryonic pole of the placenta (Boyd and Hamilton, 1970). Ki67-positive trophoblast was consistently detected in chorion laeve cytotrophoblast in all placentae, although the transferrin receptor was not detected. The chorion laeve forms a major materno-fetal tissue interface between fetal amnion and maternal decidua and it is possible that this trophoblast retains its capacity to proliferate and repair defects, thus maintaining an unbroken layer. Endometrial gland epithelial cells in early pregnancy decidua showed expression of the transferrin receptor although they were negative for the Ki67 antigen. In contrast, in non-pregnant endometrium from the secretory phase of the menstrual cycle, a small proportion of glandular epithelial cells show nuclear reactivity with Ki67 (D. Pace and J.N. Bulmer, unpublished observations). These data are supported by recent reports of proliferative activity in secretory phase endometrial glands detected by flash labelling with bromodeoxyuridine as well as uptake of tritiated thymidine (Thornton et al., 1988). Endometrial glands exhibit decreased expression of class I MHC antigens in early pregnancy and are associated with an unusual mononuclear cell response (Johnson and Bulmer, 1984; Bulmer and Johnson, 1985). In later pregnancy, many glandular structures in the placental bed are identified only as attenuated channels (Bulmer et al., 1986). The function of the endometrial glands in pregnancy is not known, although immunological suppressor-type activity has been reported in epithelial cells separated from non-pregnant endometrium (Johnson et al., 1987). In a recent study of murine pregnancy, uterine glandular epithelial cells were shown to express M-CSF mRNA, which was regulated by steroid hormones (Pollard et al., 1987); there are no comparable data for humans but endometrial glands in pregnancy are worthy of further investigation. In summary, the expression of the proliferation markers, transferrin receptor and Ki67, has been documented for fetal trophoblast populations in early pregnancy. Proliferative capacity was retained by villous cytotrophoblast throughout pregnancy and the major site of proliferative activity was localised to the cytotrophoblast columns.

301

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