Reactivity of human trophoblast with an antibody to the HLA class II antigen, HLA-DP

Reactivity of human trophoblast with an antibody to the HLA class II antigen, HLA-DP

Journal of Reproductive Immunology, 11 (1987) 63-70 63 Elsevier Scientific Publishers Ireland Ltd. JRI 00476 Brief Communication Reactivity of hu...

2MB Sizes 0 Downloads 32 Views

Journal of Reproductive Immunology, 11 (1987) 63-70

63

Elsevier Scientific Publishers Ireland Ltd.

JRI 00476

Brief Communication

Reactivity of human trophoblast with an antibody to the HLA class II antigen, HLA-DP Phyllis M. S t a r k e y Nuffield Department of Obstetrics and Gynaecology, John Radcliffe Hospital, Headington,

Oxford OX3 9DU (U.K.) (Accepted for publication 24 March 1987)

Summary Cytotrophoblast cells in term amniochorion and in first trimester chorionic villi were shown by immunohistology of frozen tissue sections to bind B7/21, an antibody specific for the MHC Class II antigen, HLA-DP. This binding was shown to be specific, as adsorption of the B7/21 antibody with a B cell line expressing HLA-DP prevented subsequent binding to trophoblast. When tested with a variety of other antibodies reacting with HLA-DR, HLA-DQ or the common sequences of HLA-DR, -DQ and -DP, trophoblast was negative, thus confirming previous reports. The significance of this unique pattern of reactivity of trophoblast is discussed. Key words: human trophoblast, MHC-Class H, HLA-DP, immunohistology In early human pregnancy, cytotrophoblast cells invade the maternal decidua and remain in intimate contact with maternal tissues throughout gestation (Boyd and Hamilton, 1970). Despite their potential for recognition by the maternal immune system, the placenta and fetus survive as a semi-allograft. It was previously thought that trophoblast cells did not express the major histocompatibility complex (MHC) antigens and thereby avoided immune rejection (Goodfellow et al., 1976). It is now known, however, that the extra-villous cytotrophoblast cells which invade the maternal tissues express HLA-Class I antigen from as early as 6 weeks of gestation (Sunderland et al., 1981a; Redman et al., 1984). The trophoblast Class I M H C antigen is different to that of adult cells, however, in 0165-0378/87/$03.50 ¢~) Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

64

that it has a heavy chain of M r about 40 000 which does not appear to express the polymorphic HLA-A, -B or -C determinants (Redman et al., 1984; Ellis et al., 1986). Trophoblast has been shown, using CR3/43 (an antibody which recognises an epitope common to HLA-DR, -DQ and -DP), not to express HLA-Class II antigens (Sunderland et al., 1981a,b; Redman et al., 1984). Brami et al. (1983), using tissue sections of first trimester chorionic sac that had been pretreated with trypsin, have reported faint binding of an anti-HLA-DR antibody to trophoblast, but the physiological significance of this observation is doubtful. The present study demonstrates that some trophoblast populations react with B7/21, an antibody specific for HLADP (Watson et al., 1983). Tissues were obtained within 1 h following first or second trimester therapeutic abortions, or at term after normal vaginal delivery, and blocks were snap-frozen in liquid nitrogen and stored at -70°C. Cryostat sections (7-8 ~m) were cut, acetone fixed, and stained with mouse monoclonal antibodies (see Table 1 for details), followed by peroxidase-conjugated rabbit anti (mouse immunoglobulin) IgG, sometimes enhanced by an additional step of peroxidase-conjugated swine anti (rabbit immunoglobulin) IgG, as previously described (Sutton et al., 1986). Both conjugated antibodies were obtained from Dakopatts a/s, Copenhagen, DenTABLE 1

Monoclonal antibodies used in immunohistology. Antibodies were obtained from the following sources: CR3/43, Dr. D.Y. Mason, Dept. of Histopathology, John Radcliffe Hospital, Oxford, UK; DA6/164, Dr. M. Steel, MRC Clinical and Population Cytogenetics Unit, Edinburgh, UK; Leu 10, Laboratory Impex Ltd., Twickenham, Middlesex, UK; B7/21, Dr. A.J. Watson, Immunobiology Research Centre, University of Minnesota, Minneapolis, MN 55455; L243, Dr. G.M. Taylor, Department of Medical Genetics, St Mary's Hospital, Manchester, UK; NFK1, NFK2, NDS21, Dr. S.V. Fuggle, and JMB2, Prof. J.O.'D. McGee, Nuffield Department of Surgery, John Radcliffe Hospital, Oxford, UK. Antibody

Specificity

Reference

CR3/43 DA6/164 Anti-Leu 10 B7/21 a L243 NFK1 NFK2 NDS21 JMB2

D R / D P / D Q /3, a/3 DR a/3 DQ DP DR DR/DP/?DQ DR/DP/?DQ

Sunderland et al., 1981b Van Heyningen et al., 1982 Chen et al., 1984 Watson et al., 1983 Lampson and Levy, 1980 Fuggle et al., 1983 Fuggle et al., 1983 S.V. Fuggle (pers. commun.) McGee et al., 1982

DR/DP/?DQ Intermediate filament

a This antibody is unrelated to the B721 antibody reCently described as reacting with a 75 000 M r membrane glycoprotein present on syncytiotrophoblast, vascular endothelium and smooth muscle cells (Voland et al., 1986).

65

66

Fig. 1. Reactivity of membrane rolls with antibodies to HLA-Class II antigens. Indirect two-stage immunoperoxidase staining of serial sections of membrane roll taken from term placenta and labelled with (a) CR3/43, binding to macrophages in the decidua and amniotic connective tissue, (b) B7/21, labelling cytotrophoblast in the chorion in addition to macrophages as above, and (c) no first antibody as a control: D, decidua; C, chorion; A, amnion. All sections were counterstained with haematoxylin ( x 220).

mark. Cytotrophoblast and syncytiotrophoblast were identified by staining with the antibody JMB2 (Redman et al.. 1984). When term amniochorion and chofionic villous tissues were incubated with the anti-HLA-Ctass II antibodies listed in Table 1, all, including B7/21, gave a positive reaction with macrophages in the maternal decidua and in the fetal amniotic connective tissue and chorionic villi, but. with the exception of B7/21, none reacted with the cytotrophoblast layer of the fetal chorion (see Fig. la), or with syncytiotrophoblast. In contrast, B7/21 gave a strong positive reaction with cytotrophoblast cells either in the fetal chorion, or invading the maternal decidua (Fig. lb), and with the extra-villous cytotrophoblast cells found in the chorionic plate at the base of the chorionic villi (Fig. 2a). The cytotrophoblast cells of the amniochorion were also positive with B7/21 in three second trimester samples. Syncytiotrophoblast in term chofionic villi gave a weak positive reaction with B7/21, but did not react with other antibodies to HLA-Class II.

In both first and second trimester samples of chorionic sac the extravillous cytotrophoblast of the cell islands was found to be labelled strongly with B7/21 (Fig. 2b), whilst showing no reaction with the other anti-HLAClass II antibodies. In sections from first trimester tissue of 9-11 weeks, B7/21 also gave a weak reaction with the cytotrophoblast layer that surrounds the villi, although the overlying syncytiotrophoblast was negative. In contrast, in sections from a second trimester pregnancy of 24 weeks, the syncytiotrophoblast of the chorionic villi showed weak positive staining with B7/21; by this stage the underlying cytotrophoblast layer had virtually disappeared. To show that the labelling of trophoblast by B7/21 is due to the antibody to HLA-DP, and not to a contaminating immunoglobulin, the B7/21 supernatant (1.0 ml diluted 1 to 400) was depleted of antibodies to HLA-DP by consecutive incubations, each for 2 h at 4°C, with two batches of 4.85 × 10 7 EBV-transformed human B cells. These cells express high levels of the HLA-DP antigen. The adsorbed B7/21 was confirmed to contain no detectable antibody to HLA-DP by measuring binding to fresh EBV-transformed B cells, followed by detection of any bound antibody with FITC-labelled rabbit anti (mouse immunoglobulin) F(ab')2 and analysis in a Coulter EPICS 541 flow cytometer. When this adsorbed antibody was used to stain sections of membrane roll, term chorionic villous tissue or first trimester chorionic sac, there was no binding to macrophages, syncytiotrophoblast or cytotrophoblast, indicating that binding of B7/21 to trophoblast is a property of the anti-HLA-DP antibody itself. The general unreactivity of trophoblast with a number of monoclonal antibodies directed against HLA-DR or HLA-DQ, or HLA-DR/DQ/DP described here, confirms and extends previous reports (Sunderland et al., 1981a,b; Redman et al., 1984). In contrast, the binding of the B7/21 antibody to extra-villous cytotrophoblast in term chorion and chorionic plate, to cytotrophoblast islands and villous cytotrophoblast in first trimester chorionic sac, and weakly to syncytiotrophoblast at 24 weeks and at term, was clearly demonstrated by adsorption experiments to be due to the specific anti-HLA-DP antibody. Partial amino acid sequences of the o~and/3 chains of the MHC-Class II antigens show considerable homology between HLA-DR, -DQ and -DP, Fig. 2. Reactivity of chorionic villous tissue with B7/21. Indirect three-stage immunoperoxidase staining of sections labelled with B7/21 from (a) term placenta, strong staining of the cytotrophoblast of the chorionic plate (CP) and weak staining of the syncytiotrophoblast around the chorionic villi (CV), (b) first trimester chorionic sac, cytotrophoblast cells in the cell island (CI) stained, with weaker staining of the cytotrophoblast layer (CL) underlying the syncytiotrophoblast. All sections were counterstained with haematoxylin (×220).

68

69

with differences specific for each chain type scattered throughout (Figueroa and Klein, 1986). The trophoblast antigen seems to express the epitope detected by B7/21, whose precise localisation on the HLA-DP molecule is not known, whilst not expressing any of the epitopes detected by the other anti-HLA-Class II antibodies included in this study. This unique trophoblast reactivity may represent a chance cross-reaction with a non-MHC protein unrelated to HLA-DP. Alternatively, it may be due to an unusual HLA-Class II antigen, analogous to the apparently unique HLA-Class I antigen known to be expressed on extra-villous cytotrophoblast. Further studies are in progress to investigate the structure of the trophoblast antigen precipitated by B7/21 and to compare it with that of HLA-DP. Acknowledgements. Dr. S.V. Fuggle is thanked for her gift of antibodies and EBV-transformed B cells, Mrs. L, Clover for her tolerance and expert technical assistance, and my colleagues for helpful discussions. References Boyd, J.D. and Hamilton, W.J. (1970) The Human Placenta. Heifer & Sons, Cambridge. Brami, C.J., Sanyal, M.K., Dwyer, J.M., Johnson, C.C., Kohorn, E.I. and Naftolin, F. (1983). HLA-DR antigen on human trophoblast. Am. J. Reprod. Immunol. 3, 165-174. Chen, Y.-X., Evans, R.L., Pollack, M.S., Lanier, L.L., Phillips, J.H., Rousso, C., Warner, N.L. and Brodsky, F.M. (1984) Characterization and expression of the HLA-DC antigens defined by anti-Leu 10. Hum. Immunol. 10, 221-235. Ellis, S.A., Sargent, I.L., Redman, C.W.G. and McMichael, A.J. (1986) Evidence for a novel HLA antigen found on human extra-villous trophoblast and choriocarcinoma cell line. Immunology 59, 595-601. Figueroa, F. and Klein, J. (1986) The evolution of MHC class II genes. Immunol. Today 7, 78-81. Fuggle, S.V., Errasti, E, Daar, A.S., Fabre, J.W., Ting, A. and Morris, EJ. (1983) Localization of major histocompatibility complex (HLA-ABC and DR) antigens in 46 kidneys. Transplantation 35, 385-390. Goodfellow, EN., Barnstable, C.J., Bodmer, W.F., Snary, D. and Crumpton M.J. (1976) Expression of HLA system antigens on placenta. Transplantation 22, 595-603. Lampson, L.A. and Levy, R. (1980) Two populations of Ia-like molecules on a human B cell line. J. Immunol. 125, 293-299. McGee, J.O'D., Morton, J.A., Barbatis, C., Bradley, J.F., Fleming, K.A., Goate, A.M., and Burns, J. (1982) Monoclonal antibodies to Mallory bodies/intermediate filaments and HLA (class 1) antigens in human liver disease. In: Monoclonal Antibodies in Clinical Medicine (eds. A.J. McMichael and J.W. Fabre), p. 431. Academic Press, London. Redman, C.W.G., McMichael, A.J., Stirrat, G.M., Sunderland, C.A. and Ting, A. (1984) Class I major histocompatibility complex antigens on human extravillous trophoblast. Immunology 52, 457-468. Sunderland, C.A., Redman, C.W.G. and Stirrat, G.M. (1981a) HLA-A, B, C antigens are expressed on non-villous trophoblast of the early human placenta. J. Immunol. 127, 2614-2615.

70 Sunderland, C.A., Naiem, M., Mason, D.Y., Redman, C.W.G. and Stirrat, G.M. (1981b) The expression of major histocompatibility antigens by human chorionic villi. J. Reprod. Immunol. 3, 323-331. Sutton, L., Gadd, M., Mason, D.Y., and Redman, C.W.G. (1986) Cells bearing class II MHC antigens in the human placenta and amniochorion. Immunology 58, 23-29. Van Heyningen, V., Guy, K., Newman, R. and Steel, C.M. (1982) Human MHC class II molecules as differentiation markers. Immunogenetics 16, 459-469. Voland, J.R., Frisman, D.M. and Baird, S.M. (1986) Presence of an endothelial antigen on the syncytiotrophoblast of human chorionic villi: detection by a monoclonal antibodyl Am. J. Reprod. Immunol. 11, 24-30. Watson, A.J., DeMars, R., Trowbridge, I.S. and Bach, F.H. (1983) Detection of a novel human class II HLA antigen. Nature 304, 358-361.