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Electron microscopic localization of the Pa and RT1.Aa antigens on the placenta of the rat
Immunology Letters, 16 (1987) 273-276 Elsevier IML 00966
Electron microscopic localization of the Pa and RT1.Aa antigens on the placenta of the rat A m a l Kanbour, Trevor A. M a c p h e r s o n , Heinz W. Kunz a n d T h o m a s J. Gill III Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, U.S.A. (Received 21 July 1987; accepted 10 August 1987)
1. Summary
A major factor in the ability of the placenta to avoid allograft rejection is the differential expression of MHC class I antigens on its surface. Using monoclonal antibodies and the electron microscopic immunogold technique, we have demonstrated that only the pregnancy-associated (Pa) antigen, which carries a broadly shared antigenic determinant, is expressed on the placental surface in the rat, whereas the allele-specific classical transplantation antigens are not. Both types of antigens are, however, present in the cytoplasm of the basal trophoblast but completely absent from the labyrinthine trophoblast.
2. Introduction
The success of viviparous reproduction depends upon the ability of the placenta to survive in a female who has a fully functional immune system. This apparent paradox remains a central issue in transplantation immunology, and it has been addressed from a variety of points-of-view [1-8]. Amongst the potential mechanisms considered are blocking antibodies, immunosuppressive antibody-antigen complexes, non-antibody suppressor molecules of a wide variety, and specific and nonspecific suppres-
Key words: Placenta; MHC antigens; Monoclonal antibodies; Pregnancy-associated (Pa) antigen; Transplantation antigens
Correspondence to: A. Kanbour, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, U.S.A.
sor cells; the idea of maternal immunoincompetence, even in a selective way, is no longer tenable. A persistent, and attractive, idea has been that the placenta itself is poorly immunogenic, and a number of interesting ideas have been proposed to account for such a putative lack of immunogenicity (reviewed in [3]). The experimental approach to this problem in humans has been confounded by the transplacental passage of fetal lymphocytes and red blood cells which elicit an immune response in the mother that is directed against paternal antigens, especially the major histocompatibility complex (MHC)-encoded transplantation antigens. This makes it difficult to dissect the antipaternal antibody response into the component elicited by the fetal cells and the component elicited by antigens on the placental surface; the latter is the one of interest in studying the transplantation immunology of the fetal placental unit. Also, the human MHC is highly polymorphic, so it is difficult to assign a given specificity to a particular molecule without unrealistically extensive immunochemical investigation. The placenta of the mouse is essentially impermeable to fetal cells, but the mouse has an even higher level of MHC polymorphism than the human. By contrast, the rat has a unique advantage for studying the immunochemical structure of the placental surface: it has a very low level of MHC polymorphism, and the appropriate reagents and strains for such a study are available [9]. The placenta of the rat [10-12], mouse [13, 14], human [15-18] and baboon [19] all express MHC class I antigens in the trophoblast. There is evidence in the rat [10], human [20, 21] and baboon [19] that
0165-2478 / 87 / $ 3.50 © 1987 Elsevier Science Publishers BN. (Biomedical Division)
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these class I antigens are i m m u n o c h e m i c a l l y u n i q u e in t h a t they c a r r y b r o a d l y shared antigenic specificities different f r o m t h o s e t h a t characterize the classical, allele-specific class I t r a n s p l a n t a t i o n antigens. In the m o u s e a n d the h u m a n , the p l a c e n t a does n o t c a r r y class II antigens [ 1 6 - 1 8 , 2 2 - 2 6 ] , a n d recent d a t a f r o m o u r l a b o r a t o r y (unpubl.) indicate t h a t the rat also does n o t have class II antigens on its p l a c e n ta.
3. Materials and Methods The most potent maternal anti-paternal antibody
response in the rat occurs in the u x a m a t i n g c o m b i n a t i o n [10, 27], a n d analysis o f this response shows t h a t it is directed against a unique p l a c e n t a l class I antigen, the p r e g n a n c y - a s s o c i a t e d (Pa) antigen [10, 28]. This a n t i g e n is c o n t r o l l e d by a diallelic system: the a, d, f, b a n d m h a p l o t y p e s c a r r y the Pa antigen, b u t the n, c, l, u, g, k a n d h h a p l o t y p e s d o not. Using a l l o a n t i s e r a a n d the a v i d i n - b i o t i n c o m p l e x imm u n o c y t o c h e m i c a l technique, the Pa antigen a n d RT1.A a antigen, which is the m a j o r class I transp l a n t a t i o n in the rat, were d e m o n s t r a t e d to be on the b a s a l t r o p h o b l a s t but n o t on the l a b y r i n t h i n e t r o p h o b l a s t [11, 29]. However, only the Pa antigen a n d n o t the A a a n t i g e n - elicited an a n t i b o d y re-
Fig. 1. Electron photomicrographs showing staining of the basal trophoblast of the rat placenta (day 17 of gestation) using the single-label protein A-gold technique with anti-Aa (A, B) and anti-Pa (C, D) monoclonal antibodies. B = basal trophoblast; M = cell membrane; S = maternal sinus. The WF x DA placenta (A) but not the WF × WF placenta (B) stains with the anti-Aa monoclonal antibody (arrow). The A a antigen is in the cell cytoplasm, but none is on the cell membrane. The WF x DA placenta (C) stains with the anti-Pa monoclonal antibody, and it is both on the cell membrane and in the cell cytoplasm (arrows). The WF × WF placenta (D) does not stain, x40,600. 274
sponse during pregnancy, and this observation suggests that there is differential regulation of the expression o f these two antigens: Pa is on the placental surface, whereas A a is not. This hypothesis was tested using electron microscopy and the immunogold technique. Monoclonal antibodies to the Pa antigen (mAb 124; [28]) and to the A a antigen (mAb 211; [9]) were labeled with 15 nm gold particles and used to stain sections of the basal and labyrinthine trophoblast [30, 31]. The anti-A a antibody stained only molecules in the cytoplasm o f the WF (u) x DA (a) placenta (Fig. 1, A), whereas the anti-Pa antibody stained molecules both on the cell membrane and in the cell cytoplasm of the WF x DA placenta (Fig. I, C).
Neither antibody stained the WF x WF placenta (Fig. 1, B and 1, D). The absence of both class I molecules in or on the labyrinthine trophoblast was also demonstrated using this technique (Fig. 2), and this finding confirms our previous light microscopic studies [11, 29].
4. Discussion The electron microscopic studies show that only the Pa antigen is expressed on the surface of the basal trophoblast in the WF × DA placenta and that both the Pa and A a antigens are present in the cytoplasm o f these cells. This finding provides substantial sup-
Fig. 2. Electron photomicrographs showing staining of the labyrinthine trophoblast of the rat placenta (day 17 of gestation) using the single-label protein A-gold technique with anti-Aa (A, B) and anti-Pa (C, D) monoclonal antibodies, L = labyrinthine trophoblast; M = cell membrane; S = maternal sinus. Neither the WF x DA (A, C) nor the WF x WF (B, D) labyrinthine trophoblast stains with either antibody, x40,600. 275
p o r t for o u r h y p o t h e s i s t h a t t h e m e c h a n i s m by w h i c h t h e p l a c e n t a e s c a p e s i m m u n e r e j e c t i o n is t h e d i f f e r e n t i a l e x p r e s s i o n o f class I a n t i g e n s o n its surface. O n l y class I a n t i g e n s w i t h b r o a d l y s h a r e d a n t i g e n i c d e t e r m i n a n t s are expressed, e.g., t h e P a a n t i g e n in t h e rat, w h i l e t h e a l l e l e - s p e c i f i c class I t r a n s p l a n t a t i o n a n t i g e n s are n o t expressed.
Acknowledgement T h i s w o r k was s u p p o r t e d b y G r a n t s H D 08662 a n d H D 09880 f r o m t h e N a t i o n a l I n s t i t u t e s o f H e a l t h o f t h e U n i t e d States P u b l i c H e a l t h Service.
References [1] [2] [3l [4] [5] [6] [7] [8]
[9] [10] [11]
276
Medawar, P. B. (1953) Symp. Soc. Exp. Biol. 7, 320. Billingham, R. E. (1964) N. Eng. J. Med. 270, 667. Gill, T. J. III, and Repetti, C. E (1979) Am. J. Path. 95,465. Gill, T. J. III. (1985) CRC Crit. Rev. Immunol. 5, 201. Hunziker, R. D. and Wegmann, T. G. (1986) CRC Crit. Rev. Immunol. 6, 245. Wegmann, T. G. and Gill, T. J. III, Eds. (1983) Immunology of Reproduction, Oxford University Press, New York. Clark, D. A. and Croy, B. A., Eds. (1986) Reproductive Immunology, 1986, Elsevier Science Publishers, Amsterdam. Gill, T. J. III and Wegmann, T. G., Eds. (1987) Immunoregulation and Fetal Survival, Oxford University Press, New York. Gill, T. J. III, Kunz, H. W., Misra, D. N. and Cortese Hassett, A. L. (1987) Transplantation 43, 773. Ghani, A. M., Gill, T. J. Ill, Kunz, H. W. and Misra, D. N. (1984) Transplantation 37, 187. Macpherson, T. A., Ho, H-N., Kunz, H. W. and Gill, T. J. IlI. (1986) Transplantation 41, 392.
[12] Billington, W. D. and Burrows, E J. (1986) J. Reprod. Immunol. 9, 155. [13] Wegmann, T. G. and CarLson, G. A. (1977) J. immunol. 119, 1659. [14] Chatterjee-Hasrouni, S. and Lala, P. K. (1979) J. Exp. Med. 149, 1238. [15] Goodfellow, P. N., Barnstable, C. J., Bodmer, W. E, Snary, D. and Crumpton, M. J. (1976) Transplantation 22, 595. [16] Faulk, W. P., Sanderson, A. R. and Temple, A. (1977) Transplant. Proc. 9, 1379. [17] Sunderland, C. A., Naiem, N., Mason, D. Y., Redman, C. W. G. and Stirrat, G. (1981) J. Reprod. Immunol. 3, 323. [18] Adinolfi, M., Akle, C. A., McColl, I., Fensom, A. H., Tansley, L., Connolly, P., Hsi, B. L., Faulk, W. P., Travers, P. and Bodmer, W. (1982) Nature 295, 325. [19] Stern, P. L., Beresford, N., Friedman, C. I., Stevens, V. C., Risk, J. M. and Johnson, P. M. (1987) J. Immunol. 138, 1088. [20] Mclntyre, J. A. and Faulk, W. P. (1982) Hum. Immunol. 4, 27. [21] Ellis, S. A., Sargent, 1. L., Redman, C. W. G. and McMichael, A. J. (1986) Immunology 59, 595. [22] Chatterjee-Hasrouni, S. and Lala, P. K. (1981) J. Immunol. 127, 2070. [23] Raghupathy, R., Singh, B., Leigh, J. B. and Wegmann, T. G. (1981) J. Immunol. 127, 2074. [24] Galbraith, R. M., Kantor, R. R. S., Ferrara, G. B., Ades, E. W. and Galbraith, G. M. (1981) Am. J. Reprod. Immunol. 1, 331. [25] Brami, C. J., Sanyal, M. K., Dwyer, J. M., Johnson, C. C., Kohorn, E. I. and Naftolin, E (1983) Am. J. Reprod. Immunol. 3, 165. [26] Redman, C. W. G., McMichael, A. J., Stirrat, G. M., Sunderland, C. A. and Ting, A. (1984) Immunology 52, 457. [27] Smith, R. N., Sternlicht, M. and Butcher, G. W. (1982) J. Immunol. 129, 771. [28] Ghani, A. M., Kunz, H. W. and Gill, T. J. III. (1984) Transplantation 37, 187. [29] Ho, H.-N., Macpherson, T. A., Kunz, H. W. and Gill, T. J. III. (1987) Am. J. Reprod. Immunol. Microbiol. 13, 51. [30] Bendayan, M. and Zollinger, M. (1983) J. Histochem. Cytochem. 31, 101. [31] Bendayan, M., Nanci, A., Herber, G. H., Gregoire, S. and Duhr, M. A. (1986) Am. J. Anat. 175, 379.
Electron microscopic localization of the Pa and RT1.Aa antigens on the placenta of the rat A m a l Kanbour, Trevor A. M a c p h e r s o n , Heinz W. Kunz a n d T h o m a s J. Gill III Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, U.S.A. (Received 21 July 1987; accepted 10 August 1987)
1. Summary
A major factor in the ability of the placenta to avoid allograft rejection is the differential expression of MHC class I antigens on its surface. Using monoclonal antibodies and the electron microscopic immunogold technique, we have demonstrated that only the pregnancy-associated (Pa) antigen, which carries a broadly shared antigenic determinant, is expressed on the placental surface in the rat, whereas the allele-specific classical transplantation antigens are not. Both types of antigens are, however, present in the cytoplasm of the basal trophoblast but completely absent from the labyrinthine trophoblast.
2. Introduction
The success of viviparous reproduction depends upon the ability of the placenta to survive in a female who has a fully functional immune system. This apparent paradox remains a central issue in transplantation immunology, and it has been addressed from a variety of points-of-view [1-8]. Amongst the potential mechanisms considered are blocking antibodies, immunosuppressive antibody-antigen complexes, non-antibody suppressor molecules of a wide variety, and specific and nonspecific suppres-
Key words: Placenta; MHC antigens; Monoclonal antibodies; Pregnancy-associated (Pa) antigen; Transplantation antigens
Correspondence to: A. Kanbour, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, U.S.A.
sor cells; the idea of maternal immunoincompetence, even in a selective way, is no longer tenable. A persistent, and attractive, idea has been that the placenta itself is poorly immunogenic, and a number of interesting ideas have been proposed to account for such a putative lack of immunogenicity (reviewed in [3]). The experimental approach to this problem in humans has been confounded by the transplacental passage of fetal lymphocytes and red blood cells which elicit an immune response in the mother that is directed against paternal antigens, especially the major histocompatibility complex (MHC)-encoded transplantation antigens. This makes it difficult to dissect the antipaternal antibody response into the component elicited by the fetal cells and the component elicited by antigens on the placental surface; the latter is the one of interest in studying the transplantation immunology of the fetal placental unit. Also, the human MHC is highly polymorphic, so it is difficult to assign a given specificity to a particular molecule without unrealistically extensive immunochemical investigation. The placenta of the mouse is essentially impermeable to fetal cells, but the mouse has an even higher level of MHC polymorphism than the human. By contrast, the rat has a unique advantage for studying the immunochemical structure of the placental surface: it has a very low level of MHC polymorphism, and the appropriate reagents and strains for such a study are available [9]. The placenta of the rat [10-12], mouse [13, 14], human [15-18] and baboon [19] all express MHC class I antigens in the trophoblast. There is evidence in the rat [10], human [20, 21] and baboon [19] that
0165-2478 / 87 / $ 3.50 © 1987 Elsevier Science Publishers BN. (Biomedical Division)
273
these class I antigens are i m m u n o c h e m i c a l l y u n i q u e in t h a t they c a r r y b r o a d l y shared antigenic specificities different f r o m t h o s e t h a t characterize the classical, allele-specific class I t r a n s p l a n t a t i o n antigens. In the m o u s e a n d the h u m a n , the p l a c e n t a does n o t c a r r y class II antigens [ 1 6 - 1 8 , 2 2 - 2 6 ] , a n d recent d a t a f r o m o u r l a b o r a t o r y (unpubl.) indicate t h a t the rat also does n o t have class II antigens on its p l a c e n ta.
3. Materials and Methods The most potent maternal anti-paternal antibody
response in the rat occurs in the u x a m a t i n g c o m b i n a t i o n [10, 27], a n d analysis o f this response shows t h a t it is directed against a unique p l a c e n t a l class I antigen, the p r e g n a n c y - a s s o c i a t e d (Pa) antigen [10, 28]. This a n t i g e n is c o n t r o l l e d by a diallelic system: the a, d, f, b a n d m h a p l o t y p e s c a r r y the Pa antigen, b u t the n, c, l, u, g, k a n d h h a p l o t y p e s d o not. Using a l l o a n t i s e r a a n d the a v i d i n - b i o t i n c o m p l e x imm u n o c y t o c h e m i c a l technique, the Pa antigen a n d RT1.A a antigen, which is the m a j o r class I transp l a n t a t i o n in the rat, were d e m o n s t r a t e d to be on the b a s a l t r o p h o b l a s t but n o t on the l a b y r i n t h i n e t r o p h o b l a s t [11, 29]. However, only the Pa antigen a n d n o t the A a a n t i g e n - elicited an a n t i b o d y re-
Fig. 1. Electron photomicrographs showing staining of the basal trophoblast of the rat placenta (day 17 of gestation) using the single-label protein A-gold technique with anti-Aa (A, B) and anti-Pa (C, D) monoclonal antibodies. B = basal trophoblast; M = cell membrane; S = maternal sinus. The WF x DA placenta (A) but not the WF × WF placenta (B) stains with the anti-Aa monoclonal antibody (arrow). The A a antigen is in the cell cytoplasm, but none is on the cell membrane. The WF x DA placenta (C) stains with the anti-Pa monoclonal antibody, and it is both on the cell membrane and in the cell cytoplasm (arrows). The WF × WF placenta (D) does not stain, x40,600. 274
sponse during pregnancy, and this observation suggests that there is differential regulation of the expression o f these two antigens: Pa is on the placental surface, whereas A a is not. This hypothesis was tested using electron microscopy and the immunogold technique. Monoclonal antibodies to the Pa antigen (mAb 124; [28]) and to the A a antigen (mAb 211; [9]) were labeled with 15 nm gold particles and used to stain sections of the basal and labyrinthine trophoblast [30, 31]. The anti-A a antibody stained only molecules in the cytoplasm o f the WF (u) x DA (a) placenta (Fig. 1, A), whereas the anti-Pa antibody stained molecules both on the cell membrane and in the cell cytoplasm of the WF x DA placenta (Fig. I, C).
Neither antibody stained the WF x WF placenta (Fig. 1, B and 1, D). The absence of both class I molecules in or on the labyrinthine trophoblast was also demonstrated using this technique (Fig. 2), and this finding confirms our previous light microscopic studies [11, 29].
4. Discussion The electron microscopic studies show that only the Pa antigen is expressed on the surface of the basal trophoblast in the WF × DA placenta and that both the Pa and A a antigens are present in the cytoplasm o f these cells. This finding provides substantial sup-
Fig. 2. Electron photomicrographs showing staining of the labyrinthine trophoblast of the rat placenta (day 17 of gestation) using the single-label protein A-gold technique with anti-Aa (A, B) and anti-Pa (C, D) monoclonal antibodies, L = labyrinthine trophoblast; M = cell membrane; S = maternal sinus. Neither the WF x DA (A, C) nor the WF x WF (B, D) labyrinthine trophoblast stains with either antibody, x40,600. 275
p o r t for o u r h y p o t h e s i s t h a t t h e m e c h a n i s m by w h i c h t h e p l a c e n t a e s c a p e s i m m u n e r e j e c t i o n is t h e d i f f e r e n t i a l e x p r e s s i o n o f class I a n t i g e n s o n its surface. O n l y class I a n t i g e n s w i t h b r o a d l y s h a r e d a n t i g e n i c d e t e r m i n a n t s are expressed, e.g., t h e P a a n t i g e n in t h e rat, w h i l e t h e a l l e l e - s p e c i f i c class I t r a n s p l a n t a t i o n a n t i g e n s are n o t expressed.
Acknowledgement T h i s w o r k was s u p p o r t e d b y G r a n t s H D 08662 a n d H D 09880 f r o m t h e N a t i o n a l I n s t i t u t e s o f H e a l t h o f t h e U n i t e d States P u b l i c H e a l t h Service.
References [1] [2] [3l [4] [5] [6] [7] [8]
[9] [10] [11]
276
Medawar, P. B. (1953) Symp. Soc. Exp. Biol. 7, 320. Billingham, R. E. (1964) N. Eng. J. Med. 270, 667. Gill, T. J. III, and Repetti, C. E (1979) Am. J. Path. 95,465. Gill, T. J. III. (1985) CRC Crit. Rev. Immunol. 5, 201. Hunziker, R. D. and Wegmann, T. G. (1986) CRC Crit. Rev. Immunol. 6, 245. Wegmann, T. G. and Gill, T. J. III, Eds. (1983) Immunology of Reproduction, Oxford University Press, New York. Clark, D. A. and Croy, B. A., Eds. (1986) Reproductive Immunology, 1986, Elsevier Science Publishers, Amsterdam. Gill, T. J. III and Wegmann, T. G., Eds. (1987) Immunoregulation and Fetal Survival, Oxford University Press, New York. Gill, T. J. III, Kunz, H. W., Misra, D. N. and Cortese Hassett, A. L. (1987) Transplantation 43, 773. Ghani, A. M., Gill, T. J. Ill, Kunz, H. W. and Misra, D. N. (1984) Transplantation 37, 187. Macpherson, T. A., Ho, H-N., Kunz, H. W. and Gill, T. J. IlI. (1986) Transplantation 41, 392.
[12] Billington, W. D. and Burrows, E J. (1986) J. Reprod. Immunol. 9, 155. [13] Wegmann, T. G. and CarLson, G. A. (1977) J. immunol. 119, 1659. [14] Chatterjee-Hasrouni, S. and Lala, P. K. (1979) J. Exp. Med. 149, 1238. [15] Goodfellow, P. N., Barnstable, C. J., Bodmer, W. E, Snary, D. and Crumpton, M. J. (1976) Transplantation 22, 595. [16] Faulk, W. P., Sanderson, A. R. and Temple, A. (1977) Transplant. Proc. 9, 1379. [17] Sunderland, C. A., Naiem, N., Mason, D. Y., Redman, C. W. G. and Stirrat, G. (1981) J. Reprod. Immunol. 3, 323. [18] Adinolfi, M., Akle, C. A., McColl, I., Fensom, A. H., Tansley, L., Connolly, P., Hsi, B. L., Faulk, W. P., Travers, P. and Bodmer, W. (1982) Nature 295, 325. [19] Stern, P. L., Beresford, N., Friedman, C. I., Stevens, V. C., Risk, J. M. and Johnson, P. M. (1987) J. Immunol. 138, 1088. [20] Mclntyre, J. A. and Faulk, W. P. (1982) Hum. Immunol. 4, 27. [21] Ellis, S. A., Sargent, 1. L., Redman, C. W. G. and McMichael, A. J. (1986) Immunology 59, 595. [22] Chatterjee-Hasrouni, S. and Lala, P. K. (1981) J. Immunol. 127, 2070. [23] Raghupathy, R., Singh, B., Leigh, J. B. and Wegmann, T. G. (1981) J. Immunol. 127, 2074. [24] Galbraith, R. M., Kantor, R. R. S., Ferrara, G. B., Ades, E. W. and Galbraith, G. M. (1981) Am. J. Reprod. Immunol. 1, 331. [25] Brami, C. J., Sanyal, M. K., Dwyer, J. M., Johnson, C. C., Kohorn, E. I. and Naftolin, E (1983) Am. J. Reprod. Immunol. 3, 165. [26] Redman, C. W. G., McMichael, A. J., Stirrat, G. M., Sunderland, C. A. and Ting, A. (1984) Immunology 52, 457. [27] Smith, R. N., Sternlicht, M. and Butcher, G. W. (1982) J. Immunol. 129, 771. [28] Ghani, A. M., Kunz, H. W. and Gill, T. J. III. (1984) Transplantation 37, 187. [29] Ho, H.-N., Macpherson, T. A., Kunz, H. W. and Gill, T. J. III. (1987) Am. J. Reprod. Immunol. Microbiol. 13, 51. [30] Bendayan, M. and Zollinger, M. (1983) J. Histochem. Cytochem. 31, 101. [31] Bendayan, M., Nanci, A., Herber, G. H., Gregoire, S. and Duhr, M. A. (1986) Am. J. Anat. 175, 379.