- Email: [email protected]
Ontogeny and distribution of cells expressing HLA-B locus-specific determinants in the placenta and extraplacental membranes
Journal of Reproductive Immunology, 15 (1989) 21--30 Elsevier Scientific Publishers Ireland Ltd.
21
JR100583
Ontogeny and distribution of cells expressing HLA-B locus-specific determinants in the placenta and extraplacental membranes J o a n S. H u n t a, D i a n n a L. Lessin a a n d C h a r l e s R. King b *Department of Pathology and Oncology and bDepartment of Obstetrics and Gynecology, University of Kansas Medical Center, Kansas City, Kansas (U.S.A.) (Accepted for publication 19 December 1988)
Summary Both the trophectoderm and the inner cell mass of the blastocyst contribute to the cell populations found in the placenta and extraplacental membranes. Previous studies have shown differences between those two embryologically distinct populations of cells in their expression of class I HLA, and further differences among trophectoderm-derived trophoblast cell subpopulations. Binding patterns for antibodies to both monomorphic and allotypic determinants on class I heavy chains have been reported. In the present study, extraembryonic cells were evaluated by immunohistology for binding of the monoclonal antibody 4E, which identifies locus-specific determinants on HLA-B. Some inner cell mass-derived cells (mesenchymal cells) acquired high levels of HLA-B as gestation progressed and others continued to express low levels at late stages of gestation (amnion cells). In contrast, throughout gestation both villous and extravillous trophoblast cells failed to express detectable HLA-B. The binding patterns for 4E followed the patterns established by a monoclonal antibody to class I HLA heavy chains (61D2), and those reported for antisera to allotypic determinants. The findings support the suggestion that trophoblast cells forming the fetal component of the maternal-fetal interface exert highly effective regulation over the expression of class I HLA. Key words: human; major histocompatibility antigens; placenta; pregnancy;
trophoblast cells.
Correspondence to: Joan S. Hunt, Ph.D., Department of Pathology and Oncology, University of Kansas Medical Center, 39th Street & Rainbow Blvd., Kansas City, KS 66103, U.S.A. 0165-0378/89/$03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
22
Introduction
The expression of class I HLA by extraembryonic cells is of interest because maternal and fetal cells are intimately associated throughout pregnancy. Mothers would be expected to mount a massive immune response to fetal cells bearing disparate HLA, yet there is little evidence for that event. Two embryologically distinct populations of cells are present in the placenta and extraplacental membranes. One of those populations, the mesenchymal cells that arise from the inner cell mass of the blastocyst, gradually acquire the ability to bind a monoclonal antibody to monomorphic determinants on class I HLA heavy chains as gestation progresses (Lessin et al., 1988). The second population, trophoblast cells that arise from the trophectoderm of the blastocyst, forms the active interface between the fetus and the mother, secluding mesenchymal cells from maternal blood and tissues. One explanation for the failure of mothers to reject their semi-allogeneic fetuses is that the trophoblast cells of the placenta and extraplacental membranes do not express a normal complement of mature class I HLAA,B,C (Beer and Sio, 1982). Placental trophoblast is invariably described as class I HLA negative (Fanlk and Temple, 1976; Goodfellow et ai., 1976; Galbraith et al., 1981; Hunt et al., 1987; Hunt et al., 1988b). However, trophoblast cells exposed to decidual cells are recognized as class I H L A positive by some anti-class I reagents (Sunderland et al., 1981; Wells et al., 1984; Hsi et al., 1984; Hunt et al., 1988b). H L A positive trophoblast cells bind the monoclonal antibody (mAb) W6/32 but not the mAb 61D2, both of which identify HLA-A,B,C. Anti-beta2-microglobulin gives positive results, but antisera directed toward maternally-derived allotypic determinants of class I antigens do not (Redman et al., 1984). Lower than normal molecular weight antigens with unusual isoelectric points are immunoprecipitated from some trophoblast cells by W6/32 (Ellis et al., 1986). It is not clear if the antigens detected on some trophoblast cells are immature class I HLA lacking a full complement of determinants or if those antigens are dictated by developmentally regulated class I HLA-A,B,C-Iike genes (Orr and DeMars, 1983; Geraghty et al., 1987, Koller et al., 1988; Mizuno et al., 1988). The mAb that does bind to some trophoblast cells, W6/32, requires beta-2-microglobulin for recognition (Parham et al., 1979) and both HLA and HLA-like molecules associate with that non-polymorphic molecule. Allotypic determinants and the locus-specific determinants that define the subclasses of class I HLA, HLA-A and HLA-B, are interspersed in the alphal-alpha2 domains of class I HLA heavy chains. The mAb 4E identifies HLA-B locus specific determinants on heavy chains and does not require
23
beta2-microglobulin for binding (Yang et al., 1984). 4E is therefore a useful reagent for further definition of the structure of the class I antigens expressed by trophoblast cells. Since HLA-B would not be immunogenic to the mother, expression of that epitope by trophoblast cells would not facilitate graft rejection. Positive identification of HLA-B would, however, firmly identify the class I antigens on trophoblast cells as arising from the H L A gene complex. The results of the present study demonstrate that HLA-B is never expressed by either villous or extravillous trophoblast cells although mesenchymal cells exhibit increased HLA-B positivity as gestation progresses. Thus, the genes coding for the class I antigens on some trophoblast cells remain to be established. Materials and methods Tissues First trimester (n, 3) and term tissues (n, 3) from elective pregnancy terminations or normal deliveries were obtained courtesy of the Department of Obstetrics and Gynecology of this institution, and were processed within 30 min. Portions of tissue (3--5 m m 3) were flash frozen in liquid N 2 and stored at - 70°C until sectioned by cryostat and tested. A n tibodies Culture supernatant containing the mouse m A b 4E, which binds to locusspecific determinants on HLA-B (Yang et al., 1984) was used at a 1:2 dilution. 4E was a generous gift from Drs. S.Y. Yang (Memorial Sloan Kettering Cancer Center) and E.J. Yunis (Dana-Farber Cancer Institute). The m A b to m o n o m o r p h i c determinants on class I H L A heavy chains 61D2 (Ugolini et al., 1980) was obtained from Bethesda Research Laboratories, Gaithersburg, MD, and was used at a 1:100 dilution. W6/32 (Barnstable et al., 1978), which recognizes a combinatorial determinant of class I H L A heavy chains and beta2-microglobulin (Parham et al., 1979) was generated as ascites from the American Type Culture Collection hybridoma HB95, and was used at a 1:400 dilution. Immunohistochemistry Sections of frozen tissues were air-dried and fixed in cold acetone. Sections were either used immediately or were stored at - 70°C (< 5 days) before testing. Binding of the m A b was detected using an avidin-biotin immunoperoxidase staining system for mouse lgG from Vector Laboratories (Vectastain ABC, Burlingame, CA). Quenching of endogenous peroxidase was performed following incubation with the biotinylated anti-mouse reagent
24
rather than prior to incubation with the primary mAb in order to maintain antigen integrity. Following incubation with the substrate, diaminobenzidine tetrahydrachloride (0.5 mg/ml, Sigma, St. Louis), the tissue sections were counterstained with Gill's hematoxylin. Positive cells bore a brown reaction product, and the nuclei of both positive and negative cells were blue. Results
First trimester tissue Figure 1 shows first trimester placental tissue stained with anti-HLA-B (4E). Maternal ceils in the fibrinoid tissue adjacent to fetal trophoblast cells were strongly positive with 4E. Mesenchymal cells within the placental villi were weakly positive for HLA-B, and trophoblast cells were negative. The same general pattern of staining was obtained with the anti-class I HLA reagents 61D2 and W6/32, although staining was invariably more intense with W6/32 than with either 4E or 61D2 (not shown). Term placenta In Fig. 2, cross-sections of term placental villi are shown that contain mesenchymal cells that were strongly positive with the mAb to HLA-B, 4E.
::~.-
:
: i "~
~.:
:i
!:
:::: ,~:?i~ili: ii :
i
i ! ~
•
I~ i
.
i
..:.
Fig. 1. First trimester placenta stained with the monoclonal antibody 4E (anti-HLA-B). Maternal cells in fibrous tissue adjacent to placental tissue are strongly positive (arrows), placental villous mesenchynml cells (VM) are weakly positive, and trophoblast cells (TB, arrowheads) are negative. × 400.
25 = "": i
'
~
• ~: ~
.~ ~ ~ '~::" -::;ii~ :: "
i~'~, i ".
.::
! ; ! i ~ : " .... ,..
!:~:
~'i::'~
~~
, ,
~
~
.
~
.
.
.
.
.
.
.
~: !~!i
.
:i
: :
:
.
:i
::
:i~
• .:
:':ii!~
" .i, ".::i.
"
"
~"/~,i ! ~.ii~ .... i . ~. ~ . . i:~.~,i;~i~ ~. . . . . .
i[i:
i:/~ ° .::,~:ii i
i,:.,]
:~:
:
.'!~]~:::i
.?
::
..~.'
. ~:,
~~""~ ! . ~ . . . ~ .~r' ~ ..... ~
•
: .. ,:'~. : ~ .
....~.~ :
:
• [:
] ,~
, ~ , ~ ~ ~ i l
.~
Fig. 2. Term placenta stained with the monoclonal antibody 4E (anti-HLA-B). Mesenchymal cells in the centers of placental villi are strongly positive with 4E whereas trophoblast cells lining the maternal blood spaces (arrows) are negative. × 250.
Trophoblast cells lining the maternal blood spaces were negative with 4E. The same results were obtained with 61D2 and W6/32 (not shown). Figure 3 shows cross-sections o f a few placental villi and large areas of fibrinoid tissue. In Fig. 3a, the tissue is stained with the m A b 61D2, which binds to mesenchymal cells in placental villi (arrows), but does not bind to cells with cytotrophoblastic characteristics in the fibrinoid tissue (arrowhead). Figure 3b shows that the binding patterns for 4E were essentially the same as for 61D2. A cluster o f 4E-negative cells with cytotrophoblast cell m o r p h o l o g y is shown in Fig. 3c.
, .~, Fig. 3a
'
~'" "~.
~i,
~ :
'~'~.~;
i~i~,~ "~'~.~
26
, :'i
:i ¸
i ¸ ••
•
i
:
i!r- ~
Fig. 3. Serial sections of term placenta stained with two monoclonal antibodies to class I HLA. (a) Staining with 61 D2 (anti-HLA-A,B,C). Mesenchymal cells in the centers o f chorionic villi are strongly positive (arrows) whereas cells with cytotrophoblast morphology in adjacent fibrous tissue (arrowhead) were negalive with 61D2, showing only nuclear staining with the Gill's hematoxylin ¢ounterstain. x 100. (b) Staining with 4E (anti-HLA-B). The staining pattern for 4E resembles that obtained with 61D2. Mesenchymal cells in the chorionic villi were strongly positive (arrows), but cells with cytotrophoblast morphology were negative with 4E (arrowhead). x 100. (c) Higher magnification of the tissue in (b) showing a nest of 4E negative cells with cytotrophoblast cell morphology (arrows). x 250.
Term extraplacental membranes Figure 4A demonstrates that maternal decidual cells were strongly positive with the mAb to HLA-B, 4E, as were the fetal cells in the mesenchyme between the amnion and chorion membranes. In contrast, 4E was essentially negative with chorion membrane cytotrophoblast cells. The amnion membrane (Fig. 4b) usually stained very weakly with 4E, but there was some variability among the membranes tested and occasional amnion cells were more strongly positive (Fig. 4b). Serial sections stained with 61D2 showed the
27
~ " •,~ ,! •i!•!~:~i~•~i~i~,!II~~•ii~~ •
•
: i
•: ~!• i i'i~,~ii~~i•~~ •
~ .... ~ i ¸
b
:ii!i
i
ii~i:
:
¸
i: ~!?i: i
..
: iUi, ¸ i~: ~ i~. ~
Fig. 4. Term extraplacental m e m b r a n e s stained with the monoclonal antibody 4E (anti-HLA-B). (a) Maternal cells in the decidua (D) are strongly positive, chorion m e m b r a n e cytotrophoblast cells (C) are negative, and fetal mesenchymal cells (FM) in the fibrous layers between the amnion and chorion membranes are positive, x 250. (b) A m n i o n m e m b r a n e epithelial cells stained weakly with 4E although occasional cells (arrow) were m o r e strongly positive, x 400.
28
same pattern of staining as with 4E whereas sections stained with W6/32 demonstrated positive chorionic cytotrophoblast and amnion epithelial cells, as expected from previous reports (Hsi et al., 1984; Hunt et al., 1988a; Hsi et al., 1988). Discussion
A comprehensive study of tissues from first, second and third trimesters established that class I HLA, as identified by the binding of the mAb 61D2, is acquired gradually by fetal mesenchymal cells as gestation progresses (Lessin et al., 1988). The results of the present study show that the binding patterns for 61D2 and 4E are similar in first trimester and term tissues. Fetal mesenchymal cells that bind 4E very weakly in comparison to maternal cells in first trimester tissues have, at term, densities of the HLA-B antigens equivalent to those in maternal cells. Since the original observations by Faulk and Temple (1976) and Goodfellow and co-workers (1976), numerous investigators have tested villous trophoblast cells and have failed to detect binding of anti-class I H L A reagents. The anti-HLA-B-specific m A b 4E used in the present study also failed to bind to villous trophoblast cells in both early and late gestational stages. At the molecular level, term syncytial trophoblast has been shown to contain very low levels of m R N A capable of hybridizing to a class I heavy chain probe (Hunt et al., 1988b), an observation that may well explain the failure of syncytial trophoblast at term to synthesize class I antigens. In contrast to the consistent immunohistologic results with syncytial trophoblast, cytotrophoblast cells have been tested with a number of anticlass I reagents, with conflicting results. The binding patterns with the mAb 61D2 and polyclonal reagents to allotypic determinants suggest that fully mature class I H L A are not expressed by trophoblast cells whereas the results with W6/32 suggest that some type of class I antigen is expressed by both extravillous cytotrophoblast cells in situ (Sunderland et al., 1981; Hsi et al., 1984; Wells et al., 1984) and villous cytotrophoblast cells that are cultured in vitro (Feinman et al., 1987; Loke and Burland, 1988). The binding patterns for the anti-HLA-B-specific m A b 4E obtained in the present study matched those reported for 61 D2 and allospecific reagents rather than those for W6/ 32. Had 4E bound to 61D2-/W6/32 ÷ cytotrophoblast cells, the antigens would have been clearly identified as HLA. However, trophoblast cells in all locations and in both early and term tissues were HLA-B negative. Preliminary experiments suggest that in tissue explants, trophoblast cells remain HLA-B negative even when exposed to interferon-gamma, just as they remain negative for 61 D2 (Hunt et al., 1987). Given the results'with 4E, arguments can still be made for trophoblast cell
29
class I antigens as truncated versions of classical I HLA as well as for trophoblast cell class I antigens as arising from non-HLA-A,B,C class I genes. In support of the first postulate, JEG-3 trophoblast-derived choriocarcinoma cells contain mRNA for HLA-B (Hunt et al., 1987), and villous cytotrophoblast cells from term placentas tested with a different class I HLA probe also contain specific message (Feinman et al., 1987). The postulate that novel HLA-A,B,C-Iike antigens are expressed by chorionic cytotrophoblast cells is supported by evidence that the antigens vary too greatly in their isoelectric point from classical antigens to be simply truncated versions of H L A (Ellis et al., 1986). Genes coding for HLAA,B,C-like antigens have recently been reported (Geraghty et al., 1987; Koller et al., 1988; Mizuno et al., 1988). In situ hybridization studies have not yet resolved the issue. Chorion membrane cytotrophoblast cells that are 61D2-/4E-/W6/32 ÷ contain class I mRNA (Hunt et al., 1988b), but because of the methodology used, the probe could have hybridized to mRNA from both HLA-B and other class I genes (Orr and Demars, 1983). Previous experiments suggest that amnion cells may express both lymphocyte-like (61D2÷/W6/32 ÷) and trophoblast-like (61D2-/W6/32 ÷) antigens, but predominantly the latter (Hunt et al., 1988a; Hsi et al., 1988). As was observed with other types of cells in extraembryonic tissues, the binding of 4E by amnion cells followed the general pattern established by 61D2 rather than W6/32. Externally derived molecules are known to influence the expression of class I HLA and the synthesis of class I HLA mRNA by amnion cells (Hunt and Wood, 1986; Hunt et al., 1988a). Precise biochemical and molecular approaches, which ideally would include cloning the class I genes expressed by some trophoblast cells and identifying their chromosomal location(s), are required to elucidate the nature of the class I antigens expressed by extraembryonic cells, and to progress with experimentation designed to determine the functions of those gene products.
Acknowledgements This study was supported by a grant from the E. Speas Foundation to J.S.H. and by a Pathology Fellowship to D.L.L. The authors wish to thank D. Friesen for expert assistance with photography.
References Barnstable, C.J., Bodmer, W.F., Brown, G., Galfre, G., Milstein, C., Williams, A.F. and Ziegler, A. (1978) Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens. Cell 14, 9--20. Beer, A.E. and Sio, J.O. (1982) Placenta as an immunological barrier. Bio. Reprod. 26, 15--27.
30 Ellis, S.A., Sargent, I.L., Redman, C.W. and McMichael, A.J. (1986) Evidence for a novel HLA antigen found on human extravillous trophohlast and a choriocarcinoma cell line. Immunology 59, 595--601. Faulk, W.P. and Temple, A. (1976) Distribution of heta-2-microglobulin and HLA in chorionic villi of human placentae. Nature (London) 262,799--802. Feinman, M.A., Kliman, H.J. and Main, E.K. (1987) HLA antigen expression and induction by gammainterferon in cultured human trophoblasts. Am. J. Ohstet.. Gynecol. 157, 1429--1434. Galbraith, R.M., Kantor, R.R.S., Ferrara, G.B., Ades, E.W. and Galbraith, G.M.P. (1981) Differential expression of transplantation antigens within the normal placental chorionic villus. Am. J. Reprod. Immunol. 1,331--335. Geraghty, D.E., Koller, B.H. and Orr, H.T. (1987) A human major histocompatibility complex class I gene that encodes a protein with a shortened cytoplasmic segment. Proc. Natl. Acad. Sci. USA 84, 9145--9149. Goodfellow, P.N., Barnstable, C.J., Bodmer, W.F., Snary, D.E. and Crumpton, M.J. 0976) Expression of H L A system antigens on placentae. Transplantation 22, 555--603. Hsi, B.-L., Grivanx, C., Fenichel, P., Hunt, J.S., Samson, M. and Yeh, C.-J. (1988) Topographical expression of class I major histocompatibilitycomplex antigens on human amniotic epithelium. J. Reprod. Immunol. 13,183--191. Hsi, B.-L., Yeh, C.-J. G. and Faulk, W.P. (1984) Class I antigens of the major histocompatibilitycomplex on cytotrophoblast of human chorion laeve. Immunology 52, 621--629. Hunt, J.S. and Wood, G.W. 0986) G a m m a interferon induces class I H L A and beta-2-microglobulin expression by human amnion cells.J. Immunol. 136, 364--367. Hunt, J.S., Andrews, G.K. and Wood, G.W. (1987) Normal trophoblasts resist induction of class I H L A . J. Immunol. 138, 2481--2487. Hunt, J.S., Andrews, G.K., Fishback, J.L., Feess, M. and Wood, G.W. (1988a) Amnion membrane epithelialcellsexpress class I H L A and contain class I H L A m R N A . J. Immunol.. 140, 2790--2795. Hunt, J.S., Fishback, J.L., Andrews, G.K. and Wood, G.W. (1988b) Expression of class I H L A genes by trophoblast cells: analysis by in situ hybridization. J. lmmunol. 140, 1293--1299. Koller, B.H., Geraghty, D.E., Shimizu, Y., DeMars, R. and Orr, H.T. (1988) HLA-E. A novel HLA class I gene expressed in resting T lymphocytes. J. Immunol. t41,897--904. Lessin, D.L., Hunt, J.S., King, C.R. and Wood, G.W. (1988) Antigen expression by cells near the maternai-fetal interface. Am. J. Reprod. Immunol. Microhiol. 16, 1--7. Loke, Y.W. and Burland, K. (1988) Human trophoblast cells cultured in modified medium and supported by extracellular matrix. Placenta 9, 173-- 182. Mizuno, S., Trapani, J.A., Koller, B.H., Dupont, B. and Yang, S.Y. (1988) Isolation and nucleotide sequence of a cDNA clone encoding a novel HLA class I gene. J. Immunol. 140, 4024--4030. Orr, H.T. and DeMars, R. 0983) Class I-like HLA genes map telomeric to the HLA-A2 locus in human cells. Nature 302, 534--536. Parham, P., Barnstahle, C.J. and Bodmer, W.F. (1979) Use of a monoclonal antibody (W6/32) in structural studies of HLA-A,B,C antigens. J. Immunol. 123,342--349. 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. (1981) HLA-A,B,C antigens are expressed on nonvillous trophoblasts of the early human placenta. J. Immunol. 127, 2614--2615. Ugolini, V., Nunez, G., Smith, R.G., Stastny, P. and Capra, J.D. (1980) Initial characterization of monoclonal antibodies against human monocytes. Proc. Nat. Acad. Sci. USA 77, 6764--6768. Wells, M., Hsi, B.-L. and Faulk, W.P. (1984) Class I antigens of the major histocompatibility complex on cytotrophoblast of the human placental basal plate. Am. J. Reprod. I mmunol. 6, 167--174. Yang, S.Y., Morishima, Y., Collins, N.H., Alton, T., Pollack, M.S., Yunis, E.J. and Dupont, B. (1984) Comparison of one-dimensionai IEF patterns for serologically detectable HLA-A and B allotypes. Immunogenetics 19, 217--231.
21
JR100583
Ontogeny and distribution of cells expressing HLA-B locus-specific determinants in the placenta and extraplacental membranes J o a n S. H u n t a, D i a n n a L. Lessin a a n d C h a r l e s R. King b *Department of Pathology and Oncology and bDepartment of Obstetrics and Gynecology, University of Kansas Medical Center, Kansas City, Kansas (U.S.A.) (Accepted for publication 19 December 1988)
Summary Both the trophectoderm and the inner cell mass of the blastocyst contribute to the cell populations found in the placenta and extraplacental membranes. Previous studies have shown differences between those two embryologically distinct populations of cells in their expression of class I HLA, and further differences among trophectoderm-derived trophoblast cell subpopulations. Binding patterns for antibodies to both monomorphic and allotypic determinants on class I heavy chains have been reported. In the present study, extraembryonic cells were evaluated by immunohistology for binding of the monoclonal antibody 4E, which identifies locus-specific determinants on HLA-B. Some inner cell mass-derived cells (mesenchymal cells) acquired high levels of HLA-B as gestation progressed and others continued to express low levels at late stages of gestation (amnion cells). In contrast, throughout gestation both villous and extravillous trophoblast cells failed to express detectable HLA-B. The binding patterns for 4E followed the patterns established by a monoclonal antibody to class I HLA heavy chains (61D2), and those reported for antisera to allotypic determinants. The findings support the suggestion that trophoblast cells forming the fetal component of the maternal-fetal interface exert highly effective regulation over the expression of class I HLA. Key words: human; major histocompatibility antigens; placenta; pregnancy;
trophoblast cells.
Correspondence to: Joan S. Hunt, Ph.D., Department of Pathology and Oncology, University of Kansas Medical Center, 39th Street & Rainbow Blvd., Kansas City, KS 66103, U.S.A. 0165-0378/89/$03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
22
Introduction
The expression of class I HLA by extraembryonic cells is of interest because maternal and fetal cells are intimately associated throughout pregnancy. Mothers would be expected to mount a massive immune response to fetal cells bearing disparate HLA, yet there is little evidence for that event. Two embryologically distinct populations of cells are present in the placenta and extraplacental membranes. One of those populations, the mesenchymal cells that arise from the inner cell mass of the blastocyst, gradually acquire the ability to bind a monoclonal antibody to monomorphic determinants on class I HLA heavy chains as gestation progresses (Lessin et al., 1988). The second population, trophoblast cells that arise from the trophectoderm of the blastocyst, forms the active interface between the fetus and the mother, secluding mesenchymal cells from maternal blood and tissues. One explanation for the failure of mothers to reject their semi-allogeneic fetuses is that the trophoblast cells of the placenta and extraplacental membranes do not express a normal complement of mature class I HLAA,B,C (Beer and Sio, 1982). Placental trophoblast is invariably described as class I HLA negative (Fanlk and Temple, 1976; Goodfellow et ai., 1976; Galbraith et al., 1981; Hunt et al., 1987; Hunt et al., 1988b). However, trophoblast cells exposed to decidual cells are recognized as class I H L A positive by some anti-class I reagents (Sunderland et al., 1981; Wells et al., 1984; Hsi et al., 1984; Hunt et al., 1988b). H L A positive trophoblast cells bind the monoclonal antibody (mAb) W6/32 but not the mAb 61D2, both of which identify HLA-A,B,C. Anti-beta2-microglobulin gives positive results, but antisera directed toward maternally-derived allotypic determinants of class I antigens do not (Redman et al., 1984). Lower than normal molecular weight antigens with unusual isoelectric points are immunoprecipitated from some trophoblast cells by W6/32 (Ellis et al., 1986). It is not clear if the antigens detected on some trophoblast cells are immature class I HLA lacking a full complement of determinants or if those antigens are dictated by developmentally regulated class I HLA-A,B,C-Iike genes (Orr and DeMars, 1983; Geraghty et al., 1987, Koller et al., 1988; Mizuno et al., 1988). The mAb that does bind to some trophoblast cells, W6/32, requires beta-2-microglobulin for recognition (Parham et al., 1979) and both HLA and HLA-like molecules associate with that non-polymorphic molecule. Allotypic determinants and the locus-specific determinants that define the subclasses of class I HLA, HLA-A and HLA-B, are interspersed in the alphal-alpha2 domains of class I HLA heavy chains. The mAb 4E identifies HLA-B locus specific determinants on heavy chains and does not require
23
beta2-microglobulin for binding (Yang et al., 1984). 4E is therefore a useful reagent for further definition of the structure of the class I antigens expressed by trophoblast cells. Since HLA-B would not be immunogenic to the mother, expression of that epitope by trophoblast cells would not facilitate graft rejection. Positive identification of HLA-B would, however, firmly identify the class I antigens on trophoblast cells as arising from the H L A gene complex. The results of the present study demonstrate that HLA-B is never expressed by either villous or extravillous trophoblast cells although mesenchymal cells exhibit increased HLA-B positivity as gestation progresses. Thus, the genes coding for the class I antigens on some trophoblast cells remain to be established. Materials and methods Tissues First trimester (n, 3) and term tissues (n, 3) from elective pregnancy terminations or normal deliveries were obtained courtesy of the Department of Obstetrics and Gynecology of this institution, and were processed within 30 min. Portions of tissue (3--5 m m 3) were flash frozen in liquid N 2 and stored at - 70°C until sectioned by cryostat and tested. A n tibodies Culture supernatant containing the mouse m A b 4E, which binds to locusspecific determinants on HLA-B (Yang et al., 1984) was used at a 1:2 dilution. 4E was a generous gift from Drs. S.Y. Yang (Memorial Sloan Kettering Cancer Center) and E.J. Yunis (Dana-Farber Cancer Institute). The m A b to m o n o m o r p h i c determinants on class I H L A heavy chains 61D2 (Ugolini et al., 1980) was obtained from Bethesda Research Laboratories, Gaithersburg, MD, and was used at a 1:100 dilution. W6/32 (Barnstable et al., 1978), which recognizes a combinatorial determinant of class I H L A heavy chains and beta2-microglobulin (Parham et al., 1979) was generated as ascites from the American Type Culture Collection hybridoma HB95, and was used at a 1:400 dilution. Immunohistochemistry Sections of frozen tissues were air-dried and fixed in cold acetone. Sections were either used immediately or were stored at - 70°C (< 5 days) before testing. Binding of the m A b was detected using an avidin-biotin immunoperoxidase staining system for mouse lgG from Vector Laboratories (Vectastain ABC, Burlingame, CA). Quenching of endogenous peroxidase was performed following incubation with the biotinylated anti-mouse reagent
24
rather than prior to incubation with the primary mAb in order to maintain antigen integrity. Following incubation with the substrate, diaminobenzidine tetrahydrachloride (0.5 mg/ml, Sigma, St. Louis), the tissue sections were counterstained with Gill's hematoxylin. Positive cells bore a brown reaction product, and the nuclei of both positive and negative cells were blue. Results
First trimester tissue Figure 1 shows first trimester placental tissue stained with anti-HLA-B (4E). Maternal ceils in the fibrinoid tissue adjacent to fetal trophoblast cells were strongly positive with 4E. Mesenchymal cells within the placental villi were weakly positive for HLA-B, and trophoblast cells were negative. The same general pattern of staining was obtained with the anti-class I HLA reagents 61D2 and W6/32, although staining was invariably more intense with W6/32 than with either 4E or 61D2 (not shown). Term placenta In Fig. 2, cross-sections of term placental villi are shown that contain mesenchymal cells that were strongly positive with the mAb to HLA-B, 4E.
::~.-
:
: i "~
~.:
:i
!:
:::: ,~:?i~ili: ii :
i
i ! ~
•
I~ i
.
i
..:.
Fig. 1. First trimester placenta stained with the monoclonal antibody 4E (anti-HLA-B). Maternal cells in fibrous tissue adjacent to placental tissue are strongly positive (arrows), placental villous mesenchynml cells (VM) are weakly positive, and trophoblast cells (TB, arrowheads) are negative. × 400.
25 = "": i
'
~
• ~: ~
.~ ~ ~ '~::" -::;ii~ :: "
i~'~, i ".
.::
! ; ! i ~ : " .... ,..
!:~:
~'i::'~
~~
, ,
~
~
.
~
.
.
.
.
.
.
.
~: !~!i
.
:i
: :
:
.
:i
::
:i~
• .:
:':ii!~
" .i, ".::i.
"
"
~"/~,i ! ~.ii~ .... i . ~. ~ . . i:~.~,i;~i~ ~. . . . . .
i[i:
i:/~ ° .::,~:ii i
i,:.,]
:~:
:
.'!~]~:::i
.?
::
..~.'
. ~:,
~~""~ ! . ~ . . . ~ .~r' ~ ..... ~
•
: .. ,:'~. : ~ .
....~.~ :
:
• [:
] ,~
, ~ , ~ ~ ~ i l
.~
Fig. 2. Term placenta stained with the monoclonal antibody 4E (anti-HLA-B). Mesenchymal cells in the centers of placental villi are strongly positive with 4E whereas trophoblast cells lining the maternal blood spaces (arrows) are negative. × 250.
Trophoblast cells lining the maternal blood spaces were negative with 4E. The same results were obtained with 61D2 and W6/32 (not shown). Figure 3 shows cross-sections o f a few placental villi and large areas of fibrinoid tissue. In Fig. 3a, the tissue is stained with the m A b 61D2, which binds to mesenchymal cells in placental villi (arrows), but does not bind to cells with cytotrophoblastic characteristics in the fibrinoid tissue (arrowhead). Figure 3b shows that the binding patterns for 4E were essentially the same as for 61D2. A cluster o f 4E-negative cells with cytotrophoblast cell m o r p h o l o g y is shown in Fig. 3c.
, .~, Fig. 3a
'
~'" "~.
~i,
~ :
'~'~.~;
i~i~,~ "~'~.~
26
, :'i
:i ¸
i ¸ ••
•
i
:
i!r- ~
Fig. 3. Serial sections of term placenta stained with two monoclonal antibodies to class I HLA. (a) Staining with 61 D2 (anti-HLA-A,B,C). Mesenchymal cells in the centers o f chorionic villi are strongly positive (arrows) whereas cells with cytotrophoblast morphology in adjacent fibrous tissue (arrowhead) were negalive with 61D2, showing only nuclear staining with the Gill's hematoxylin ¢ounterstain. x 100. (b) Staining with 4E (anti-HLA-B). The staining pattern for 4E resembles that obtained with 61D2. Mesenchymal cells in the chorionic villi were strongly positive (arrows), but cells with cytotrophoblast morphology were negative with 4E (arrowhead). x 100. (c) Higher magnification of the tissue in (b) showing a nest of 4E negative cells with cytotrophoblast cell morphology (arrows). x 250.
Term extraplacental membranes Figure 4A demonstrates that maternal decidual cells were strongly positive with the mAb to HLA-B, 4E, as were the fetal cells in the mesenchyme between the amnion and chorion membranes. In contrast, 4E was essentially negative with chorion membrane cytotrophoblast cells. The amnion membrane (Fig. 4b) usually stained very weakly with 4E, but there was some variability among the membranes tested and occasional amnion cells were more strongly positive (Fig. 4b). Serial sections stained with 61D2 showed the
27
~ " •,~ ,! •i!•!~:~i~•~i~i~,!II~~•ii~~ •
•
: i
•: ~!• i i'i~,~ii~~i•~~ •
~ .... ~ i ¸
b
:ii!i
i
ii~i:
:
¸
i: ~!?i: i
..
: iUi, ¸ i~: ~ i~. ~
Fig. 4. Term extraplacental m e m b r a n e s stained with the monoclonal antibody 4E (anti-HLA-B). (a) Maternal cells in the decidua (D) are strongly positive, chorion m e m b r a n e cytotrophoblast cells (C) are negative, and fetal mesenchymal cells (FM) in the fibrous layers between the amnion and chorion membranes are positive, x 250. (b) A m n i o n m e m b r a n e epithelial cells stained weakly with 4E although occasional cells (arrow) were m o r e strongly positive, x 400.
28
same pattern of staining as with 4E whereas sections stained with W6/32 demonstrated positive chorionic cytotrophoblast and amnion epithelial cells, as expected from previous reports (Hsi et al., 1984; Hunt et al., 1988a; Hsi et al., 1988). Discussion
A comprehensive study of tissues from first, second and third trimesters established that class I HLA, as identified by the binding of the mAb 61D2, is acquired gradually by fetal mesenchymal cells as gestation progresses (Lessin et al., 1988). The results of the present study show that the binding patterns for 61D2 and 4E are similar in first trimester and term tissues. Fetal mesenchymal cells that bind 4E very weakly in comparison to maternal cells in first trimester tissues have, at term, densities of the HLA-B antigens equivalent to those in maternal cells. Since the original observations by Faulk and Temple (1976) and Goodfellow and co-workers (1976), numerous investigators have tested villous trophoblast cells and have failed to detect binding of anti-class I H L A reagents. The anti-HLA-B-specific m A b 4E used in the present study also failed to bind to villous trophoblast cells in both early and late gestational stages. At the molecular level, term syncytial trophoblast has been shown to contain very low levels of m R N A capable of hybridizing to a class I heavy chain probe (Hunt et al., 1988b), an observation that may well explain the failure of syncytial trophoblast at term to synthesize class I antigens. In contrast to the consistent immunohistologic results with syncytial trophoblast, cytotrophoblast cells have been tested with a number of anticlass I reagents, with conflicting results. The binding patterns with the mAb 61D2 and polyclonal reagents to allotypic determinants suggest that fully mature class I H L A are not expressed by trophoblast cells whereas the results with W6/32 suggest that some type of class I antigen is expressed by both extravillous cytotrophoblast cells in situ (Sunderland et al., 1981; Hsi et al., 1984; Wells et al., 1984) and villous cytotrophoblast cells that are cultured in vitro (Feinman et al., 1987; Loke and Burland, 1988). The binding patterns for the anti-HLA-B-specific m A b 4E obtained in the present study matched those reported for 61 D2 and allospecific reagents rather than those for W6/ 32. Had 4E bound to 61D2-/W6/32 ÷ cytotrophoblast cells, the antigens would have been clearly identified as HLA. However, trophoblast cells in all locations and in both early and term tissues were HLA-B negative. Preliminary experiments suggest that in tissue explants, trophoblast cells remain HLA-B negative even when exposed to interferon-gamma, just as they remain negative for 61 D2 (Hunt et al., 1987). Given the results'with 4E, arguments can still be made for trophoblast cell
29
class I antigens as truncated versions of classical I HLA as well as for trophoblast cell class I antigens as arising from non-HLA-A,B,C class I genes. In support of the first postulate, JEG-3 trophoblast-derived choriocarcinoma cells contain mRNA for HLA-B (Hunt et al., 1987), and villous cytotrophoblast cells from term placentas tested with a different class I HLA probe also contain specific message (Feinman et al., 1987). The postulate that novel HLA-A,B,C-Iike antigens are expressed by chorionic cytotrophoblast cells is supported by evidence that the antigens vary too greatly in their isoelectric point from classical antigens to be simply truncated versions of H L A (Ellis et al., 1986). Genes coding for HLAA,B,C-like antigens have recently been reported (Geraghty et al., 1987; Koller et al., 1988; Mizuno et al., 1988). In situ hybridization studies have not yet resolved the issue. Chorion membrane cytotrophoblast cells that are 61D2-/4E-/W6/32 ÷ contain class I mRNA (Hunt et al., 1988b), but because of the methodology used, the probe could have hybridized to mRNA from both HLA-B and other class I genes (Orr and Demars, 1983). Previous experiments suggest that amnion cells may express both lymphocyte-like (61D2÷/W6/32 ÷) and trophoblast-like (61D2-/W6/32 ÷) antigens, but predominantly the latter (Hunt et al., 1988a; Hsi et al., 1988). As was observed with other types of cells in extraembryonic tissues, the binding of 4E by amnion cells followed the general pattern established by 61D2 rather than W6/32. Externally derived molecules are known to influence the expression of class I HLA and the synthesis of class I HLA mRNA by amnion cells (Hunt and Wood, 1986; Hunt et al., 1988a). Precise biochemical and molecular approaches, which ideally would include cloning the class I genes expressed by some trophoblast cells and identifying their chromosomal location(s), are required to elucidate the nature of the class I antigens expressed by extraembryonic cells, and to progress with experimentation designed to determine the functions of those gene products.
Acknowledgements This study was supported by a grant from the E. Speas Foundation to J.S.H. and by a Pathology Fellowship to D.L.L. The authors wish to thank D. Friesen for expert assistance with photography.
References Barnstable, C.J., Bodmer, W.F., Brown, G., Galfre, G., Milstein, C., Williams, A.F. and Ziegler, A. (1978) Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens. Cell 14, 9--20. Beer, A.E. and Sio, J.O. (1982) Placenta as an immunological barrier. Bio. Reprod. 26, 15--27.
30 Ellis, S.A., Sargent, I.L., Redman, C.W. and McMichael, A.J. (1986) Evidence for a novel HLA antigen found on human extravillous trophohlast and a choriocarcinoma cell line. Immunology 59, 595--601. Faulk, W.P. and Temple, A. (1976) Distribution of heta-2-microglobulin and HLA in chorionic villi of human placentae. Nature (London) 262,799--802. Feinman, M.A., Kliman, H.J. and Main, E.K. (1987) HLA antigen expression and induction by gammainterferon in cultured human trophoblasts. Am. J. Ohstet.. Gynecol. 157, 1429--1434. Galbraith, R.M., Kantor, R.R.S., Ferrara, G.B., Ades, E.W. and Galbraith, G.M.P. (1981) Differential expression of transplantation antigens within the normal placental chorionic villus. Am. J. Reprod. Immunol. 1,331--335. Geraghty, D.E., Koller, B.H. and Orr, H.T. (1987) A human major histocompatibility complex class I gene that encodes a protein with a shortened cytoplasmic segment. Proc. Natl. Acad. Sci. USA 84, 9145--9149. Goodfellow, P.N., Barnstable, C.J., Bodmer, W.F., Snary, D.E. and Crumpton, M.J. 0976) Expression of H L A system antigens on placentae. Transplantation 22, 555--603. Hsi, B.-L., Grivanx, C., Fenichel, P., Hunt, J.S., Samson, M. and Yeh, C.-J. (1988) Topographical expression of class I major histocompatibilitycomplex antigens on human amniotic epithelium. J. Reprod. Immunol. 13,183--191. Hsi, B.-L., Yeh, C.-J. G. and Faulk, W.P. (1984) Class I antigens of the major histocompatibilitycomplex on cytotrophoblast of human chorion laeve. Immunology 52, 621--629. Hunt, J.S. and Wood, G.W. 0986) G a m m a interferon induces class I H L A and beta-2-microglobulin expression by human amnion cells.J. Immunol. 136, 364--367. Hunt, J.S., Andrews, G.K. and Wood, G.W. (1987) Normal trophoblasts resist induction of class I H L A . J. Immunol. 138, 2481--2487. Hunt, J.S., Andrews, G.K., Fishback, J.L., Feess, M. and Wood, G.W. (1988a) Amnion membrane epithelialcellsexpress class I H L A and contain class I H L A m R N A . J. Immunol.. 140, 2790--2795. Hunt, J.S., Fishback, J.L., Andrews, G.K. and Wood, G.W. (1988b) Expression of class I H L A genes by trophoblast cells: analysis by in situ hybridization. J. lmmunol. 140, 1293--1299. Koller, B.H., Geraghty, D.E., Shimizu, Y., DeMars, R. and Orr, H.T. (1988) HLA-E. A novel HLA class I gene expressed in resting T lymphocytes. J. Immunol. t41,897--904. Lessin, D.L., Hunt, J.S., King, C.R. and Wood, G.W. (1988) Antigen expression by cells near the maternai-fetal interface. Am. J. Reprod. Immunol. Microhiol. 16, 1--7. Loke, Y.W. and Burland, K. (1988) Human trophoblast cells cultured in modified medium and supported by extracellular matrix. Placenta 9, 173-- 182. Mizuno, S., Trapani, J.A., Koller, B.H., Dupont, B. and Yang, S.Y. (1988) Isolation and nucleotide sequence of a cDNA clone encoding a novel HLA class I gene. J. Immunol. 140, 4024--4030. Orr, H.T. and DeMars, R. 0983) Class I-like HLA genes map telomeric to the HLA-A2 locus in human cells. Nature 302, 534--536. Parham, P., Barnstahle, C.J. and Bodmer, W.F. (1979) Use of a monoclonal antibody (W6/32) in structural studies of HLA-A,B,C antigens. J. Immunol. 123,342--349. 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. (1981) HLA-A,B,C antigens are expressed on nonvillous trophoblasts of the early human placenta. J. Immunol. 127, 2614--2615. Ugolini, V., Nunez, G., Smith, R.G., Stastny, P. and Capra, J.D. (1980) Initial characterization of monoclonal antibodies against human monocytes. Proc. Nat. Acad. Sci. USA 77, 6764--6768. Wells, M., Hsi, B.-L. and Faulk, W.P. (1984) Class I antigens of the major histocompatibility complex on cytotrophoblast of the human placental basal plate. Am. J. Reprod. I mmunol. 6, 167--174. Yang, S.Y., Morishima, Y., Collins, N.H., Alton, T., Pollack, M.S., Yunis, E.J. and Dupont, B. (1984) Comparison of one-dimensionai IEF patterns for serologically detectable HLA-A and B allotypes. Immunogenetics 19, 217--231.