- Email: [email protected]
In vitro antigenicity of trophoblast
In vitro antigenicity ROSALIND G.
M.
I.
URBACH,
Toronto,
Ontario,
of trophoblast
DOUTHWAITE, M.D.,
M.Sc. F.R.C.S.(C),
F.A.C.O.G.
Canada
The addition of maternal or allogenic leukocytes to trypsinized placental monolaytr cultures resulted in severe cytotoxic efltcts to the trophoblast cells, maximal on the fourth day of incubation. These results are interpreted as indicating that trophoblast does possess antigenic determinants. It is suggested that at least two factors art involved in protecting the conctptus from maternal immunologic rejection. First, the sialomucin coating of the trofihoblast masks the underlying histocompatibility antigens and prevents their detection by mattrnal immunologically competent cells. Second, maternal leukocytes fail to cross the placental barrier early in pregnancy.
1 N s P I T E 0 F recent advances in the field of transplantation immunology over the last two decades and the vast amount of literature published, the immunologic enigma of mammalian pregnancy, the most fascinating exception to the fundamental laws of allograft rejection, remains largely unsolved. In recent studies in our laboratory,l with the use of fluorescent microscopy, no indication of an immune humoral response to placental trophoblast could be detected in either postpartum or postabortal patients. However, using an in vitro histocompatibility test based on the allogenic inhibition phenomenon,’ Currie and Bagshawe31 4 reported that the addition of maternal lymphocytes to trophoblast monolayer cultures resulted in marked cytotoxic effects, maximal on the fourth day of incubation. On the basis of the above experimental work and the continuing controversy regarding the antigenic nature of trophoblast, we have chosen the allogenic
inhibition phenomenon as a model for trophoblast antigenicity. Methods
and
for testing
materials
Trophoblast culture. Forty normal placentas from 8 to 20 week pregnancies were collected at the time of therapeutic abortion and placed immediately in ice-cold saline. After thorough washing in several changes of saline, the villi were dissected from the chorionic plate and washed in Hanks solution containing 400 units of penicillin and 200 pg of streptomycin per milliliter. The cells were then dissociated for 30 minutes in one per cent trypsin in Ca++- and Mg++-free Hanks solution at pH 7.4 at 37O C. The resulting cell suspension was filtered through for 5 minutes at 1,000 gauze, centrifuged r.p.m., washed once, and resuspended in approximately 10 ml. of Eagles basal medium in Earles supplemented with 15% heatinactivated fetal calf serum and containing 100 units of penicillin and 100 pg of streptomycin per milliliter. The final volume was adjusted to give a concentration of 0.5 x lo6 cells per milliliter. Aliquots of 2 ml. were inoculated into Leighton tubes and placed in a humidified incubator at 37’ C. in an atmosphere of 5 per cent CO, in air. The viability of the trophoblast was assessed by the semiquantitative assay of chorionic go-
From the Department of Obstetrics and Gynatcology, University of Toronto, and the Welltsley Hospital. This research project was made possible by grants from the Medical Research Council of Canada, Grant No. MA 2661, and the Department of Obstetrics and Gynaecology, the University of Toronto. Reprint requests: Dr. G. I. Urbach, The Wtlleslty Hospital, 160 Welltsley St. E., Toronto 284, Ontario, Canada.
1023
1024
Douthwaite
and
Aprii
Urbach Amer.
nadotropin in the culture medium and by extensive morphologic examination under phase-contrast microscopy with the use of time-lapse photography and vital staining with trypan blue.
Lymphocyte
and
leukocyte
cultures.
Twenty-four hours after processing the placental tissue, 50 ml. of peripheral venous blood was collected from the patient, and the lymphocytes were isolated by the method described by Thomas and colleagues.5 Apart from a few erythrocytes, the resulting white cell suspension contained at least 97 per cent lymphocyte-like cells. Allogenic lymphocytes were also harvested from postpartum or postabortal patients. Buffy coat leukocytes were obtained in the same manner with the omission of the incubation stage in cotton-packed tubes. Each batch of cells was counted and suspended in Eagles complete medium at a concentration of 1 x lo6 cells per milliliter. At this point the viability of the leukocytes was assessed by trypan blue exclusion. The culture medium from the 24 hour cultures was then gently pipetted off, the monolayers were rinsed twice to remove any remaining blood cells, and 2 ml. of the white cell suspension was added to each tube to give a ratio of 2 leukocytes or lymphocytes to each test cell. The cultures were incubated for a further 4 days and then assessed for viability.
Control cultures. Allogenic control. Samples of embryo lung tissue were obtained when available and were processed in the same way as the placental tissue. Syngeneic control. Endometrium, processed as above, was incubated with donor and allogenic leukocytes. Assay of chorionic gonadotropin. After the 4 day test period, the culture medium from each group of trophoblast cultures was assayed in duplicate by the hemagglutination reaction of human chorionic gonadotropin (HCG) -sensitized erythrocytes (Pregnosticon). The sensitivity of this immunologic reaction is about 1,000 U. of HCG per liter. Unused culture medium from the same
batch, incubated under served as a control.
identical
J. Obstet.
1, 1971 Gynec.
conditions,
Morphologic tests. Cover slips were mounted in a stainless steel diffusion chamber and examined with the microscope at 37’ C. under phase-contrast microscopy at magnifications up to 1,600. Several cells from each group were selected and filmed for 20 minutes at one frame per 4 sec. Histologic techniques. Samples of untrypsinized placental tissue and selected monolayer cultures from each group were routinely fixed in Carnoy’s solution and stained with Harris’ hematoxylin and eosin. Results Thirty-five of the original placental ceil cultures were used for the experiments. The other 5 cultures were all from early pregnancies (8 to 10 weeks) and either failed to grow or yielded too few cells to merit testing. In the successful cultures the cells quickly adhered to the glass cover slips and spread out within 16 hours. Three basic cell types with many intermediate forms were observed. These were the spindle cells, the epitheloid cells, and the multinucleated giant cells. All 3 types were present in the 16 hour cultures, the latter two being the dominant cells during the first week of culture. Placental tissue from the more advanced pregnancies (14 to 20 weeks) yielded large numbers of multinucleated giant cells which in some cultures formed a complete monolayer. Trophoblast control cultures. Following the 4 day test period, more than 90 per cent of the placental cells were still viable (Fig. 1) , and chorionic gonadotropin levels ranged from 2 to 16 units of HCG per IO0 cells per 96 hours.
Addition of “purified” lymphocytes to the monolayers. In all but 2 of 35 placental cell cultures, the addition of up to 8 x lo6 maternal or allogenic lymphocytes “purified” by the technique described,5 caused no detectable cytotoxic effects (Fig. 2)) nor were any such effects ever observed in the embryo lung cultures after the 4 day test period.
Trophoblast plus maternal or allogenic buffy coat leukocytes. The addition of 2 x
Volume Number
109 7
Fig. 1. Control placental monolayer from a 16 week pregnancy after 5 days’ incubation. (Hematoxylin and eosin. Phase contrast microscopy, original magnification x256.)
In vitro
antigenicity
of
trophoblast
1025
Fig. 2. Placental monolayer after 4 days’ incubation with 2 x 106 “purified” maternal lymphocytes showing lack of cell damage. (Hematoxylin and eosin. Phase contrast microscopy, original magnification x256.)
lo6 maternal leukocytes produced dramatic cytotoxic effects, resulting in almost complete destruction of the trophoblast monolayer by the fourth day of incubation. These cytotoxic effects were not detectable under 72 hours’ incubation. Chorionic gonadotropin was never detected in the culture medium of this group (i.e., less than 1 U. of HCG per lo6 cells per 96 hours) . Allogenic leukocytes from either postpartum or postabortal patients produced identical effects (Fig. 3).
Control cultures. Allogenic controls. The addition of maternal or allogenic buffy coat leukocytes to embryo lung cultures resulted in almost complete destruction of the monolayers by the fourth day of incubation (Figs. 4 and 5). Syngenic controls. The addition of either 2 or 4 x IO6 donor buffy coat leukocytes to endometrium monolayers caused no detectable cytotoxic effects (Fig. 6), whereas 2 x lo6 allogenic buffy coat leukocytes, from the placental donor, destroyed more than 90 per cent of the cells (Fig. 7). Comment Monolayer tissue culture of immature placental tissue provides a valuable method of studying the individual trophoblast cells in the living state. By using this technique, it is possible to observe directly the effect of maternal and allogenic lymphocytes on the trophoblast cells. Although cell morphology
Fig.
3. Placental monolayer after 4 days’ incuba2 x 10s maternal leukocytes showing almost total destruction of the trophoblast cells. (Hematoxylin and eosin. Phase contrast microscopy, original magnification x256.)
tion with
in tissue culture is not a reliable index of the generic origin of cells as they tend to become undifferentiated, by commencing immunologic studies on the trophoblast cells 24 hours after processing the placental tissue, these effects are minimized. Because of this problem, it was decided to assess cell viability of the 3 basic cell types by direct morphologic examination, with the use of phasecontrast microscopy and time-lapse photography in conjunction with chorionic gonadotropin assays of the culture medium. This combination was thought to be most suitable for detecting possible cytotoxic effects in the trophoblast/lymphocyte system. Having shown that allogenic buffy coat
1026
Douthwaite
and
Urbach Amer.
April J. Obstet.
1, 1971 Gymc.
Fig. 4. Control embryo lung culture after 5 days’ incubation. (Hematoxylin and eosin. Phase contrast microscopy, original magnification x256.)
Fig. 5. Embryo lung culture 4 days after incubation with 2 x 106 maternal leukocytes showing almost total destruction of the monolayer. (Hematoxvlin and eosin. Phase contrast microscopy, original magnification x256.)
Fig. 6. Syngenic control. Endometrium monolayer after 4 days’ incubation with 4 x 106 donor leukocytes. (Phase contrast microscopy, original magni-
Fig. 7. Endometrium culture after 4 days’ incubation with 2 x 106 allogenic (placental donor) leukocytes showing the destruction of the monolayer. (Phase contrast microscopy, original magnification x380.)
fication x380.) leukocytes cause severe cytotoxic effects when incubated for 4 days with endometrium monolayers whereas syngenic leukocytes do not, it was decided to use buffy coat leukocytes in all future experiments. In the light of recent developments, the failure of the “purified” lymphocytes to elicit similar cytotoxic effects in our early experiments can be explained by the fact that the separation technique used removed some other cell type or types necessary for the reaction. There have been many recent reports that the blastogenic response of lymphocytes to antigen in vitro is reduced or prevented by the removal of the phagocytic cells from the leukocyte suspension,s and it is now certain
that at least one other cell type, a “glassadherent cell,” possibly the macrophage, is necessary for this reaction.7 In our experiments, apart from a few erythrocytes, the 3 per cent or less of the contaminating cells in the lymphocyte suspension were mostly neutrophils. Although Currie and Bagshawed were able to produce cytotoxic effects on the trophoblast with “purified” lymphocytes, the separation technique used by them resulted in a suspension which was less “pure” and in all probability contained sufficient “glassadherent cells” to result in a cytotoxic effect. The reason for the moderate cell damage to trophoblast and embryo lung monolayers by “purified” lymphocytes in 2 of 35 cultures
Volume Number
109 7
In vitro
is not clear, particularly as differential counts of the lymphocytes, based on 500 cells, revealed them to be more than 97 per cent pure. Our results demonstrate that trypsinizcd trophoblastic cells are capable of expressing allogenic histocompatibility antigens detectable by nonimmune maternal and allogenic buffy coat leukocytes in vitro. This finding is in agreement with the work of Currie and Bagshawe.33 4 Since these experiments demonstrate that trypsinized trophoblast is antigenic to the maternal host in vitro (i.e., removing the sialomucin unmasks the trophoblast antithat the genicity) , it must be concluded failure of placental/fetal rejection by the maternal host must be attributed, at least in part, to the presence of a noncellular barrier which would prevent the maternal leukocytes and lymphocytes from coming into close contact with the placental tissue. Histochemical analysis has since shown this barrier layer to be a tryptophane-rich sialomucopeptide containing sialic and hyaluronic acid.s, lo Many tumor cells are also known to be coated with acidic mucopolysaccharides,ll and it has been shown that this layer is responsible for the high electronegative charge on the surface of these cells. Removal of the surface sialic acid groups by treatment with neuraminidase reduces this electronegative chargel and reveals the antigenicity of Ehrlich ascites tumor cells, rendering them capable of immunizing host animals.13 As lymphocytes are also highly negatively charged’* and can be repelled by electronegativity,15 it was suggested by Currie and Bagshawe4 that in vivo this peritrophoblastic fibrinoid layer acts as an electrostatic barrier which prevents maternal lymphocytes from coming into close contact with the fetalderived trophoblast and hence allows the
antigenicity
of trophoblast
1027
antigens to escape detection. Currie and his colleagueP have since modified this theory, suggesting that the sialomucin may act by steric hindrance to prevent the maternal lymphocytes from gaining access to the underlying antigenic groups. Other contributory evidence supporting the action of sialomucin was the recent report of Lippman17 showing that the expression of certain mouse tumor antigens can be suppressed by coating the tumor cells in vitro with various acid mucopolysaccharides. A second protective mechanism preventing fetal rejection concerns the failure of maternal lymphocytes to cross the placental barrier early in pregnancy, when rejection of fetal tissue is possible, as opposed to late in pregnancy, when the fetus is well equipped immunologically to destroy such cells. Although it has been shown that fetal cells cross into the maternal circulation,18, Is only one report of significant numbers of maternal cells crossing into the fetus in early pregnancy has been reported. This recent claim by Tuffrey and colleaguesZO of large numbers of maternal cells crossing into the mouse fetus has not yet been confirmed, and indeed two independent research teams21s 22 have been unable to reproduce these results. It was suggested by Seller2” that the porosity of the placenta claimed by Tuffrey and colleagues could perhaps be the result of the particular strain combination used by these workers. It would thus appear that at least two factors are involved in protecting the conceptus from maternal rejection: ( 1) the protective effect of the sialomucin coating of the trophoblast cells which screens the antigenic determinants from maternal immunologically competent cells, and (2) the failure of maternal leukocytes to cross the placental barrier early in pregnancy.
REFERENCES
1. Green, L., and Urbach, G. I.: Canad. Ass. J. 99: 469, 1968. 2. Moller, G., Beckman, V., and Lundgren, Nature 212: 1203, 1966.
Med. G.:
3. 4.
Currie, G. A.: J. Obstet. Gynaec. 74: 841, 1967. Currie, G. A., and Bag&we, vances Transpl. 34: 523, 1967.
Brit. K.
Comm. D.:
Ad-
1028
5.
Douthwaite
and Urbach
Thomas, J. W., Boldt, W., Horrocks, G., and Low, B.: Canad. Med. Ass. J. 97: 832, 1967. 6. Hersh, E. M., and Harris, J. E.: J. Immun. 100: 1184, 1968. 7. Seeger, R. C., and Oppenheim, J. J.: Fed. Proc. Amer. Sot. Exp. Biol. 29: 324, 1970. 8. Coulson, A. S., and Chalmers, D. G.: Lancet 1: 468, 1964. 9. Kirby, D. R. S., Billington, W. D., and Bradbury, S.: Nature 204: 548, 1964. 10. Bradbury, S., Billington, W. D:, and Kirby, D. R. S.: T. Rov. Micr. Sot. 84: 199. 1965. 11. Franks, L: M., -O’Shea, J. D., and Thomson, A. E. R.: Cancer 117: 983, 1964. 12. Ruhenstroth-Bauer, G., Fiehrmann, G. F., Kubler, W., Rueff, F., and Munk, K.: Z. Krebsforsch. 65: 37, 1962. 13. Lindenmann, J., and Klein, P. A.: Immunological Aspects of Viral Oncolysis, New York, N. Y., 1967, Springer-Verlag.
Amer.
14. 15. 16. 17. 18.
19. 20. 21.
22.
April J. Obstet.
1, 1971 Gynec.
Robinaux, R., and Baxin, S.: VOX. Sang. 22: 241, 1951. Carey, L. C., and Lepley, D.: Surg. Forum 13: 33, 1962. Currie, G. A., Van Doorninck, W., and Barcshawe. K. D.: Nature 219: 191. 1968. Lippman,‘M.: Nature 219: 33, 1968. Zipursky, A., Pollock, J., Neelands, P., Chown, B., and Israels, L. G.: Lancet 2: 489, 1963. Walknowska, J., Conte, F. A., and Grumbach, M. M.: La&et I: 1119; 1969: Tuffrev. M.. Bishun. N. P.. and Barnes. R. D.: Nature 221:’ 1029, 1969. ’ Billington, W. D., Kirby, D. R. S., Owen, J. J. T., Ritter, M. A., -Burtonshaw; M. D., Evans, E. P.. Ford. Cl. E.. Gauld. I. R.. and McLaren, A.1 Nature 224:‘ 704, 1969. ’ Seller, Mary J.: Nature 225: 1254, 1970.
M.
I.
URBACH,
Toronto,
Ontario,
of trophoblast
DOUTHWAITE, M.D.,
M.Sc. F.R.C.S.(C),
F.A.C.O.G.
Canada
The addition of maternal or allogenic leukocytes to trypsinized placental monolaytr cultures resulted in severe cytotoxic efltcts to the trophoblast cells, maximal on the fourth day of incubation. These results are interpreted as indicating that trophoblast does possess antigenic determinants. It is suggested that at least two factors art involved in protecting the conctptus from maternal immunologic rejection. First, the sialomucin coating of the trofihoblast masks the underlying histocompatibility antigens and prevents their detection by mattrnal immunologically competent cells. Second, maternal leukocytes fail to cross the placental barrier early in pregnancy.
1 N s P I T E 0 F recent advances in the field of transplantation immunology over the last two decades and the vast amount of literature published, the immunologic enigma of mammalian pregnancy, the most fascinating exception to the fundamental laws of allograft rejection, remains largely unsolved. In recent studies in our laboratory,l with the use of fluorescent microscopy, no indication of an immune humoral response to placental trophoblast could be detected in either postpartum or postabortal patients. However, using an in vitro histocompatibility test based on the allogenic inhibition phenomenon,’ Currie and Bagshawe31 4 reported that the addition of maternal lymphocytes to trophoblast monolayer cultures resulted in marked cytotoxic effects, maximal on the fourth day of incubation. On the basis of the above experimental work and the continuing controversy regarding the antigenic nature of trophoblast, we have chosen the allogenic
inhibition phenomenon as a model for trophoblast antigenicity. Methods
and
for testing
materials
Trophoblast culture. Forty normal placentas from 8 to 20 week pregnancies were collected at the time of therapeutic abortion and placed immediately in ice-cold saline. After thorough washing in several changes of saline, the villi were dissected from the chorionic plate and washed in Hanks solution containing 400 units of penicillin and 200 pg of streptomycin per milliliter. The cells were then dissociated for 30 minutes in one per cent trypsin in Ca++- and Mg++-free Hanks solution at pH 7.4 at 37O C. The resulting cell suspension was filtered through for 5 minutes at 1,000 gauze, centrifuged r.p.m., washed once, and resuspended in approximately 10 ml. of Eagles basal medium in Earles supplemented with 15% heatinactivated fetal calf serum and containing 100 units of penicillin and 100 pg of streptomycin per milliliter. The final volume was adjusted to give a concentration of 0.5 x lo6 cells per milliliter. Aliquots of 2 ml. were inoculated into Leighton tubes and placed in a humidified incubator at 37’ C. in an atmosphere of 5 per cent CO, in air. The viability of the trophoblast was assessed by the semiquantitative assay of chorionic go-
From the Department of Obstetrics and Gynatcology, University of Toronto, and the Welltsley Hospital. This research project was made possible by grants from the Medical Research Council of Canada, Grant No. MA 2661, and the Department of Obstetrics and Gynaecology, the University of Toronto. Reprint requests: Dr. G. I. Urbach, The Wtlleslty Hospital, 160 Welltsley St. E., Toronto 284, Ontario, Canada.
1023
1024
Douthwaite
and
Aprii
Urbach Amer.
nadotropin in the culture medium and by extensive morphologic examination under phase-contrast microscopy with the use of time-lapse photography and vital staining with trypan blue.
Lymphocyte
and
leukocyte
cultures.
Twenty-four hours after processing the placental tissue, 50 ml. of peripheral venous blood was collected from the patient, and the lymphocytes were isolated by the method described by Thomas and colleagues.5 Apart from a few erythrocytes, the resulting white cell suspension contained at least 97 per cent lymphocyte-like cells. Allogenic lymphocytes were also harvested from postpartum or postabortal patients. Buffy coat leukocytes were obtained in the same manner with the omission of the incubation stage in cotton-packed tubes. Each batch of cells was counted and suspended in Eagles complete medium at a concentration of 1 x lo6 cells per milliliter. At this point the viability of the leukocytes was assessed by trypan blue exclusion. The culture medium from the 24 hour cultures was then gently pipetted off, the monolayers were rinsed twice to remove any remaining blood cells, and 2 ml. of the white cell suspension was added to each tube to give a ratio of 2 leukocytes or lymphocytes to each test cell. The cultures were incubated for a further 4 days and then assessed for viability.
Control cultures. Allogenic control. Samples of embryo lung tissue were obtained when available and were processed in the same way as the placental tissue. Syngeneic control. Endometrium, processed as above, was incubated with donor and allogenic leukocytes. Assay of chorionic gonadotropin. After the 4 day test period, the culture medium from each group of trophoblast cultures was assayed in duplicate by the hemagglutination reaction of human chorionic gonadotropin (HCG) -sensitized erythrocytes (Pregnosticon). The sensitivity of this immunologic reaction is about 1,000 U. of HCG per liter. Unused culture medium from the same
batch, incubated under served as a control.
identical
J. Obstet.
1, 1971 Gynec.
conditions,
Morphologic tests. Cover slips were mounted in a stainless steel diffusion chamber and examined with the microscope at 37’ C. under phase-contrast microscopy at magnifications up to 1,600. Several cells from each group were selected and filmed for 20 minutes at one frame per 4 sec. Histologic techniques. Samples of untrypsinized placental tissue and selected monolayer cultures from each group were routinely fixed in Carnoy’s solution and stained with Harris’ hematoxylin and eosin. Results Thirty-five of the original placental ceil cultures were used for the experiments. The other 5 cultures were all from early pregnancies (8 to 10 weeks) and either failed to grow or yielded too few cells to merit testing. In the successful cultures the cells quickly adhered to the glass cover slips and spread out within 16 hours. Three basic cell types with many intermediate forms were observed. These were the spindle cells, the epitheloid cells, and the multinucleated giant cells. All 3 types were present in the 16 hour cultures, the latter two being the dominant cells during the first week of culture. Placental tissue from the more advanced pregnancies (14 to 20 weeks) yielded large numbers of multinucleated giant cells which in some cultures formed a complete monolayer. Trophoblast control cultures. Following the 4 day test period, more than 90 per cent of the placental cells were still viable (Fig. 1) , and chorionic gonadotropin levels ranged from 2 to 16 units of HCG per IO0 cells per 96 hours.
Addition of “purified” lymphocytes to the monolayers. In all but 2 of 35 placental cell cultures, the addition of up to 8 x lo6 maternal or allogenic lymphocytes “purified” by the technique described,5 caused no detectable cytotoxic effects (Fig. 2)) nor were any such effects ever observed in the embryo lung cultures after the 4 day test period.
Trophoblast plus maternal or allogenic buffy coat leukocytes. The addition of 2 x
Volume Number
109 7
Fig. 1. Control placental monolayer from a 16 week pregnancy after 5 days’ incubation. (Hematoxylin and eosin. Phase contrast microscopy, original magnification x256.)
In vitro
antigenicity
of
trophoblast
1025
Fig. 2. Placental monolayer after 4 days’ incubation with 2 x 106 “purified” maternal lymphocytes showing lack of cell damage. (Hematoxylin and eosin. Phase contrast microscopy, original magnification x256.)
lo6 maternal leukocytes produced dramatic cytotoxic effects, resulting in almost complete destruction of the trophoblast monolayer by the fourth day of incubation. These cytotoxic effects were not detectable under 72 hours’ incubation. Chorionic gonadotropin was never detected in the culture medium of this group (i.e., less than 1 U. of HCG per lo6 cells per 96 hours) . Allogenic leukocytes from either postpartum or postabortal patients produced identical effects (Fig. 3).
Control cultures. Allogenic controls. The addition of maternal or allogenic buffy coat leukocytes to embryo lung cultures resulted in almost complete destruction of the monolayers by the fourth day of incubation (Figs. 4 and 5). Syngenic controls. The addition of either 2 or 4 x IO6 donor buffy coat leukocytes to endometrium monolayers caused no detectable cytotoxic effects (Fig. 6), whereas 2 x lo6 allogenic buffy coat leukocytes, from the placental donor, destroyed more than 90 per cent of the cells (Fig. 7). Comment Monolayer tissue culture of immature placental tissue provides a valuable method of studying the individual trophoblast cells in the living state. By using this technique, it is possible to observe directly the effect of maternal and allogenic lymphocytes on the trophoblast cells. Although cell morphology
Fig.
3. Placental monolayer after 4 days’ incuba2 x 10s maternal leukocytes showing almost total destruction of the trophoblast cells. (Hematoxylin and eosin. Phase contrast microscopy, original magnification x256.)
tion with
in tissue culture is not a reliable index of the generic origin of cells as they tend to become undifferentiated, by commencing immunologic studies on the trophoblast cells 24 hours after processing the placental tissue, these effects are minimized. Because of this problem, it was decided to assess cell viability of the 3 basic cell types by direct morphologic examination, with the use of phasecontrast microscopy and time-lapse photography in conjunction with chorionic gonadotropin assays of the culture medium. This combination was thought to be most suitable for detecting possible cytotoxic effects in the trophoblast/lymphocyte system. Having shown that allogenic buffy coat
1026
Douthwaite
and
Urbach Amer.
April J. Obstet.
1, 1971 Gymc.
Fig. 4. Control embryo lung culture after 5 days’ incubation. (Hematoxylin and eosin. Phase contrast microscopy, original magnification x256.)
Fig. 5. Embryo lung culture 4 days after incubation with 2 x 106 maternal leukocytes showing almost total destruction of the monolayer. (Hematoxvlin and eosin. Phase contrast microscopy, original magnification x256.)
Fig. 6. Syngenic control. Endometrium monolayer after 4 days’ incubation with 4 x 106 donor leukocytes. (Phase contrast microscopy, original magni-
Fig. 7. Endometrium culture after 4 days’ incubation with 2 x 106 allogenic (placental donor) leukocytes showing the destruction of the monolayer. (Phase contrast microscopy, original magnification x380.)
fication x380.) leukocytes cause severe cytotoxic effects when incubated for 4 days with endometrium monolayers whereas syngenic leukocytes do not, it was decided to use buffy coat leukocytes in all future experiments. In the light of recent developments, the failure of the “purified” lymphocytes to elicit similar cytotoxic effects in our early experiments can be explained by the fact that the separation technique used removed some other cell type or types necessary for the reaction. There have been many recent reports that the blastogenic response of lymphocytes to antigen in vitro is reduced or prevented by the removal of the phagocytic cells from the leukocyte suspension,s and it is now certain
that at least one other cell type, a “glassadherent cell,” possibly the macrophage, is necessary for this reaction.7 In our experiments, apart from a few erythrocytes, the 3 per cent or less of the contaminating cells in the lymphocyte suspension were mostly neutrophils. Although Currie and Bagshawed were able to produce cytotoxic effects on the trophoblast with “purified” lymphocytes, the separation technique used by them resulted in a suspension which was less “pure” and in all probability contained sufficient “glassadherent cells” to result in a cytotoxic effect. The reason for the moderate cell damage to trophoblast and embryo lung monolayers by “purified” lymphocytes in 2 of 35 cultures
Volume Number
109 7
In vitro
is not clear, particularly as differential counts of the lymphocytes, based on 500 cells, revealed them to be more than 97 per cent pure. Our results demonstrate that trypsinizcd trophoblastic cells are capable of expressing allogenic histocompatibility antigens detectable by nonimmune maternal and allogenic buffy coat leukocytes in vitro. This finding is in agreement with the work of Currie and Bagshawe.33 4 Since these experiments demonstrate that trypsinized trophoblast is antigenic to the maternal host in vitro (i.e., removing the sialomucin unmasks the trophoblast antithat the genicity) , it must be concluded failure of placental/fetal rejection by the maternal host must be attributed, at least in part, to the presence of a noncellular barrier which would prevent the maternal leukocytes and lymphocytes from coming into close contact with the placental tissue. Histochemical analysis has since shown this barrier layer to be a tryptophane-rich sialomucopeptide containing sialic and hyaluronic acid.s, lo Many tumor cells are also known to be coated with acidic mucopolysaccharides,ll and it has been shown that this layer is responsible for the high electronegative charge on the surface of these cells. Removal of the surface sialic acid groups by treatment with neuraminidase reduces this electronegative chargel and reveals the antigenicity of Ehrlich ascites tumor cells, rendering them capable of immunizing host animals.13 As lymphocytes are also highly negatively charged’* and can be repelled by electronegativity,15 it was suggested by Currie and Bagshawe4 that in vivo this peritrophoblastic fibrinoid layer acts as an electrostatic barrier which prevents maternal lymphocytes from coming into close contact with the fetalderived trophoblast and hence allows the
antigenicity
of trophoblast
1027
antigens to escape detection. Currie and his colleagueP have since modified this theory, suggesting that the sialomucin may act by steric hindrance to prevent the maternal lymphocytes from gaining access to the underlying antigenic groups. Other contributory evidence supporting the action of sialomucin was the recent report of Lippman17 showing that the expression of certain mouse tumor antigens can be suppressed by coating the tumor cells in vitro with various acid mucopolysaccharides. A second protective mechanism preventing fetal rejection concerns the failure of maternal lymphocytes to cross the placental barrier early in pregnancy, when rejection of fetal tissue is possible, as opposed to late in pregnancy, when the fetus is well equipped immunologically to destroy such cells. Although it has been shown that fetal cells cross into the maternal circulation,18, Is only one report of significant numbers of maternal cells crossing into the fetus in early pregnancy has been reported. This recent claim by Tuffrey and colleaguesZO of large numbers of maternal cells crossing into the mouse fetus has not yet been confirmed, and indeed two independent research teams21s 22 have been unable to reproduce these results. It was suggested by Seller2” that the porosity of the placenta claimed by Tuffrey and colleagues could perhaps be the result of the particular strain combination used by these workers. It would thus appear that at least two factors are involved in protecting the conceptus from maternal rejection: ( 1) the protective effect of the sialomucin coating of the trophoblast cells which screens the antigenic determinants from maternal immunologically competent cells, and (2) the failure of maternal leukocytes to cross the placental barrier early in pregnancy.
REFERENCES
1. Green, L., and Urbach, G. I.: Canad. Ass. J. 99: 469, 1968. 2. Moller, G., Beckman, V., and Lundgren, Nature 212: 1203, 1966.
Med. G.:
3. 4.
Currie, G. A.: J. Obstet. Gynaec. 74: 841, 1967. Currie, G. A., and Bag&we, vances Transpl. 34: 523, 1967.
Brit. K.
Comm. D.:
Ad-
1028
5.
Douthwaite
and Urbach
Thomas, J. W., Boldt, W., Horrocks, G., and Low, B.: Canad. Med. Ass. J. 97: 832, 1967. 6. Hersh, E. M., and Harris, J. E.: J. Immun. 100: 1184, 1968. 7. Seeger, R. C., and Oppenheim, J. J.: Fed. Proc. Amer. Sot. Exp. Biol. 29: 324, 1970. 8. Coulson, A. S., and Chalmers, D. G.: Lancet 1: 468, 1964. 9. Kirby, D. R. S., Billington, W. D., and Bradbury, S.: Nature 204: 548, 1964. 10. Bradbury, S., Billington, W. D:, and Kirby, D. R. S.: T. Rov. Micr. Sot. 84: 199. 1965. 11. Franks, L: M., -O’Shea, J. D., and Thomson, A. E. R.: Cancer 117: 983, 1964. 12. Ruhenstroth-Bauer, G., Fiehrmann, G. F., Kubler, W., Rueff, F., and Munk, K.: Z. Krebsforsch. 65: 37, 1962. 13. Lindenmann, J., and Klein, P. A.: Immunological Aspects of Viral Oncolysis, New York, N. Y., 1967, Springer-Verlag.
Amer.
14. 15. 16. 17. 18.
19. 20. 21.
22.
April J. Obstet.
1, 1971 Gynec.
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