Antigenicity of trophoblastic cells indicated by fluorescein technique

FETUS AND NEWBORN

Antigenicity of trophoblastic cells indicated by fluorescein technique Z.

KOREN.

M.D.

S. ].

BEHRJ.\L\!\<.

P.

PAl);E

].

MD

Ann Arbor. Michigan

By the direct and indirect fluorescein technique, it was shown that mice trophoblastic cells are capable of expressing antigenic properties, both in 1•il•o and in l'itro. The absence uf trophobla1tic tissue antigenic actil•ity during dez·eloping pregnancy was discuued. The possible u1e of immunologic methods in the control of trophoblastic tissue growth was suggested.

A K E Y T o the understanding of immunologic problems of pregnancy lies within, and emphasizes the importance of, tlw trophoblastic cell. At present. opinions still diffcr regarding the role that trophoblastic tissue plays in the immunologic mechanism where the rnothPr dors not reject, but instead, is able to accept the fetoplacental unit'· ''· ~. 1 " Thus, one of thf' most important and as y1·1 unsolved problf'ms at present is proof of the antigenicity of trophoblastic cells and is tlw purpose of this presentation. Simmons ;md Russell'' claimed that histocomnatihilit\• ;mtiP"Pns arP not exoressed b\· trophoblastic tissue. Kirby and associates,' on the other hand, suggest that histocompatibility antigens are present in trophoblastic tissue. but that thev are prevented from -

~

--

I

-

-

0

--

~

reaching, and thereby sensitizing, the host by a fibrinoid layer surrounding the trophoblast layer. Identification by in vivo studies of this pcritrophoblastic coat, or sialomucin, was made by Currie and Bagshawe. 1 They su.e;gcsted that because this layer possPssed an electronegative surface charge~ the trophoblastic cells escape attack by maternal lymphocytes in vivo because they also carry a negative charge, and thus an electrochemical repulsion occurs. Currie and Bagshawe 1 claim that at the same time maternal lymphocytes do. howe\·er, react with trophoblast in \ itro as well as with any allogenic tissue. This suggests that cytolysis of normal and malignant trophoblast occurs in the' presence of host or maternal lymphocytes in tissue cultun'. but not in vivo, because the fibrinoid coat around the trophoblastic cells is removed during trypsinization before culturing·. To investigate the problem of trophoblastic tissue antigenicity, pure trophoblast organ n·ll cu lturp as pn·viously described by 11s was

I

From the Center fv1 Reiearch in Reprvductil•e BivloRy, Department of Obstetrics and (;,•necolorn_ Unil•ersitv of Michigan Medical Ce;-;ter. -

This il'or!.. zt•as .IUpfJorted by Ford Foundation Grant No. 37482_

50

Antigenicity of trophoblastic cells 51

Volume 104 Number 1

prepared. 8 In this method, no trypsinization of tissue is carried out, nor are other enzymes used, making this method most suitable for immunologic experiments. Then in the second step, pregnant mice were treated with antiplacental serum as described below. To

placental cells. Booster injections of saline cell suspension ( 10 x 106 cells) without adjuvant were given intravenously on 3 successive days, 4 weeks later; and the rabbits were then bled 4 days after the last injection. Whole placental homogenate. Homogeni-

trace the localization of the rabbit anti-

zation of tissue \r\/as performed in normal

serum and thus antigenicity of trophoblastic cells a fluorescent protein tracing technique was employed.

saline solution using glass tissue grinders (Homogenizers Bodine Electric Company). The final solution of homogenate contained 25 mg. of protein per milliliter and w::~s

Materia! and methods

emulsified with an equal volun1e of Freund·s

Animals. Inbred C57BL/6J mice from the Jackson Laboratory, Bar Harbor, Maine, and a strain of albino mice from the Spartan Research Animals, Inc., Haslett, Michigan, were used. Organ-cell culture. The technique of obtaining pure trophoblastic tissue free of other placental components has been described elsewhere.'· 13 Tissue culture from mouse liver, placenta, and endometrium prepared in exactly the same way as the trophoblastic

incomplete adjuvant (Difco). The latter procedure was identical to the one described for placental cell suspension. The blood drawn from rabbits immunized by Method A or Method B was aliowed to clot at room temperature and then stored at +4° C. for 24 hours. Each serum was separated, pooled, and inactivated at 56° C. for 30 minutes. The serum was stored in small aliquots at -20° C. until used. The titer and the characteristics of the antibodies were ex-

endo~

arnined using the hemagglutination test, in1-

metrium was obtained from pseudopregnant mice 4 days after mating with vasectomized males.

munodiffusion test, and by electrophoresis. The hemagglutination technique employed was that of Boyden as modified by Stavitsky.1n. 17 Microimmunodiffusion and immunoelectrophoresis techniques were preYiously described in detail. 0

cC'll cultures '\vas used as controls. The

Immunization and preparation of antiserum

The placenta of the albino mice at 16 ± 2 days of gestation were used. Under general ether anesthesia, the abdomen was open and the uterus exposed. The placentas were removed, pooled, cut into small pieces, and washed with cold normal saline solution (0.9 per cent NaCl). From this stage the antigen was prepared in two ways. Placental cell homogenate. The tissue was pressed through a stainless steel mesh (U. S. Standard Sieve Series Opening 250 p., Tyler equivalent 60 mesh) into normal saline solution. The resulting cell suspension was washed three times in saline. The final suspension ( 10 x 10 6 placental cells per milliliter) was en1ulsified with an equal volurne of Freund's

incomplete adjuvant (Difco) and 0.50 ml. of this mixture was injected into each of four foot pads of rabbits such that each animal received a total of approximately 10 x 10"

Procedure of fluorescence

Fresh antiserum and normal serum were prepared for fluorochrome conjugation by inactivation at 56° C. for 30 minutes, followed by globulin fractionation repeated three times with saturated ammonium sulfate. Precipitates after each fractionation collected by centrifugation at 27,000 g and 4° C. were resuspended in Tris buffer, pH 7.2 (0.04~y1 Trizma Base, 0.0051Vf Trizn1a HCl). Dialysis of the resuspended precipitates against phosphate-buffered saline, pH 7.6 (0.15M KH 2 P0 4 , 0.15M Na 2 HPO,), was completed after each fractionation for the removal of excess ammonium sulfate. The fractionated serum and commercially prepared sheep antirabbit gamma globulin* *pH 7.6, Hyland Laboratorirs, Los Angeles. California.

52

~t;,,

Koren, Behrman, and Pame

were adjusted to 10 mg. of protein per milliliter by dilution in a carbonate-bicarbonat1· buffer, pH 9.0 (0.045M Na~CO::, 0.45M NaHCO,,*). Protein determinations wnc made using the Lowrv method. 11 All scr;1 were conjugate to fluorescein isothiocyanak using the dialysis method dt'Scribed bv Clark and Shepard.~ Thf~ conjugated materials welT dialyzed against phosphate-buffered sali1v·. pH 7.6, for the rcmoy;~ 1 of cxcc'SS tluorc,;cein.1" Previously prepared cultun~s of trophoblast ,,·ere collectl'd and quick frozen at - 70" C. for futun~ staining· or ether-alcohol fixed for 1.5 minutes, followed by 15 minute washin~ in phosphate-buffered saline, pH 7.6. and immediate fluorochronw staining. Direct staining of each culture with fluorescein isothiocyanatc---b bclcd serum mctlhatcd at :l7" C. for 30 minutes and !" ( :. for 30 minutes was completed by a V> mimtte washing of dH' Ct!ltures in tlm·e changes of barbital buffer (0.1 M sodilllll barbital, 0.02M acid barbital), pH H.6, at l C:. Stained cultures wNe mounted on alcoholcleaned g·lass microslides with Elvanol prcservatiw ( poly\·inyl alcohoL Grade 51 09.'it . Indirect staining of tlw cultmes with fluorescein isothiocyanate-conjugatcc! sllf't'p anri. rabbit gamma globulin was completed in th!' same manner as th(' direct method." Prior to the addition of the 1luorochrome-lahckcl indirect serum, tissue extract or serum anti• 1 , 1 · ,1 1 1, . n-:· .[!J'Il was mctmmeu wnn cacn cunure ar .11 C. for L'i minutes and :t t t C. for 15 minutes, n'SJWclin'ly. followed bv phosphatr·bufff'red saline. pH 7.6. \\:Jshing· for 15 minutes at room tf'mperatun·. Experimental and control stains :m· listed in Table I for the direct method and T:thl!' II for the indirPct method. In the second skp of our study, mice W<'IT maintained in groups of ~ in stainless steel cag<·s and W('H' allowed free ;1ccess to Purin
Table I. Direct tiuorescent staining A. Experimental: I. CulturP fluorescein isothiocyanatt' 'unjugated rabbit antimous!" trophoblast ., Culture fluorescein isothiocyanat<' conjugatet! mouse antirabbit antirnouse trophoblast

B. Control: l. Culture fluorescein isothiocyanat<· < oujugated normal rabbit sera ·> Culture fluorescein isothit•cyanalt' conjugated normal mouse sera >. Cultun· • no stain

Table II. Indirect fluorescent staining :\. Experimental: I. Culture ~ rabbit anti mouse tmphoblast ' fluorescein isothiocyanatc- conj ugatccl antirahhit .Qattuna globulin

B. Control: I. Culture + normal rabbit sera • flw,rcsu·in isothiocyanate--n >nj uga ted sht>cp an tirahbi t g·amrna globulin ' 1 Culture norma I nwusc sna ' fluorcS Culturr· - liver tissue extract ' fluorcsn·in isothiocyanatc-conj ugated sheep antirabhi t ~amma g-lobulin -{. Culture ., fluorescein isothiocyanatt- <"•niugatPd sheep antirahbit gamma globulin

1

·:<-pH i .6, Hyland

Laborat(ldc~.

tDuPont, Wilmington, Dela\van·.

Los Angdes.

I 1'1119

.\Jn . ./. Ub~!. & (_;ytu·c.

CaJiforni<~

animals recr·in·d antisera obtairwd frorn Libbits imrrmnizPcl with placental cdl suspension. Control animals were injected \1 ith equal ;~mounts oJ normal rabbit senllll. Antiserunr was injected intran·nously into the tail \Tins for :1 consecutive davs in dost·s of 0.25 to 0.5 ml. Four to 6 houn• aft<·r rlw last injection. the animals were killed. Till' utnus with conceptus. as wdl as splct•rJ. li\·er. and kidney tissue. was n·mn\Td R:mdorn cross frozen at -70" C. in 2-methyl butane and stored in tlw dcPp frer·zc for future frozeu sectioninc;. The section' of tissues wt·n· cut at -1 to h at ~0° (~. \Yith a L.ab-'rck cryostat and dry mounted on alc-ohol-cleant·d cover slips. Sections \\'ere ethcr-:dcohol fixed and washed for 15 minutes in phosphatc-bufferPd saline. pH 7.6. at room temperature. Adjacent se(_11

Antigenicity of trophoblastic cells 53

Volume 104 Number I

Fig. I

Fig. 2

Fig. 3

Fig. 4

Fig. 1. Indirect fluorescent staining of 3-day-old mouse trophoblastic cell cultures. Most of the trophoblastic cells are round and relatively compact. (x200.) Fig. 2. Indirect fluorescent staining of 12-day-old mouse trophoblastic cell cultures. Most of the trophoblastic cells are giant cells. Note fluorescent localization mainly in the nucleus. (x200.) Fig. 3. Photomicrograph of placenta from a mouse injected with antiplacental serum, and then killed 6 hours after the last injection. Indirect fluorescent staining localized in largely necrotic and hemorrhagic placenta. (x100.) Fig. 4. Photomicrograph of fetus (approximately 14 days of gestation) from a mouse injected with antiplacental serum, and then killed 6 hours after the last injection. Indirect fluorescent staining localized in largely necrotic and hemorrhagic fetal tissues. (x100.)

Volume 104

Antigenicity of trophoblastic cells 55

l~umber j

Table III. Indirect fluorescent staining of female mouse tissues (fluorescein isothiocyanate-conjugated sheep antirabbit globulin) Staining antigen Rabbit

Normal rabbit sera

Normal mouse sera

Negative Negative Negative +3 +2

Negative Negative Negative Negative Negative

Negative Negative Negative Negative Negative

Tissues Liver Spleen Uterus Implant Conceptus

Table IV. Indirect fluorescent staining of organ-cell cultures*

Staining material

Rabbit antimouse placenta Normal mousP sera

Normal rabbit sera

Fluorescein isothiocyanateconjugated sheep antirabbit globulin culture Endometrium l'I.T~~•

..~..,...,oa.-

+3

tive Nega-

Negative

Negative

tive

Negative

Negative

Negative

Negative

*The lis.ted fluoresc("nt rpsults are the numerical of eacl1 cultun· stained.

::.vPragp

Results

The sera obtained from the immunized rabbits had an antiplacental titer ranging from 1: 1,920 to 1: 15,360 as tested by hemagglutination technique. These results were positively verified by immunodiffusion and immunoelectrophoresis. 9 No significant difference in titer was observed in antiserum obtained by either of the above described immunization methods; nor was there any difference in antibody characterization. Therefore the results discussed belo"v pertain to

both types of antisera. By the direct stain, we observed (Table III) that all antiplacental sera showed positive staining with pure trophoblastic cell culture 3 to 12 days old, ranging from +1 to +3. Control placental culture also gave positive results. Negative results were obtained when control normal rabbit serum or normal mouse serum was used. Using the indirect stain, the rabbit antimouse placental serum gave +3 fluon·scence in both young and old trophoblast cultures (Figs. 1 and 2). No fluorescence was observed with control normal mouse and normal rabbit serum staining. Endometrium and liver control cell cultures were also negative.

Table V. Fluorescent staining of organ-cell cultures

Stain,ing rnaterial

Rabbit antimouse placenta Normal mouse sera Normal rabbit sera

+3 Negative Negative

+2 Negative Negative

In vivo. Indirect positive fluorescence was localized only in the placenta and conceptus (Figs. 3 and 4). The greatest positive fluorescent localization was found to locate in the peripheral placenta; specifically, in the trophoblastic cells. Sections of liver, kidney, and spleen were negative throughout the fluorescent staining procedure. Commeni

tions were stained with hematoxylin and eosin for morphologic comparisons. Each tissue section was fluorescent stained with the direct or indirect method as described above. Tissue and staining controls for the indirect method are summarized in Table III. Dark-field determinations of fluorescence as well as photomicrographs were completed as described above on a Zeiss photomicroscope. (Results of all fluorescence determinations are listed in Tables III, IV, and V.)

The results of this study demonstrate that at ]east under certain conditions, placental tissue and, particularly, trophoblastic cells are capable of expressing antigenic properties as indicated by fluorescein technique in vitro and in vivo. The antisera obtained from the rabbit immunized with placental cell suspension, or placental homogenate, contained a mixture of antibodies against mouse placental tissue, which is composed of mesenchymal and fetal

56

Koren, Behrman, and Paine

tissur· in addition to trophoblastic crlls. Howthe antig-enic matf'rial used in vitro consisted of almost pure trophoblastic cells. so that its properties in culture would not be confusrd with otht>r placrntal elrmrnts. Bv this means. ewn if the antiserum consists of polyvalent antibodies, the demonstration of positive fluorescent stainin,g of the trophoblastic cC'l!s both by the direct and indirect methods clearly indicates that the trophoblastic cells can express antigenic proprrtie~ l'\'('!'.

in vitro. In vivo the clinical and pathologic pictttn' in pregnant mice injectfcl with antiplacPntal srrum has been dr·scribed in detail in a previous study." The fact that placental tissurs are potrntially antigenic was shown by the rapid intfrruption of prfgnancies in mice in jectcd with the describ(•d antisera. In this particular study the positive Hnorcscent stain was localized only in the placenta and in the conceptus, and the most dense staining was locatrd in trophoblastic n•lls of partially twcrotic placentas. Tlw presence of positivt• staining in the conceptus shows that either antifetal antibodies must be present in our sera or antipbcental serum is hmvily clamaging the trophoblastic cdl barrier, enabling the free passage of antibodies into the rells. In any event, it w:1s possible to drmonstrate the localization of rabbit globulin in partially necrotic placentas, and to show that at least under crrtain conditions. trophoblastic tissnc is also antigenic in \'ivo vVith passive ir:nmunization and iuununohistorhemical techniques, in a previous study'' it was shown that plact•ntal components arc antigt~nic and kad to the formation (in a :-;enogeneic host1 of antiplacental antibodit·s demonstrable in hemagglutination. immunoelectrophoretic. and immunodiffusion systems. The most important point in this study was the biologic dfect of interruption of pregnancy in passin· immunized min·. The results thus obtained here an· in accord with those. of Simmons and Russell,' 1-1ho showed by transplantation techniques that mouse trophoblast is antigenic in the xenogeneic. host. These results underscon' tlw importancr of determining whv in normallY

.\lay I. 1%'1 .\m.

J, Oh..,t.

& (;ynt·r

developing pregnancy. the trophoblastic antigt•nicity is not expressed. Currie,c using tissue culture methods, wa,; able to show that normal and malignant trophobbst was destroyed in \ itro by tht'ir respectiv<' host lymphocytes. However. m~~­ lignant trophoblast cannot sC'rve as an t'xample of normal trophoblast antigenicity. taking into consideration the involved immunologic changes occurring during mali.gnant transformation of cells. Furthermon>, tlw technique of normal trophoblast tisstw culturt> ust'd by Currie givrs rise to a mixed population of cPlls, part of which is not trophoblastic but of fHal origin. Thus tlw cytolytic action of lymphocytf's on cC'\l population cannot set'\T as an example of trophoblastic antigenicity in \·ivo. It is clear tlwt fetal ct'lls are destroyed and in this way toxic st!bstances ap))('ar ;u1d influence trophoblastic cells. In \·ivo. the trophoblastic n·lls serve as a barriPr between the conceptus and the maternal circub tion. Simmons and Russell' 1 separated 7-day-old mouse conn·ptust·s into their trophoblastic and embryonic portions and implanted them beneath kidn<·~ capsules. TlH'n' was prompt destmctiou ol P!llbryonir cells, while tlw trophobla~tic n·lls proliferated and sun·in·d for the usual p<·riod of gestation in hosts sensitized against male strain antigens. This <'xperiment fmtber providPs suppmt of tlw role of trophoblast as an immunologic barrier. The problem of how the trophoblastic cells escape thC' immunologic attack in \·ivo in ;1 dcvt~loping pregnancy is still unsolvt~d. It is possiblt' that under normal physiologic conditions. th<' trophoblast is incapable of manti· facturin,g or ,·xpressing on the cell stuLJcl' histocompatibility antigens. 11 Howe\'er, this theory is not in keeping with the finding·s presented here or the n•sults of others. Trophoblasts, like all nudeated <'<'lb. <'nsitizing the maternal host by a layn of surrounding fibrinoid material.

Volume 104 Number 1

Under normal physiologic conditions this could act by preventing the escape of antigens into the maternal system. However, although fibrinoid material has been demonstrated in the placenta of many mammalian species/ 9 Wynn 18 states that while fibrinoid may serve an ancillary function as an antigen barrier in the hemochorial placenta, its apparent absence in other placentas argues against a general immunologic significance. It has also been speculated that a high local concentration of steroid hormones pro= duced by the placenta may act to prevent sensitization by the trophoblast. 20 These explanations, however, should not be considered to rationalize the acceptance by the mother of the very early trophoblast in a species where little or no placental steroid production occurs. One must then envoke

Antigenicity of trophoblastic cells 57

speculation about the possible protective role of enzymes present in young invasive trophoblast, or the possible protective mechanism embodied in the phagocytic activity of such trophoblast. In conclusion, it appears that, for reasons to this day not completely clarified, the trophoblast is not antigenically active in the developing pregnancy. Using the fluorescein technique, it was proved that at least under certain conditions the trophoblastic tissue is capable of expressing its antigenicity in vivo and in vitro. These results emphasized the fact that trophoblastic tissue is potentially antigenic and support our hypothesis that by the use of immunologic methods we might be able to control the growth of this tissue.

REFERENCES

1. Billingham. R. E.: New England T. Med. 270:

2. 3.

4. 5.

6. 7. 8. 9.

667, l964.' ~ Clark, M. F., and Shepard, C. C.: Virology 20: 642, 1963. Coons, A. M.: In Danielli, J. F., editor: Fluorescent Antibody Methods in General Cytochemical Methods, New York, 1958, Academic Press, Inc., p. 399. Currie, G. A., and Bagshawe, K. D.: Lancet 1: 708, 1967. Currie, G. .A.: J. Obst. & Gynaec. Brit. Comm. 74: 841, 1968. Douglas, G. W.: Obst. & Gynec. Surv. 20: 442, 1965. Kirby, D. R. S., Billington, W. D., Bradbury, S., and Goldstein, D. J.: Nature 204: 548, 1964. Koren, Z., and Behrman, S. J.: AM. J. 0BST. & GYNEC. In press. Koren, Z., Abrams, G., and Behrman, S. J.: AM. j. 0BsT. & GYNEC. In press.

10. Lanman, J. T., Dinnerstein, J., and Firrig, S.: Ann. New York Acad. Sc. 99: 706, 1962. 11. Lowry, 0. H., and Hunter, T. H.: ]. Bioi. Chern. 159: 465, 1945. 12. Nairn, R. C.: Fluorescent Protein Tracing, ed. 2, Baltimore, The Williams & Wilkins Company. 13. Schlesinger, M., and Koren, Z.: Fertii. & Steril. 18: 95, 1967. 14. Simmons, R. L., and Russell, P. S.: Ann. Ne·w York A.cad. Sc. 99: 717, 1962. 15. Simmons, R. L., and Russell, P. S.: Transplantation 4: 713, 1967. 16. Stavitsky, A. B.: J. Immunol. 72: 360, 1954. 17. Stavitsky, A. B.: J. Immunol. 72: 368, 1954. 18. Wynn, R. M.: AM. J. OnsT. & GYNEC. 97: 832, 1967. 19. Wynn, R. M.: Obst. & Gynec. 29: 644, 1967. 20. Zipper, J., Ferrando, G., Saez, G., and Tchernitchin, A.: AM. J. 0BsT. & GvNEc. 94: 105, 1966.