placenta, transcortin, and localized immune...

Placenta, Transcortin, and Localized Immune Response

SEYMOURWERTHAMER,SHANTHI GOViNDARAJ, and LEONARDAMARAL

From the Department of Pathology and Clinical Laboratories, The MethodistHospital, Brooklyn, NewYork 11215 and the Department of Pathology,State University of New York Downstate Medical Center, Brooklyn, NewYork 11203

A B S T R A CT The syncytiotrophoblastic cells of thehuman placenta contain a cytoplasmic protein recog-nized by fluorescein-labeled transcortin-specific anti-body. Purification of this protein from human placenta,by those methods employed for the purification of hu-man plasma transcortin, yielded a protein that exhibitedantigenic and biochemical similarity to plasma trans-cortin. Placental transcortin differs from plasma trans-cortin in that it has a smaller sedimentation coefficient(3S vs 3.75S) and binds cortisol less strongly. Thispurified protein is able to block the phytohemagglutininresponse of maternal lymphocytes even more than serumtranscortin. It is postulated that the biological role maybe that of inhibiting the maternal cell-mediated immuneresponse to the presence of the antigenic conceptus.

INTRODUCTION

The mammalian conceptus is antigenically differentfrom the mother, and yet immunological rejection ofthe fetus does not normally occur. Lack of immun-ological rejection has generally been attributed to de-pressed cell-mediated phenomena of the mother duringpregnancy (1-5). Evidence has been presented demon-strating factors present in pregnancy plasma capable oflimiting as well as inhibiting those in vitro responsesrepresentative of cell-mediated immunity (6-9). Hor-mones such as gonadotropin (10, 11), corticosteroids(12), progesterone (13), plasma proteins (14, 15), andserum blocking antibodies (16, 17) are synthesized bythe placenta and have been demonstrated to suppress invitro responses associated with cell-mediated immunity.

The steroid hormone, cortisol, elevated to very highlevels with continuation of pregnancy, is a potent sup-pressor of cell-mediated phenomena. The repressiveaction of cortisol on the lymphocyte is considered to bedependent upon the presence of a specific cortisol recep-tor protein of the lymphocyte's cytoplasm (18-20). We

Received for publication 31 December 1974 and in revisedform 21 November 1975.

have recently demonstrated that the maj or cortisol-binding protein of the human lymphocyte is chemico-physically similar and immunologically identical totranscortin, the cortisol-binding globulin of humanplasma (21). Furthermore, this protein is activelytransported into the lymphocyte' and is postulated tointerfere with the protein-synthetic apparatus of thecell (22). Transcortin is markedly and continuouslyincreased during pregnancy (23, 24). This increase isdue to increased synthesis induced by elevations ofplasma estrogens (25, 26) and occurs exclusively in theliver (27, 28). It is possible, therefore, that increasedlevels of transcortin could result in the suppression ofcell-mediated immunity. If transcortin has a centralrole in dampening the cell-mediated immune activity ofthose lymphocytes responding to the presence of theantigenic fetus, it seemed logical to us to expect thatperhaps the action of transcortin might be more im-mediately potent if its site of synthesis and release werethe placenta. Placental synthesis of transcortin wouldalso result in delimiting the activity of T lymphocytesin the vicinity of the placenta, with perhaps lesser sys-temic effects on the mother, for she must continue tohave operable "immunological surveillance."

This study investigated the question of whether trans-cortin is present in the fetal placental unit and whetherthe purified placental transcortin had any inhibitoryaction on the phytohemagglutinin (PHA)2-induced blasttransformation of human lymphocytes. Direct measure-ments of lymphocyte intracellular transcortin of thenormal and pregnant woman correlate well with in-creases in plasma transcortin in pregnancy and, coupledwith the findings that the placenta contains a proteinsimilar to plasma transcortin and capable of inhibitingthe lymphocyte's response to PHA, the important roleof transcortin in the prevention of immunological re-

jection of the human fetus receives considerable support.

'Werthamer, S., R. Khaund, and L. Amaral. Submittedfor publication.

2Abbreviations used in this paper: HAP, hydroxylapatite;PHA, phytohemagglutinin.

The Journal of Clinical Investigation Volume 57 April 1976 1000-10081000

METHODS

The purification of human plasma transcortin, its charac-terization, and the preparation of a transcortin-specific anti-body have been previously described (21).

In situ localization of intracellular transcortin. Smearsof human lymphocytes or frozen sections of human placentawere air-dried, immersed in ice-cold acetone, and coatedwith fluorescein-labeled transcortin-specific antibody (21).After suitable washing with phosphate-buffered saline, theslides were mounted with a mixture of 50% glycerol-phos-phate saline solution and studied with a Leitz fluorescencemicroscope (E. Leitz, Inc., Rockleigh, N. J.).

Quantitation of lymphocyte intracellular transcortin.Smears of peripheral blood of normal healthy women andpregnant women of well-defined trimesters were made andcoated with serial dilutions of the fluorescein-labeled trans-cortin antibody. Microscopic fields were first studied underdark field illumination and the number of lymphocytes pres-ent was recorded. This same field was then examined underultraviolet illumination and the number of the lymphocytesof that field exhibiting positive fluorescence was recorded.The highest dilution of the fluorescein-labeled antibody yield-ing fluorescence in 50% of the lymphocytes examined (D50)was employed as an index denoting the relative amount oflymphocytes intracellular transcortin. In this manner, nor-mal, pregnant and post-partum peripheral lymphocytes wereexamined.

Preparation of placental cytosol. Full-term placentaswere cut into slices approximately 100 g each, the slices werethinly minced, and the minced pieces (about 1-2 mm inaverage thickness) were rinsed eight times with ice-coldsaline or until the last rinse exhibited no erythrocytes andthe tissues contained no demonstrable hemoglobin. Mincedplacental tissues were homogenized at 40C in 1 mMphos-phate, pH 7.4, with a conical hand-driven homogenizer. Thecrude homogenate was then filtered through two layers ofcheesecloth and then centrifuged at 10,000 g. The supernatewas then centrifuged at 105,000 g for 3 h, thus yielding aplacental cytosol. This cytosol was lyophilized, dissolved inwater, and dialyzed against two changes of 4 liters of 1 mMphosphate, pH 7.4, and the protein concentration was deter-mined by the method of Lowry et al. (29).

Determination of placental intracellular transcortin. Cy-tosol was assayed for transcortin by the Ouchterlony double-diffusion procedures (21).

Determination of cortisol-binding activity by placentalcytosol. 0.5-ml aliquots of placental cytosol (approximately1 mg of protein/ml) were incubated with 106 cpm of [3H]-cortisol, sp act 5,000 mCi/mM, (Schwarz/Mann Div., Bec-ton, Dickinson & Co., Orangeburg, N. Y.) for 1 h at 4°C.Determination of specific cortisol-binding activity was de-termined by passing the above incubation mixture througha buffer-equilibrated G-25 Sephadex column (45 X 0.5 cm)in accordance with the method of DeMoor et al. (30).Radioactivities of the fractions collected were assayed by aPicker Ansitron II liquid scintillation counter (PickerCorp., Cleveland, Ohio) after the addition of 15 ml ofBray's scintillant to the vials containing the fractions.

Purification of placental transcortin. The entire cytosolpreparation of one full-term placenta was incubated with[3H]cortisol under conditions previously stated. Purificationof placental transcortin was accomplished by the same pro-cedure employed and described by Muldoon and Westphal(31) for the purification of plasma transcortin. Details per-taining to the various steps of purification are given in thetext and appropriate legends of the figures.

Behavior of placental transcortin in ultracentrifugation in

sucrose gradients. Sucrose gradients of 5-20% were pre-pared in 1 mMphosphate, pH 7.4. Aliquots of 0.5 ml ofvarious preparations of placental transcortin obtained fromeach step of the purification procedures were carefully lay-ered on top of the gradients and the gradients were centri-fuged in a SW-65 rotor at 60,000 rpm for 21 h at 50C ina Beckman L-2-75 B ultracentrifuge (Beckman Instruments,Inc., Spinco Div., Palo Alto, Calif.). Fractionation of thegradient into 30 equal fractions was performed with theaid of an Isco sucrose gradient-ultraviolet monitoring sys-tem (Instrumentation Specialties Co., Lincoln, Neb.) (at280 nm), and analysis of radioactivity representing [3H]-cortisol bound to protein was obtained after the additionof 15 ml of Bray's scintillant. Standardization of the pro-cedure required for the estimation of sedimentation co-efficients has been previously described (21).

Disc-acrylamide electrophoresis of purified placental trans-cortin. 0.1 mg of the purified hydroxylapatite (HAP) IIfraction was layered on top of disc acrylamide electropho-retic columns (0.5 X 7.0 cm) containing a 10% acrylamideseparation gel. Electrophoresis was conducted as describedby Ornstein (32) and Davis (33).

The effect of placental transcortin on PHA-induced blasttransformation by human lymphocytes. Human lympho-cytes, purified by methods previously described (22, 34)were suspended in autologous plasma at a concentration of25 X 106 cells/ml of plasma. 0.1 ml of these suspensions wasadded to 1 ml of TC-199 medium containing penicillin (100U/ml) and streptomycin (10 mg/ml) and 0.25 ml ofPHA-P (Difco Laboratories, Detroit, Mich.). To half ofthese cultures, approximately 0.1 mg of purified placentaltranscortin was added. All cultures were incubated for upto 3 days at 370C in a 5% C02 atmosphere mixture. Atthe end of designated intervals each of the cultures received1 ,uCi of [3H]thymidine (sp act 33 Ci/mM) and the cultureswere incubated for 2 h at 37°C. Incorporation of [3H]thy-midine by cultured cells was determined by methods previ-ously described (22). In addition, the cultured cells weresmeared, and the percentage of lymphocytes transformedwas determined by microscopic evaluation by two inde-pendent investigators. In all cases, agreement of such analy-sis did not deviate by more than ±3% of the mean betweenthe two sets of analysis.

RESULTS

Quantitation of intracellular lymphocytetranscortin

Previously we demonstrated that the human lympho-cyte contains within its cytoplasm a protein specieswith many of the physicochemical properties of trans-cortin (21). In situ identification of this transcortin-like protein was afforded by the application of a flu-orescein-labeled transcortin antibody to smears ofperipheral blood lymphocytes. It was noted that almostall of the fluorescence was limited to the cytoplasmicrim of the lymphocyte. Controls demonstrated that suchfluorescence was not due to membrane-bound trans-cortin (21).

We have attempted to measure the level of intra-cellular transcortin of peripheral blood lymphocytesduring pregnancy. Our reasons for attempting thisanalysis stem from studies demonstrating that trans-

Placenta, Transcortin, and Localized Immune Response 1001

FIGURE 1 In situ localization of transcortin within the cytoplasm of placental cells. Frozensections, approximately 5 /Am thick, were prepared from pieces of fresh, extensively saline-washed, full-term placenta. Diluted fluorescein-labeled transcortin antibody was layered asdescribed previously. Note that fluorescence is primarily found in the cytoplasm of the syncytio-trophoblastic cells.

cortin levels are markedly elevated during pregnancy(23, 24). Since transcortin is found within the lym-phocyte and since lymphocytes do not appear to synthe-size transcortin, the possibility that increased plasmalevels of transcortin would result in increases of intra-cellular lymphocyte transcortin seemed worthy of in-vestigation. The use of serial dilutions of the fluorescein-labeled transcortin antibody to quantitate the relativelevel of lymphocyte intracellular transcortin illustratesthat this level increases with continuation of pregnancyand reaches a maximum during the third trimester(Table I). It should be noted from the data presentedthat the range of D5o is rather narrow within the normal(nonpregnant) group of women (1 :40-1:80). How-ever, during the first trimester the D5o increases as wellas its range. The range of D50 continues to increaseduring the second trimester. It is during the thirdtrimester, however, that the levels of intracellular trans-cortin are truly significantly different from those of the

normal. Within this group, 12 of the 14 individuals hada D50 of 1:640. Immediately before parturition, the in-tracellular transcortin level drops to within the normalrange.

In situ localization of transcortinwithin placental cellsBiosynthetically plasma transcortin has been con-

sidered to originate exclusively in the liver (27, 28).Furthermore, administration of estrogens or their syn-thetic analogues results in a maximal twofold increasein plasma transcortin levels (35), suggesting that themaximum synthesis in the liver might be limited. How-ever, transcortin levels in pregnant women reach muchhigher levels (23, 24), suggesting in turn that perhapsanother organ or organs might be synthesizing thisprotein as well. To determine whether the placentamight synthesize and/or contain transcortin, we appliedthe fluorescein-labeled transcortin antibody to frozen

1002 S. Werthamer, S. Govindaraj, and L. Amaral

sections of a full-term human placenta. Fig. 1 illustratesthe presence of transcortin within the cytoplasm of thesyncytiotrophoblastic cells lining the maternal sinus.No other cells of the placenta, except an occasionallymphocyte, exhibit any fluorescence. Wehave similarlytreated a 15-wk placenta obtained surgically and nofluorescence was detected.

Determination of cortisol bindingby placental cytosolThe above studies on the human placenta suggest that

placental tissue contains a protein(s) recognized by thetranscortin-specific antibody. Additional evidence sug-gesting that the placenta indeed contains transcortin isprovided by the determination of cortisol binding bythe G-25 Sephadex filtration of DeMoor et al. (30).From the data presented in Fig. 2, it is clear that theplacental cytosol contains species capable of rigorouslybinding cortisol (front-running radioactivity peak).

Purification of placenta transcortin

The above findings suggest that the placenta containsspecies of protein that bind cortisol and are recognizedby a transcortin-specific antibody. To determine whetherthe above species are indeed transcortin, their purifica-tion was attempted via the methods used for the puri-fication of transcortin. Passage of ['H]cortisol-placentalcytosol through a water-equilibrated DEAE columnand subsequent washing of the column with water,followed by elution with buffer, yielded an ['H]cortisolelution profile depicted in Fig. 3. Under the elutionconditions listed in the legend of Fig. 3, a single radio-active peak coinciding with an optical density peak at

TABLE IQuantitation of Lymphocyte Intracellular Transcortin

of Normal and Pregnant Females

Numberof

individuals Range of D5o*

Normal 34 1:40-1:80First trimester 26 1:80-1:320Second trimester 25 1:80-1:640Third trimester 34t 1:320-1:640At parturition 3 1:80-1:1602 days after delivery 9 1:40-1 :80

The significant difference between normal and 3rd trimestervalues as determined by Student t test is P < 0.02.* Range of dilution of fluorescein-labeled antibody that yieldedapproximately 50% of the lymphocytes demonstrating fluores-cence. Lymphocytes of a single field were first examined underdark field microscopy and then under ultraviolet excitation.t 20 of 34 individuals had a D5o of 1:640.

0

2-

10 20 30 40FRACTION

FIGURE 2 G-25 Sephadex assay of cortisol binding byplacental cytosol. 1 mg of placental cytosol was incubatedwith 10' cpm of ['H]cortisol (sp act 5,000 Ci/mM) at 4°Cfor 60 min. The mixture was passed through a column ofG-25 Sephadex (45 X 0.5 cm) and elution was conductedwith mMphosphate, pH 7.4. Fractions of 1 ml were col-lected and assayed for radioactivity. Controls lacking pla-cental cytosol, but instead containing 1 mg of heat-denaturedbovine albumin were similarly processed. The fractions be-tween the arrows denote the elution of free cortisol. Thefront-running radioactive peak represents ['H]cortisolboundto some species of the cytosol (--A). Incubations con-taining the heat-denatured albumin (0-0-0) exhibit nofront-running radioactive peak.

280 nm was obtained. After the pooling of the fractionsunder the radioactive peak, the pooled fractions (afterDEAE) were lyophilized and dialyzed against mMphosphate, pH 7.4. G-25 Sephadex filtration proceduresdemonstrated that the radioactivity of the post-DEAEpooled fraction represented protein-bound ['H]cortisol.This pooled post-DEAE fraction was split into twoportions, one rapidly frozen and stored at - 700C, theother portion passed through a HAP column. Elutionof the applied material yielded two radioactivity peakslabeled HAP I and HAP II in Fig. 4. Only the HAPII fraction exhibited strong cortisol-binding activity,demonstrable via G-25 Sephadex methods, as previouslydescribed. This HAP II fraction was passed throughtwo additional HAP columns, the last of which ex-hibited a single ['H]cortisol peak coinciding with asingle optical density peak at 280 nm. Lyophilizationwith subsequent dialysis against saline was carried out.


v 3 9 IS 21 27 33 39 45

FRACTIONFIGuRE 3 Elution of [3H]cortisol-placental cytosol fromwater-equilibrated DEAE columns. Placental cytosol (ap-proximately 300 ml) was incubated with 109 cpm of [8H]-cortisol for 1 h at 4VC. This mixture was loaded onto awater-equilibrated DEAE column (Pharmacia DEAE-52)(70 X 1.5 cm) and subsequently washed with water until

little radioactivity was present in the eluate. Elution wasconducted via a linear gradient formed with 1 liter each of1 mMphosphate, pH 8, and 0.5 M phosphate, pH 5.5. Frac-tions consisting of about 10 ml were collected and radio-activity was determined.

Comparison of purified placental cortisol-bindingprotein to purified plasma transcortin and itspartial characterizationOuchterlony studies. That this post-HAP fraction

contained transcortin is demonstrated by the results ofdouble diffusion-Ouchterlony procedures. Fig. 5 illus-trates that purified human plasma transcortin formstwo precipitin lines. The existence of a second pre-cipitin is not due to an impurity but to the formationof a dimer by the transcortin molecule (21). The wellscontaining HAP II fractions demonstrated the forma-tion of precipitin lines contiguous with those formedbetween the antibody- and transcortin-containing wells.The wells containing HAP I pooled fractions failed todevelop any precipitin. The well containing the placentalcytosol yielded multiple precipitin lines. Whether theseprecipitin lines are due to polymers or variations in thetranscortin-like molecule due to degradation or addi-tional transcortin-like species cannot at this time bedetermined.

Sucrose gradient centrifugation analyses. Determin-

4- ~ ~ I

2-

3 9 15 21 27 33 39 45 51

FRACTION

FIGURE 4 Application and elution of post-DEAE [8H]-cortisol placental mixture from HAP columns. Lyophilizedpost-DEAE fraction, dissolved in 50 ml of 1 mMphos-phate was loaded onto an HAP column (70 X 0.5 cm) andeluted with 1 mMphosphate, pH 7.4. Fractions of about 5ml were collected and assayed for radioactivity.

ation of sedimentation coefficients for the placental in-tracellular transcortin during various stages of itspurification was accomplished. Fig. 6 illustrates thatplasma transcortin contains a single [8H]cortisol peakcorresponding to a sedimentation coefficient of 3.6-3.8.Similar treatment of the placental cytosol yields tworadioactive peaks of 3 and 4.5S (Fig. 7). The 3S peakis very broad and hence any minor species sediment-

*I

FIGURE 5 Demonstration of transcortin-like protein in thepost-HAP fraction of placental cytosol by transcortin-spe-cific antibody. Central well contains transcortin-specificantibody. Well 1 contains placental cytosol; well 2 con-tains purified transcortin; well 3 contains purified, post-HAP II placental fraction. Well 4 contains HAP I fraction.Note the continuous precipitin line closest to central well.Additional outer precipitin lines are visible. Such precipitinlines are not due to impurities but rather to degrees of poly-merization of transcortin-like proteins (see reference 21).


ing as a plasma transcortin component would be masked.However, the sedimentation profile obtained after thefirst passage through HAP columns yields a profilesimilar to that of the cytosol (Fig. 8). It should benoted that no sedimentation activity peak correspondingto that of plasma transcortin was evident at any pointduring purification.

Disc acrylamide electrophoretic analysis. In accord-ance with differences in sedimentation behavior insucrose gradients between the HAP II fraction andplasma transcortin, Fig. 9 illustrates that HAP II frac-tion and transcortin are electrophoretically dissimilar.It is clear that HAP II fraction migrates in this sys-tem with more mobility than purified transcortin.

Effect of purified placental transcortinon the PHA response

To determine whether the placental fraction (ex-hibiting many of the characteristics of transcortin)inhibits in vitro responses associated with cell-mediatedimmunity, human lymphocytes, obtained from normalwomen, were incubated with and without placental frac-tion HAP II or with purified human transcortin towhich cortisol was tightly bound. The results, presented

6 ~ 3.6-3.8S

PLASMA

5-

(I

0 4 -

(3-

2 6 10 14 IS 22 26 30

TOP FRACTION BTOP ~~~~BOTTOMFIGuRE 6 Sucrose gradient centrifugation of [8H]cortisol-plasma transcortin. 0.5 ml of a mixture of plasma trans-cortin and ['H] cortisol was layered on top of a 5-20%sucrose gradient (1 mMphosphate, pH 7.4) and centri-fuged at 60,000 rpm for 21 h. Fractionation and analysisof [PH] cortisol activity of these gradients have been pre-viously described (21).

500r

400-

(a

'a0

(3

t

TRANSCORTIN3S + 4.5S

300F

2001

100

2 6 10 14 18 22 26 30

FIGURE 7 Ultracentrifugation of placental cytosol in su-crose gradients. Conditions are as previously described.Note that two radioactive peaks, corresponding to sedi-mentation constants of 3 and 4.5 S, are present. The posi-tion fraction where transcortin is expected contains noradioactive peak.

in Table II, illustrate that the transformation of lym-phocytes by PHA is inhibited by placental factor HAPII. The effect of the above on the incorporation of['H]thymidine into DNA is presented in Table III. Itis evident from the data presented that the placentalfactor HAP II inhibits the incorporation of [3H]thy-midine by 78%. Control experiments demonstrate thatthe inhibition of PHAwas not due to the precipitationof PHA by the protein added, as shown in Table III.

4 -

TRANSCORTIN3 - 3S * 4.5qCO2-A

2 6 10 14 18 22 26 304 FRACTIONTOP BOTTOM

FIGuRE 8 Sucrose gradient ultracentrifugation of post-HAP fraction II.


TABLE IIIThe Effect of Purified Placental Transcortin on the

Incorporation of [3H]Thymidine by LymphocytesCultured in the Presence of PHA

a b C

FIGURE 9 Disc-acrylamide electrophoresis of purified trans-cortin and post-HAP fraction II. Approximately 300 usg ofpurified transcortin (gel a) and post-HAP fraction II (gelb) were electrophorised. A mixture of 150 ,ug each ofpurified transcortin and post-HAP fraction II were alsoelectrophorised (gel c). Note that the placental fractionhas a greater mobility than purified transcortin.

In addition, the presence of transcortin inhibits suchincorporation by only 35%.

DISCUSSION

The results presented in this study demonstrate that thecytoplasm of syncytiotrophoblastic cells of the humanplacenta contains a protein with immunologic andphysicochemical characteristics similar to that of plasmatranscortin. However, this molecule differs from trans-

TABLE IIThe Effect of Purified Placental Transcortin on the

Transformation of HumanLymphocytes by PHA

Blast transformed

Day I Day 2 Day 3

Control PHA 16 51 68Placental transcortin 8 34 40

Human lymphocytes were cultured in medium containingPHA for intervals of 1, 2, and 3 days in the presence andabsence of purified placental transcortin (HAP II fraction)at a protein concentration of 0.1 mg/ml of medium. At theend of 1, 2, and 3 days of culture, aliquots of 0.1 ml of theculture were smeared onto glass slides, air-dried, and stained.All immature lymphocytes were designated as "transformed."The above experiments were repeated twice with lymphocytesobtained from two other healthy pregnant women. Similarresults were obtained.

[3H]Thymidine Inhibition

cPm/10 %lymphocytes

Control (no PHA) 104PHA 2,430PHA& placental transcortin 535 78%PHA& transcortin 1,580 35%PHAfor 2 h 2,515PHAfor 2 h, then placental

transcortin added 475 81%

Human lymphocytes were incubated in medium with andwithout PHA. To other cultures containing PHA eitherpurified placental transcortin or purified plasma transcortinwas added to a final concentration of 0.1 mg/ml of medium.In addition, other cultures were incubated with PHAfor 2 hbefore the cultures were centrifuged, and the cells suspendedwith fresh medium. To experimental cultures, purifiedplacental transcortin was added to a final concentration of0.1 mg/ml of medium. At the end of 3 days of incubation, thecultures were agitated, and aliquots transferred to culturetubes containing 1 /ACi of [3H]thymidine. These cultures wereincubated for an additional 2 h and then centrifuged, and thepellets were suspended in 0.5 ml of saline. To each, ice-cold10% trichloracetic acid was added, and the precipitates werecollected by Millipore filtration, dried, and immersed intovials containing Liquiflor, and the activities of tritium weredetermined with a scintillation counter. All cultures wereperformed in triplicate. The maximum deviation did notexceed 4%. The above data was obtained from one of foursuch experiments.

cortin in that it has a smaller sedimentation coefficient(3S) than plasma transcortin (3.75S). In crude prep-arations an additional cortisol-binding component witha sedimentation coefficient of 4.5S is present. (This4.5S species, we feel, is probably a dimer). The pres-ence of this partially purified protein was also shownin this study to suppress blast transformation of normallymphocytes.

That the protein purified is not a plasma transcortincontaminant, due to inadequate mincing and washingprocedures, and is indeed obtained from placental tis-sues may be concluded from: In situ localization of themolecule within the syncytiotrophoblastic cells of frozenplacental tissues sectioned at a thickness of 5 Am. Atthis thickness the cells depicted are true sections of cells.The physicochemical characteristics, such as behaviorin sucrose gradients and electrophoretic fields, aredifferent from purified plasma transcortin. And ob-served differences that cannot be due to the purificationprocedure, since such procedures in our hands do not

- 1006 S. Werthamer, S. Govindaraj, and L. Amaral- i ...

and have not altered the physicochemical of purifiedplasma transcortin.

These results suggest that the placenta contains aprotein similar if not identical to plasma transcortin,and that its function might be that of limiting cell-mediated responses of maternal lymphocytes to thepresence of the antigenic fetal placenta.

In our previous studies, we have suggested thatplasma transcortin may be transported into the humanlymphocyte (22, 34) and that the transcortin presentwithin the cytoplasm of the human lymphocyte issmaller than plasma transcortin and very similar in itsphysicochemical characteristics to partially purifiedplacental transcortin (21). Studies submitted elsewhereprovide strong support for this contention by showingthat cultured human lymphocytes of the cell line R-PMI-1788 transport this protein from the mediumto their cytoplasm.1 We have suggested that soon afterthe transport of transcortin-cortisol complexes into thelymphocyte, an enzyme acts on this complex, therebyremoving cortisol (this cortisol is rapidly and activelyextruded [34]) and thereby yielding a small polypep-tide (21). After this enzymatic action, the resultingtranscortin has a smaller cortisol-binding capacity anda smaller sedimentation coefficient. We have postulatedthat this modified transcortin inhibits the synthesis oflymphocyte protein (21, 22). We feel that, unlike thelymphocyte, biosynthesis of the placental transcortinpossibly occurs within placental cells. To date, however,only the liver has been identified as the exclusive siteof transcortin biosynthesis (27, 28).

It is presumed that placental transcortin, like trans-cortin in the liver, is induced by maternal secretions ofestrogen. Support for this hypothesis may be found inthose studies that demonstrate that the biosynthesis oftranscortin is induced by estrogen and that increases inplasma estrogen during pregnancy are paralleled byincreases in maternal plasma transcortin (25, 26). Wefeel that during the first trimester, the increased levelof transcortin in maternal plasma is the result of itsinduced biosynthesis by the liver in response to estro-genic increases. However, the absolute limit of trans-cortin levels resulting from estrogenic induction is prob-ably equal to that observed in men receiving diethylstil-bestrol, about 6-12 mg of transcortin/100 ml of plasma(35). Since the total level of maternal plasma trans-cortin is much higher during the second, and still higherduring the third trimester (23), this total level maypossibly involve transcortin molecules of placentalorigin.

Although we have not measured plasma transcortinlevels before parturition in this study, we have mea-sured the level of lymphocyte intracellular transcortinduring pregnancy. Our results suggest that during

gestation peripheral lymphocytes contain increasinglevels of cytoplasmic transcortin. These increasesparallel those observed in plasma and may result froman increase in transcortin transport into the lymphocyte.However, unlike the transport of plasma transcortin-cortisol complexes, only the modified transcortin ofplacental origin enters the lymphocyte. It is this modi-fied protein that can limit the cell-mediated immuneresponse to a much greater degree than the unadul-terated transcortin-cortisol complex.

The presence of large amounts of transcortin withinplacental cells at time of parturition might at first seemto contradict the above hypothesis. It is suggested,however, that the transcortin observed in full-termplacental cells might simply reflect an accumulation dueto diminished secretion of this protein. This diminishedsecretion might be the result of an increase in the ratethat estrogens are metabolized by the placenta. Supportfor this contention is provided by those studies thatshow that in liver, estrogen controls the release oftranscortin to the circulatory system (27). Accordingly,our hypothesis would then predict that maternal cell-mediated immunity would now be fully operational.At this point, the placenta and fetus are rejected muchas any foreign antigenic tissue would be rejected in themammal. Evidence of such cell-mediated responses oc-curring at this time are routinely found in uterinetissues (36).

The above general theory is far from proved. Ourstudies only support the general theory at critical points.It remains for future studies, some in progress, toelucidate further.

ACKNOWLEDGMENTSWewould like to thank Bennie Denson and Phyllis Seidlerfor their excellent technical assistance, Dr. Kobkul Linfor her guidance in the immunologic studies, Dr. RobindraKhaund for conducting the PHA studies, Allan Brown forthe preparation of the figures, and Janet Mena for thepreparation of the manuscript.

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