Human trophoblast interferon: pattern of response to priming and superinduction of purified term trophoblast and choriocarcinoma cells

Journal of Reproductive Immunology, 19 (1991) 55--67

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Elsevier Scientific Publishers Ireland Ltd.

JR100686

Human trophoblast interferon: pattern of response to priming and superinduction of purified term trophoblast and choriocarcinoma cells F.D. Toth a,b, N. N d r s k o v - L a u r i t s e n a, C. Juhl a and P.

Ebbesen

a

uDepartment of Virus and Cancer, The Danish Cancer Society, 10 Gustav Wieds Vej, 8000 Aarhus C (Denmark) and ~Institute of Microbiology, Medical University, 1-1-4012Debrecen (Hungary) (Accepted for publication 28 June 1990)

Summary We recently described a beta interferon response of primary cultures of term human trophoblast exposed to poly(I:C). The response pattern has now been studied further with priming and superinduction both of normal placental cell types and JAR, JEG-3 and BeWo choriocarcinoma cells. Pre-treating placental trophoblast cells, fibroblasts and macrophages with human interferon generally led to increased yields of interferon after poly(I:C) induction, whereas choriocarcinoma cells did not respond to priming. All the cells showed the superinduction phenomenon although to varying degrees. The combination of priming and superinduction conditions led to the production of very high yields of interferon in placental fibroblast cultures. The combined procedure also produced more interferon in macrophage cultures than priming or superinduction alone. Combined superinduction and priming of normal trophoblast did not produce higher yields than those obtainable by superinduction alone. These data might help to provide additional insight into the cellular control mechanisms of sensitivity of normal and malignantly transformed cells to interferon. Furthermore, the large quantity of trophoblast interferon produced by superinduction could be used for studies of its anti-viral and immunomodulatory effects. Key words: trophoblast; choriocarcinoma; interferon; priming; superinduc-

tion; human. Correspondence to: Dr. Peter Ebbesen, The Danish Cancer Society, Department of Virus and Cancer, Gustav Wieds Vej 10, DK-8000 Aarhus C, Denmark. 0165-0378/91/$03.50

© 1991 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

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Introduction

Synthetic double-stranded RNA polymers such as poly(I:C) can induce interferon production in cells in vitro (Held et al., 1973). The iaoly(I:C)induced interferon production can be enhanced by the judicious use of inhibitors of RNA or protein synthesis, interfering with the process which normally shuts off interferon production (Vilcvek and Ng, 1971). This effect is referred to as superinduction. The rate of transcription of the interferon genes can also be influenced by interferon itself. Pre-treatment of cells with interferon just before induction frequently results in enhanced interferon production, a phenomenon known as priming (Isaacs and Burke, 1958). Enhancement of interferon production of human cells by both priming (Towell and Cantell, 1971) and superinduction (Ho et al., 1972) have been described. In an earlier report the finding of the interferon producing capability of purified human term trophoblast exposed to poly(I:C) was described (Toth et al., 1990). Furthermore, we reported that the malignant choriocarcinoma cell line JAR produced less interferon than non-transformed trophoblast. Subsequently, the trophoblast interferon was purified and chemically characterized (Mathiesen et al., 1990). In the present investigation we have attempted to analyze the capacity of normal placental cells and three different choriocarcinoma cell lines to synthesize large amounts of interferon under various conditions. The various cell types studied were found to be heterogeneous with respect to their ability to respond to priming and superinduction. Materials and Methods

Cell separations The initial trophoblast isolation procedure was based on the method described by Kliman et al. (1987). The first step was followed by an additional purification using magnetic microspheres (Douglas and King, 1989). Term placentae were obtained from spontaneous deliveries. Villous tissue was dissected from the placenta, care being taken to avoid obvious connective tissue and decidua. The tissue was treated with 0.125o70 trypsin (GIBCO Labs, Paisley, Scotland) in PBS (pH 7.4) containing 5.0 mM M g 2÷ , 1.0 mM Ca 2÷ and 0.1 mg DNase I (Boehringer Mannheim GmbH, Mannheim, F.R.G.) per ml at 30 min intervals. Cells released from 30 to 60 min after start of enzyme treatment were pooled and centrifuged in a 5--70o70 percoll (Pharmacia, Uppsala, Sweden) gradient and the cells binding at 50--60°7o percoll were collected. The cells were suspended in RPMI-1640 medium containing 10o70 fetal calf serum (FCS) to give 4 X 106 cells/ml and centrifuged.

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All subsequent steps were performed in the cold. The cells were resuspended in serum-free RPMI to the same density and to 3 ml of cell suspension 50 ~l of monoclonal anti-HLA-ABC and 20/al of monoclonal anti-HLA-DR antibodies (Dakopatts, Copenhagen, Denmark) were added. The mixture was incubated on ice for 30 min with gentle shaking and then centrifuged. The supernatant was removed and the cell pellet was resuspended in serum-free RPMI. Centrifugation and resuspension was repeated twice, then the pellet was resuspended in 1 ml of medium. To this was added 100/al of a suspension of magnetic microspheres coated with goat anti-mouse IgG (Dynabeads; Dynal A.S., Oslo, Norway). The mixture was placed on a shaker for 30 min at 4°C and the tube was then clamped to a magnetic concentrator (Biomag Separator; Advanced Magnetics Inc., Cambridge, MA). After 2 min the supernatant was removed and centrifuged. The pelleted cells were resuspended in culture medium. Placental macrophages were separated from the 45--50070 percoll band after 60--90 min of enzyme treatment by the method described by Wilson et al. (1983). Suspensions were adjusted to contain 1.5 × 106 cells per ml in RPMI plus 10070 FCS. One milliliter was added to wells of 6-well tissue culture clusters (Costar, Badhoevedorp, The Netherlands). Cells were allowed to adhere for approximately 2 h at 37°C and non-adherent cells were removed by washing 6 times with PBS (pH 7.4). The monolayers were then exposed to 0.1°70 trypsin in PBS for 10 min at room temperature. The cells were then washed again 6 times with PBS. The macrophages were removed from the surface of the wells with rubber policemen, resuspended in RPMI containing 10070FCS, counted and adjusted to the appropriate density. Placental fibroblasts were separated from the 45--50070 percoll band after 30 min of enzyme treatment. The initial steps of purification were identical to those used for purification of macrophages (see above). After treatment of monolayers with 0.1 070 trypsin, the removed cells were washed twice in RPMI plus 1007oFCS, then resuspended in the same medium.

Indirect cytoplasmic immunofluorescence The purity of the placental cell cultures was tested by indirect immunofluorescence. The primary mouse antibodies and the FITC-conjugated rabbit anti-mouse immunoglobulin were purchased from Dakopatts. Trophoblast was identified by monoclonal anti-human cytokeratin IgG (Zeitler et al., 1983). Fibroblasts were revealed with monoclonal antibody to porcine vimentin (Osborn et al., 1984) and macrophages were detected with monoclonal anti-human macrophage IgG reacting with CD68 (Kelly et al., 1988). For immunofluorescence, 104 cells suspended in PBS were placed in wells of multitest slides (Flow Laboratories, McLean, VA) and fixed in acetone. In the first phase of the reaction the cells were incubated with monoclonal

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mouse antibody diluted 1:50 for 1 h at room temperature. The control samples were incubated with normal mouse serum. After incubation the cells were washed 3 times in PBS. Thereafter, FITC-labelled rabbit anti-mouseIgG serum diluted 1:20 was added to each sample. The cells were incubated for 30 min at room temperature and then washed 3 times again before evaluation in a fluorescence microscope. Cytotrophoblast cultures used for interferon induction were found to be 98m100o70 positive for cytokeratin. The purity of the fibroblast and macrophage cultures was higher than 95 °/0.

Other cells For detection and titration of interferon, human amnion (WISH) ceils were used. The WISH cells (ATCC, CCL 25) were grown in Eagle's MEM supplemented with 10o70 FCS. The human choriocarcinoma cell lines JAR (ATTC HTB 144), JEG-3 (ATTC HTB 36) and BeWo (ATTC CCL 98) were cultured in RPMI medium containing 10o70FCS. Virus Vesicular stomatitis virus (VSV), Indiana strain, was routinely maintained in our laboratory and titrated by plaque-forming unit assay (Goorha, 1981). Interferon and anti-interferon antisera H u m a n alpha- and gamma-interferon were purchased from Sigma Chemical Company (St. Louis, MO), human beta-interferon was obtained from the Green Cross Corporation (Osaka, Japan). Their interferon activities were calibrated against international reference standards and these interferons were used as internal laboratory interferon standards. H u m a n reference alpha-(69/19) and gamma-interferon (82/587) were purchased from the National Institute for Biological Standards and Controls (London, U.K.). The human beta-interferon reference reagent (G-023-902-527) originated from NIH (Bethesda, MD). Horse antisera to human alpha-, beta- and gamma-interferon were obtained from Boehringer Mannheim GmbH. Interferon induction Trophoblast, fibroblasts, macrophages and choriocarcinoma cells were seeded with 106 cells per well in 6-well tissue culture plates (Costar) and cultured in 2 ml RPMI-1640 medium supplemented with 10°70 FCS. After 48 h in culture interferon induction was carried out in 5 cultures of each cell type in a humidified 5% CO 2 incubator at 37°C. After removing the medium, the cultures were incubated with 10 /ag/ml poly(I:C) (Sigma) in serum-free RPMI for 1 h. Control cultures were incubated with serum-free medium alone. The cells were then washed three times with serum-free RPMI and the cultures were incubated with RPMI plus 5% FCS.

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Superinduction and priming The superinduction method was a modification of that used by Tan and Berthold (1977). Cultures were incubated with poly(I'C) at 10/ag/ml for 1 h at 37°C in serum-free RPMI containing cycloheximide (Sigma) at 10/ag/ml. The poly(I:C) solution was then removed and the cell cultures were washed three times with serum-free RPMI and incubated with 2 ml of RPMI containing cycloheximide (10/ag/ml) for 3 h at 37 °C. After this time, actinomycin D (Sigma) was added to a final concentration of 1/ag/ml and incubation was continued for a further 1 h. Following the 5 h induction and superinduction regimen, the medium was removed and the cultures were washed five times with serum-free RPMI and then incubated in 2 ml of RPMI plus 5% FCS. In experiments involving priming, cell cultures were incubated for 16 h before induction with 100 IU/ml of human beta-interferon (Green Cross Corporation) in maintenance medium. Total interferon yields were determined from samples collected immediately after cessation of interferon production, i.e. interferon harvests were taken at 10 h post-induction from poly(I:C)-induced and primed cultures and at 18 h from superinduced and primed plus superinduced cultures. In the kinetic experiments the supernatant fluid was withdrawn for interferon titration at 2 h intervals. The cultures were washed once with RPMI and immediately replenished with the same volume of warm (37°C) fresh medium containing 5% FCS. The number of cells in the test cultures was counted regularly at the end of experiments. The cells were removed from the wells by rubber policemen or trypsinization.

Interferon assays Interferon assays were carried out in WISH ceils in 96-well microtiter plates (Teknunc, Copenhagen, Denmark). A 100-/A volume of supernatant dilution was added to each well, after which the samples were incubated at 37 °C in a humidified CO 2 incubator. Eighteen hours later the cultures were challenged with approximately 50 plaque-forming units of VSV. Each sampie was set up in triplicate. Cell culture fluids from uninduced cultures were also included in parallel in each test. The interferon titer was taken as that dilution which gave a 50°/0 reduction in the number of viral plaques. A standard human interferon sample in known titer was included in each test. All titers were reported in international units (IU) per 1@ cells. Neutralization of interferon was carried out as follows: samples (50 IU) as well as interferon standards were incubated for 60 min at 37 °C with an equal volume of anti-interferon antisera and the residual activity was then assayed. Concurrent control studies of interferon alone and immune sera alone were included in each test. For each experiment the amounts of anti-human interferon sera were sufficient to neutralize completely the corresponding standard interferon.

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Investigation of pH stability of interferon samples was carried out according to the classical procedure using acidification of samples. Briefly, samples were dialyzed overnight at 4°C against glycine--HCl buffer pH 2.0 and redialyzed to neutrality against TrismHC1 buffer pH 7.2 for 24 h at 4°C. Results

Effect of priming and superinduction on poly(l:C) induction The effects of various treatments on the total interferon yields are shown in Table 1. Data represent the means of five experiments. It is seen that trophoblast, fibroblast and macrophage cultures established from placenta responded to priming, whereas pre-treatment of choriocarcinoma cell lines with interferon did not increase interferon yields. On the contrary, superinduction resulted in increase of interferon production in all cell cultures tested. Although the priming and superinduction effects were consistently observed in all placental cells, their magnitude varied. In superinduced trophoblast and fibroblasts the titers of interferon were greater than those produced by priming. In the macrophage, the effectiveness of priming and superinduction in enhancing interferon yields was equal. In trophoblast cultures the combination of priming and superinduction did not increase interferon yields to levels greater than those obtained after superinduction. In fibroblasts and macrophages the combined procedure produced more interferon than priming or superinduction alone. However, in macrophages the priming and superinduction effects were only additive, whereas in fibroblasts priming increased synergistically the effect of superinduction. No cytotoxicity was observed microscopically in the cultures at the time of removal of drugs or of taking samples. Hence, the differences in the effect of various treatments cannot be correlated with their cytotoxicity. The interferon produced by trophoblast, fibroblasts, macrophages and choriocarcinoma cell lines with different procedures was totally inactivated by anti-beta-interferon, but not by anti-alpha- or anti-gamma-interferon sera. All interferon samples were stable at pH 2.0 (data not shown). Therefore, we conclude that the anti-viral activity in the supernatant fluids from cultures induced by different procedures is due to beta-interferon.

Kinetics of interferon production induced by different procedures The time course of interferon production by trophoblast, fibroblasts and macrophages is shown in Figs. 1, 2 and 3. The kinetics of interferon release was similar in five experiments of interferon induction. No interferon was produced by uninduced cells incubated for as long as 24 h. The kinetics of interferon production show that in both primed and unprimed cells the onset of interferon production is rapid. Poly(I'C) induced detectable amounts of

None Priming Superinduction Priming + superinduction

Treatment

820 3200 8100 7800

_ ± ± ±

160 480 1600 1400

Trophoblast 256 1800 8600 25000

-*- 32 ± 200 ± 1800 ± 6200

Fibroblast

Total interferon yield (IU/106 ceils)

128 3400 3600 6600

__. 24 ± 520 ± 540 ± 1400

Macrophage

Effect of priming and superinduction on poly(l:C)-stimulated interferon production.

Table 1.

64 64 512 512

JAR ± ± ± ±

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± ± ± ±

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± 6 __. 6 ± 180 ± 180

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Hours Figs. 1--4. Kinetics of interferon production in trophoblast (Fig. 1), fibroblasts (Fig. 2), macrophages (Fig. 3) and by JAR cells (Fig. 4). Treatments: O--O, poly(I:C); A--A, priming; 1-1--[7, superinduction; ~7--V, combination of priming and superinduction. Each point represents mean _+ standard deviations.

64 interferon within 2 h and a maximal titer at 4 h post-induction. After this time, there was a drop in interferon activity which was particularly marked in the unprimed cultures. After 8 h the interferon production was negligible or undetectable in all systems. It is also seen that exposure of macrophage cultures to priming led to a 16-fold increase in peak interferon activity (Fig. 3). The maximum interferon titer of primed trophoblast (Fig. 1) and fibroblast (Fig. 2) cultures was 4--8 times higher than that of the same cells induced by poly(I :C) only. In superinduced cultures the interferon production reached a maximum rate at 4 h after removal of cycloheximide and actinomycin D, i.e. at 8 h after induction with poly(I:C), continued at a high rate for 2 h, and declined quite rapidly thereafter. The cessation of production did not occur as promptly in superinduced cells as in the control or primed cultures. Data shown in Figs. 2 and 3 demonstrate the similarity in kinetics of interferon synthesis after superinduction and combined treatment with interferon and metabolic inhibitors in fibroblast and macrophage cultures. The combination of priming and treatment with drugs caused a 2- to 4-fold further increase in peak values as compared to the titers obtained after superinduction alone. The peak interferon activity induced by combined treatment in trophoblast cultures did not exceed significantly that induced by superinduction alone (Fig. 1). However, the two interferon curves showed some differences: the interferon production induced by combined treatment reached a plateau 2 h earlier than the interferon production of superinduced cells. The decrease of interferon production of cells induced by the combination of priming and drugs was faster in comparison to that of superinduced cells. The results shown in Fig. 4 indicate that the kinetic patterns of interferon production by poly(I:C)-induced and superinduced JAR cells were similar to those observed for trophoblast (Fig. 1). However, the JAR cells proved poorer responders. It is also seen that pre-treatment of JAR cells with interferon had no effect on the interferon production induced by poly(I:C) alone or by combination of poly(I:C) with metabolic inhibitors. Similar results were also obtained with JEG-3 and BeWo cells (not shown). Discussion

The present data show that superinduction or priming increased interferon yields from cultured human trophoblast when applied with poly(I:C) induction. In contrast, the choriocarcinoma cell lines, JAR, JEG-3 and BeWo did not respond to priming, but superinduction led to increase of poly(I:C)-induced interferon production in all of them. This study also provides the first demonstration that poly(I:C)-stimulated interferon production of human tissue macrophages can be enhanced by priming or

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superinduction. The observation on the elevated interferon production by placental fibroblasts treated with interferon and metabolic inhibitors confirms the results of earlier reports (Havell and Vil~ek, 1972, Meager et al., 1979) that interferon production of human fibroblast cultures of different origin can exceptionally be enhanced by superinduction or a combination of priming and superinduction. Supportive evidence of the different behavior of various cell types tested for total interferon yields comes from the results of the kinetic studies. It should also be noted that the different rates of the superinducing effect of metabolic inhibitors has been observed under conditions which do not produce microscopic evidence of cytotoxicity. Burke et al. (1978) found that pluripotential embryonal carcinoma cells did not produce interferon after treatment with a wide variety of inducers, nor were they sensitive to its action. In another study, sensitivity of human embryonal cells to interferon action did not correlate with their ability to produce interferon in culture (Siewers et al., 1970). Our results demonstrate that the synthesis of interferon after priming or superinduction could be differentially enhanced in normal placental cells and choriocarcinoma cell lines. These two cellular functions, biosynthesis of and responsiveness to interferon, are apparently governed by genes located on different chromosomes (Langer and Pestka, 1988). Vandebussche et al. (1983) found two human choriocarcinoma cell lines (HCCM-5 and BeWo) to be resistant to other biological effects of human interferon, such as inhibition of VSV multiplication and inhibition of cell growth. The present work adds another type of resistance to interferon action, characteristic for malignant human trophoblast. Cells derived from human choriocarcinomas may therefore constitute an interesting system for studying the mechanism of resistance to different actions of interferon. Interferons accomplish a number of complex actions at the cellular level. Production of a novel group of alpha-interferons by trophoblast cells has been reported for domestic animals (Farin et al., 1989). Secretion of these proteins is suggested to be essential for maternal recognition of pregnancy. Interferons clearly play a key role in modulating the response to infections, particularly from viruses. The demonstration that human trophoblast is capable of producing large amounts of interferon is interesting, because the trophoblast layer of the human placenta functions as a barrier to the transmission of infection from mother to fetus. In certain cases, maternal infection may spread to the placenta but not to the fetus itself (Yamauchi et al., 1974). The high interferon yield produced by primed placental fibroblasts and macrophages suggests that the high level of interferon production by these cells after priming with trophoblast interferon may also play an important role in the protection of the fetus against intrauterine infection. Modulation of the expression of cell surface antigens of the major histo-

66

compatibility complex (MHC) is one of the essential effects by which all three interferon species influence the immune system. The synthesis of class I MHC proteins can be stimulated in normal diploid human cells by all three human interferon species (Wallach, 1983). This property has important implications as an anti-viral mechanism, since the capacity to mount a successful anti-viral cell-mediated immune response depends to a certain extent on the ability of the target cells to present the viral antigens in association with class I antigens (Zinkernagel and Doherty, 1974). Unlike most mature nucleated cells, human trophoblast cells do not express, or express only very low levels of, class I antigens (Hunt et al., 1988). This is possibly one of the reasons that the fetus is protected from any maternal cytotoxic T cells that may be induced. It has been demonstrated that treatment of mouse trophoblast cells with murine interferons results in an enhanced class I expression (Zuckermann and Head, 1986). Work in progress will determine if an increased expression of class I antigens is one of the ways by which the trophoblast interferon produces elimination of virus-infected human trophoblast. References Burke, D.C., Graham, C.F. and Lehman, J.M. (1970) Appearance of interferon inducibility and sensitivity during differentiation of murine teratocarcinoma cells in vitro. Cell 13,243--248. Douglas, G.C. and King, B.F. (1989) Isolation of pure villous cytotrophoblast from term human placenta using immunomagnetic microspheres. J. Immunol. Methods 119,259--268. Farin, C.E., Imakawa, K. and Roberts, R.M. (1989) In situ localization of mRNA for the interferon, ovine trophoblast protein-l, during early embryonic development of the sheep. Mol. Endocrinol. 3, 1099--1107. Field, A.K., Tytell, A.A., Lampson, G.P. and Hilleman, M.R. (1983) Inducers of interferon and host resistance. II. Multistranded synthetic polynucleotide complexes. Proc. Natl. Acad. Sci. U.S.A. 70, 1981--1987. Goorha, R.M. (1981) Preparation and assay of vesicular stomatitis virus. Methods Enzymol. 78, 309-312. Havell, E.A. and Vil~ek, J. (1972): Production of high-titered interferon in cultures of human diploid cells. Antimicrob. Agents Chemother. 2,476--484. Ho, M., Tan, Y.H. and Armstrong, D (1972) Accentuation of production of human interferon by metabolic inhibitors. Proc. Soc. Exp. Biol. Med. 139,259--262. Hunt, J.S., Fishback, J.L., Andrews, G.K. and Wood, G.W. (1988) Expression of class 1 HLA genes by trophoblast cells. Analysis by in situ hybridization. J. Immunol. 140, 1293-- 1299. Isaacs, A. and Burke, D.C. (1958) Mode of action of interferon. Nature 182, 1073--1074. Kelly, P.M.A., Bliss, E., Morton, J.A., Burus, J. and McGee, J.O.D. (1988) A monoclonal antibody (EBM 1 l) with high cellular specificity for human macrophages. J. Clin. Pathol. 41, 510--515. Kliman, H.J., Feinman, M.A. and Strauss, J.F. (1987) Differentiation of human cytotrophoblast into syncytiotrophoblast in culture. Trophoblast Res. 2,407--421. Langer, J.A. and Pestka, S. (1988) Interferon receptors, lmmunol. Today 9, 393--399. Mathiesen, G., Toth, F.D., Juhl, C., Norskov-Lauritsen, N., Mosborg Petersen, P. and Ebbesen, P. (1990) Purification and initial characterization of human placental trophoblast interferon induced by poly(l:C). J. Gen. Virol., in press. Meager, A., Graves, H.E., Shuttleworth, J. and Zucker, N. (1979) Interferon production: variation in yields from human cell lines. Infect. Immun. 25,658--663.

67 Osborn, M., Debus, E. and Weber, K. (1984) Monoclonal antibodies specific for vimentin. Eur. J. Cell Biol. 34, 137--143. Siewers, C.M.F., John, C.E. and Medearis, D.N. (1970) Sensitivity of human cell strains to interferon. Proc. Soc. Exp. Biol. Med. 133, 1178--1183. Tan, Y.H. and Berthold, W. (1977) A mechanism for the induction and regulation of human fibroblast interferon genetic expression. J. Gen. Virol. 34,401--411. Toth, F.D., Juhl, C., Norskov-Lauritsen, N., Mosborg-Petersen, P. and Ebbesen, P. (1990) Interferon production by cultured human trophoblast induced with double stranded polyribonucleotide. J. Reprod. Immunol. 17,217--227. Towell, D. and Cantell, K. (1971) Kinetics of interferon production in human leukocyte suspensions. J. Gen. Virol. 13,485--489. Vandebussche, P., Kuwata, T., Verhaegen-Lewalle, M. and Content, J. (1983) Effect of interferon on two human choricarcinoma-derived cell lines. Virology 128,474--479. Vil~ek, J. and Ng, M.H. (1971) Post-transcriptional control of interferon synthesis. J. Virol. 7, 555-594. Wallach, D. (1983) The HLA proteins and a related protein of 28 kDa are preferentially induced by interferon-y in human WISH cells. Eur. J. Immunol. 13,794--798. Wilson, C.B., Haas, J.E. and Weaver, W.M. (1983) Isolation, purification and characteristics of mononuclear phagocytes from human placentas. J. Immunol. Methods 56,305--317. Yamauchi, T., Wilson, C. and Geme, J.W. (1974) Transmission of live, attenuation mumps virus to the human placenta. N. Engl. J. Med. 290, 710--712. Zeitler, P., Murphy, E. and Handwerger, S. (1983) Characterization of the synthesis and release of human placental lactogen and human chorionic gonadotropin by an enriched population of dispersed placental cells. J. Clin. Endocrinol. Metab. 57, 812--818. Zinkernagel, R.M. and Doherty, P.C. (1974) Restriction of in vitro T cell-mediated cytotoxicity in lymphocytic choriomeningitis within a syngeneic or semiallogeneic system. Nature 224, 701--702. Zuckermann, F.A. and Head, J.R. (1986) Expression of MHC antigens on murine trophoblast and their modulation by interferon. J. Immunol. 137,846--853.