Journal of General Virology(1990), 71, 3067-3069. Printedin Great Britain
3067
Interferon production by cultured human trophoblasts and choriocarcinoma cell lines induced by Sendai virus Ferenc D. T6th, 1,2 Niels Norskov-Lauritsen, 1 Claus B. Juhl, 1 George Aboagye-Mathiesen I and Peter Ebbesen 1. 1Department of Virus and Cancer, The Danish Cancer Society, Gustav Wieds Vej 10, DK-8000 Aarhus C, Denmark and 2Institute of Microbiology, Medical University, H-H012 Debrecen, Hungary
Human term-placental trophoblasts in primary culture were studied for an interferon (IFN) response when challenged with Sendai virus and compared to three choriocarcinoma cell lines, placental fibroblasts and placental macrophages. Normal trophoblasts were high producers and released both IFN-u and IFN-fl. In contrast, one choriocarcinoma cell line was a low
producer and all malignant lines produced only IFN-fl. Circulating monocytes produce IFN-~ but placental macrophages secreted IFN-fl and some IFN-~, suggesting that IFN production may be dependent on the stage of differentiation. A role for trophoblast IFNs in protection of the foetus against virus infections is proposed.
In pregnancy, interferons (IFN) may accomplish a number of complex actions at the cellular level, not only in the immunological relationship between mother and foetus but also in the defence mechanism against intrauterine infection of the foetus. Indirect evidence for IFN production by the human placenta was first shown by perfusion studies (Bocci et aL, 1985) in which perfused human placentas at term released IFN in small amounts. In a previous study (T6th et al., 1990), we reported the production of IFN by different cells of the human placenta after stimulation with polyriboinosinic-polyribocytidylic acid [poly(rI)-poly(rC)]. The ability of these cells to produce IFN in response to viruses has not been determined. This may have great importance, especially in the case of trophoblasts, because the foetal component of the maternal-foetal interface during pregnancy is composed of trophoblast cells. This paper describes the IFN response, in primary cell cultures derived from human term placentas and in three different malignant trophoblast cell lines, to Sendai virus, a virus known to be a good IFN inducer in human cells (Lee, 1969). Term placentas from normal pregnancies (36 to 42 weeks gestation) were obtained within 15min of spontaneous vaginal delivery. Trophoblasts were separated essentially as described by Douglas & King (1989), whereas placental macrophages and fibroblasts were separated by the method of Wilson et al. (1983). The purity of the cell populations was tested by indirect immunofluorescence (T6th et al., 1990). Cytotrophoblast cultures used for IFN induction were found to be 98% to 100 % pure. The purity of the fibroblast and macrophage
cultures was greater than 95%. The human choriocarcinoma cell lines JAR, JEG-3 and BeWo were from the American Type Culture Collection. Sendai virus, Cantell strain, was grown on 10-day-old embryonated eggs and vesicular stomatitis virus (VSV), Indiana strain, was propagated on monolayers of L929 cells. For IFN production, cells were seeded at 106 per well in tissue culture plates and cultured in 2 ml RPMI-1640 plus 10% foetal calf serum (FCS). After 24 h the medium was removed and 200 haemagglutination (HA) units of Sendai virus in serum-free RPMI were added. Control cultures were incubated with the appropriate dilution of sterile allantoic fluid or with medium alone. After 1 h of adsorption, the virus inoculum was removed, the cells were washed with medium, refed with fresh RPMI plus 5% FCS and then incubated at 37 °C for 18 h. In the kinetic experiments the supernatant fluid was withdrawn for IFN titration at 2 h intervals and the cultures were replenished with fresh medium. To eliminate infectious virus, IFN samples were filtered through 50 nm pore size filters. IFN was assayed in triplicate using inhibition of VSV plaque formation in WISH cells grown in 96-well microtitre plates (T6th et al., 1990). IFN titres were expressed in international reference units (IU) per 106 cells, based on human IFN standard reagents. Human reference IFN-0c (69/19) and IFN-y (82/587) were purchased from the National Institute for Biological Standards and Control, London, U.K. The human IFN-fl reference reagent (G-023-902527) originated from National Institutes of Health, Bethesda, Md., U.S.A. The antiviral activity found was
0000-9671 © 1990SGM
3068
Short communication 20
12
q ....,
10
15
8 lO
56
z
5 z
4
S 0
w 0
~ 5 10 15 Time after induction (h)
O 20
Fig. 1. Kinetics of IFN production in cultures of placental cells induced with Sendai virus. ©, Trophoblast; O, macrophage; V, fibroblast. Mean _+standard deviation is indicated for each point.
Table I. Interferon yields from placenta and choriocarcinoma cells induced with Sendai virus Induced cells
IFN yield (IU/106 cells)
Trophoblast Macrophage Fibroblast JAR JEG-3 BeWo
7800 + 1600 12 800 ___2100 3100 + 400 8700 + 1400 5100 ___ 600 1500_+ 220
characterized by neutralization with specific antisera. Horse antisera to human IFNs were obtained from Boehringer Mannheim. Investigation of pH stability of IFN samples was carried out according to the classical procedure using acidification of samples. For titration of Sendai virus in the supernatant fluids of infected cultures, HA and haemagglutination inhibition (HI) tests were used. The rabbit immune serum raised against Sendai virus was a gift from Dr S. Haahr (Institute of Medical Microbiology, Aarhus University, Aarhus, Denmark). Table 1 shows the 18 h IFN yields harvested from the different cell cultures. Data represent the mean values of five independent experiments. Uninduced and mockinfected cultures failed to produce any detectable amount of IFN (not shown). All cell types were able to produce IFN after induction with Sendai virus. Macrophages were the best producers of IFN but high interferon yields were also produced by trophoblasts and JAR cells. Cultures of JEG-3 cells produced less IFN than those of either trophoblasts or JAR cells. The BeWo cells proved relatively poor producers in comparison to the other cell types tested.
0
w
0
5
10 15 Time after induction (h)
20
Fig. 2. Kinetics of IFN production in cultures of trophoblasts and choriocarcinoma cells induced with Sendai virus. O, Trophoblast; e , JAR; ~7, JEG-3; V, BeWo. Mean + standard deviation is indicated for each point.
The time course of IFN production by three subpopulations of term placental cells is shown in Fig. 1. The kinetics of IFN release was similar in five experiments. Sendai virus-induced IFN was detectable after 4 h in all cultures. IFN production by trophoblasts and macrophages peaked at 6 h and declined thereafter. The peak in the rate of IFN production by fibroblast cultures was delayed as compared to that of trophoblast and macrophage cultures. The maximum yields produced by macrophages were about twofold greater than that of trophoblasts. The maximum titre of IFN produced by fibroblasts was roughly two-thirds of the maximal yield from trophoblasts. No appreciable differences were found in the time course of IFN production by trophoblasts and choriocarcinoma cells (Fig. 2). Results of neutralization studies are shown in Table 2. IFN activities in supernatants from choriocarcinoma cell lines could be completely neutralized by antiserum against human IFN-fl, but not by anti-IFN-~ or antiIFN-~ antiserum. IFNs from trophoblast, macrophage and fibroblast cultures apparently consisted of a mixture of IFN-~ and IFN-fl. Of the total trophoblast IFN activity, 75~ was IFN-fl and 25% was IFN-ct, the macrophage IFN activity was 65% IFN-fl and 35% IFN-~ and the fibroblast IFN activity was 90~ IFN-fl and 10% IFN-~. IFNs from all sources were equally stable to pH 2.0 for 24 h (data not shown). The replication of Sendai virus in the various cells was monitored by titrating samples of cell culture medium for
Short communication
Table 2. Characterization o f Sendai virus-induced I F N using antibodies to human IFNs Residual IFN activity (~) after incubation with antibody to IFN sample
IFN-~t
IFN-fl
IFN 7
Standard ~t Standard fl Standard 7 Trophoblast Macrophage Fibroblast JAR JEG-3 BeWo
0 100 100 75 65 90 100 100 100
100 0 100 25 35 10 0 0 0
100 100 0 100 100 100 100 100 100
HA activity. The virus-specific nature of HA was checked by an HI test. Virus replication reached a maximum of 60 to 80 HA units/106 cells by 24 h in all types of cell cultures tested (data not shown). The results of the present study are the first demonstration that trophoblasts are good producers of IFN upon virus stimulation. No appreciable differences were found in the amounts of IFN detected in cultures of trophoblasts and two choriocarcinoma cell lines (JAR and JEG-3), whereas a third choriocarcinoma cell line, BeWo, studied here responded with much less IFN production than normal trophoblasts. It is not clear whether BeWo cells generally tend to produce less IFN. There was no correlation between IFN yield and replication of Sendai virus in the cell types tested. Both IFN-0t and IFN-fl can be synthesized in normal trophoblasts after induction with Sendal virus, whereas the IFN produced by different malignant trophoblast cell lines upon induction by the same inducer is of the IFN-fl type. The expression of the IFN-0t and IFN-fl genes in different cells is apparently controlled by at least two factors, the cell type and the mode of induction and the results of Lebon et al. (1982) provide evidence for the existence of different mechanisms for IFN-~ and IFN-fl induction. The IFN produced by Sendai virus-induced trophoblasts seems to be distinct from that released from Sendai virus-infected human amniotic membrane (Duc-Goiran et al., 1983) and from that detected in human placental blood (Duc-Goiran et al., 1985). The latter two IFNs consist of ~ and B species as well as three other species neutralized to almost the same extent by IFN-~ and IFN-fl antibodies. Circulating human monocytes produce IFN-~t upon induction with Sendai virus (Saksela et al., 1984). In our study, the IFN produced by placental macrophages was mainly of the fl type. Newcastle disease virus (NDV)-induced mouse peritoneal macrophages are IFN-fl producers (de Maeyer & de MaeyerGuignard, 1986) but, when the macrophages originate from bone marrow precursors in vitro, a significant
3069
fraction of the IFN produced upon induction with NDV is IFN-at (Brehm & Kirchner, 1986). Thus, our observations on the IFN production of tissue macrophages is the first indication in a human system that the stage of differentiation of the macrophage has an influence on the types of IFN produced. Our finding, that IFN-fl is the major component in virus-induced preparations from fibroblasts, agrees well with earlier results (Havell et al., 1978). The demonstration that human trophoblasts produce large amounts of IFN when exposed in vitro to Sendai virus is interesting because the trophoblast layer of the human placenta had been thought to play an important role in defence against viruses. It remains to be determined whether viruses involved in infection of the placenta are IFN inducers in trophoblasts varying in gestational age.
References Bocci, V., PAULESCU, L. & RICCI, M. G. (1985). The physiological interferon response. IV. Production of interferon by the perfused human placenta at term. Proceedings of the Society for Experimental Biology and Medicine 18ti, 137-143. BREHM, G. & KIRCHNER, H. (1986). Analysis of interferons induced in mice in vivo and in macrophages in vitro by Newcastle disease virus and by polyinosinic-polycytidylic acid. Journal oflnterferon Research 6, 21-28. DE MAEYER, E. & DE MAEYER-GUIGNARD, J. (1986). Interferon structural and regulatory genes in mouse. In lnterferons as Cell Growth lnhibitors and Antitumor Factors. UCLA Symposia on Molecular and Cellular Biology, New Series, vol. 50, pp. 435-445. Edited by R. M. Friedman, T. Merigan & T. Sreevalsan. New York: Alan R. Liss. DOUGLAS, G. C. & KING, B. F. (1989). Isolation of pure villous cytotrophoblast from term human placenta using immunomagnetic microspheres. Journal of Immunological Methods 119, 259-268. Duc-GoIRAN, P., ROBERT-GALLIOT, B., CHUDZIO, T. & CrlANY, C. (1983). Unusual human interferons produced by virus-infected amniotic membranes. Proceedings of the National Academy oJ Sciences, U.S.A. 80, 2628-2631. Duc-GOIRAN, P., ROBERT-GALLIOT,B., LOPEZ, J. & CHANY, C. (1985). Unusual apparently constitutive interferons and antagonists in human placental blood. Proceedings of the National Academy oJ Sciences, U.S.A. 82, 5010-5014. HAVELL, E. A., HAYES, T. G. & VILCEK, J. (1978). Synthesis of two distinct interferons by human fibroblasts. Virology 89, 330-334. LEBON, P., COMMOY-CHEVALIER,M. J., ROBERT-GALLIOT,B. & CHANY, C. (1982). Different mechanisms for g and fl interferon induction. Virology 119, 504-507. LEE, S. H. S. (1969). Interferon production by human leukocytes in vitro; some biological characteristics. Applied Microbiology 18, 731-739. SAKSELA,E., VIRTANEN,I., Hovl, T., SECHER, D. S. & CANTELL,K. (1984). Monocyte is the main producer of human leukocyte alpha interferons following Sendal virus induction. Progress in Medical Virology 30, 78-86. T6TH, F. D., JUHL, C., NORSKOV-LAURITSEN,N., MOSBORG-PETERSEN, P. & EBBESEN, P. (1990). Interferon production by cultured human trophoblast induced with double-stranded polyribonucleotide. Journal of Reproductive Immunology 17, 217-227. WILSON, C. B., HAAS, J. E. & WEAVER, W. M. (1983). Isolation, purification and characteristics of mononuclear phagocytes from human placentas. Journal of Immunological Methods 56, 305-317.
(Received 14 May 1990; Accepted 14 August 1990)
3067
Interferon production by cultured human trophoblasts and choriocarcinoma cell lines induced by Sendai virus Ferenc D. T6th, 1,2 Niels Norskov-Lauritsen, 1 Claus B. Juhl, 1 George Aboagye-Mathiesen I and Peter Ebbesen 1. 1Department of Virus and Cancer, The Danish Cancer Society, Gustav Wieds Vej 10, DK-8000 Aarhus C, Denmark and 2Institute of Microbiology, Medical University, H-H012 Debrecen, Hungary
Human term-placental trophoblasts in primary culture were studied for an interferon (IFN) response when challenged with Sendai virus and compared to three choriocarcinoma cell lines, placental fibroblasts and placental macrophages. Normal trophoblasts were high producers and released both IFN-u and IFN-fl. In contrast, one choriocarcinoma cell line was a low
producer and all malignant lines produced only IFN-fl. Circulating monocytes produce IFN-~ but placental macrophages secreted IFN-fl and some IFN-~, suggesting that IFN production may be dependent on the stage of differentiation. A role for trophoblast IFNs in protection of the foetus against virus infections is proposed.
In pregnancy, interferons (IFN) may accomplish a number of complex actions at the cellular level, not only in the immunological relationship between mother and foetus but also in the defence mechanism against intrauterine infection of the foetus. Indirect evidence for IFN production by the human placenta was first shown by perfusion studies (Bocci et aL, 1985) in which perfused human placentas at term released IFN in small amounts. In a previous study (T6th et al., 1990), we reported the production of IFN by different cells of the human placenta after stimulation with polyriboinosinic-polyribocytidylic acid [poly(rI)-poly(rC)]. The ability of these cells to produce IFN in response to viruses has not been determined. This may have great importance, especially in the case of trophoblasts, because the foetal component of the maternal-foetal interface during pregnancy is composed of trophoblast cells. This paper describes the IFN response, in primary cell cultures derived from human term placentas and in three different malignant trophoblast cell lines, to Sendai virus, a virus known to be a good IFN inducer in human cells (Lee, 1969). Term placentas from normal pregnancies (36 to 42 weeks gestation) were obtained within 15min of spontaneous vaginal delivery. Trophoblasts were separated essentially as described by Douglas & King (1989), whereas placental macrophages and fibroblasts were separated by the method of Wilson et al. (1983). The purity of the cell populations was tested by indirect immunofluorescence (T6th et al., 1990). Cytotrophoblast cultures used for IFN induction were found to be 98% to 100 % pure. The purity of the fibroblast and macrophage
cultures was greater than 95%. The human choriocarcinoma cell lines JAR, JEG-3 and BeWo were from the American Type Culture Collection. Sendai virus, Cantell strain, was grown on 10-day-old embryonated eggs and vesicular stomatitis virus (VSV), Indiana strain, was propagated on monolayers of L929 cells. For IFN production, cells were seeded at 106 per well in tissue culture plates and cultured in 2 ml RPMI-1640 plus 10% foetal calf serum (FCS). After 24 h the medium was removed and 200 haemagglutination (HA) units of Sendai virus in serum-free RPMI were added. Control cultures were incubated with the appropriate dilution of sterile allantoic fluid or with medium alone. After 1 h of adsorption, the virus inoculum was removed, the cells were washed with medium, refed with fresh RPMI plus 5% FCS and then incubated at 37 °C for 18 h. In the kinetic experiments the supernatant fluid was withdrawn for IFN titration at 2 h intervals and the cultures were replenished with fresh medium. To eliminate infectious virus, IFN samples were filtered through 50 nm pore size filters. IFN was assayed in triplicate using inhibition of VSV plaque formation in WISH cells grown in 96-well microtitre plates (T6th et al., 1990). IFN titres were expressed in international reference units (IU) per 106 cells, based on human IFN standard reagents. Human reference IFN-0c (69/19) and IFN-y (82/587) were purchased from the National Institute for Biological Standards and Control, London, U.K. The human IFN-fl reference reagent (G-023-902527) originated from National Institutes of Health, Bethesda, Md., U.S.A. The antiviral activity found was
0000-9671 © 1990SGM
3068
Short communication 20
12
q ....,
10
15
8 lO
56
z
5 z
4
S 0
w 0
~ 5 10 15 Time after induction (h)
O 20
Fig. 1. Kinetics of IFN production in cultures of placental cells induced with Sendai virus. ©, Trophoblast; O, macrophage; V, fibroblast. Mean _+standard deviation is indicated for each point.
Table I. Interferon yields from placenta and choriocarcinoma cells induced with Sendai virus Induced cells
IFN yield (IU/106 cells)
Trophoblast Macrophage Fibroblast JAR JEG-3 BeWo
7800 + 1600 12 800 ___2100 3100 + 400 8700 + 1400 5100 ___ 600 1500_+ 220
characterized by neutralization with specific antisera. Horse antisera to human IFNs were obtained from Boehringer Mannheim. Investigation of pH stability of IFN samples was carried out according to the classical procedure using acidification of samples. For titration of Sendai virus in the supernatant fluids of infected cultures, HA and haemagglutination inhibition (HI) tests were used. The rabbit immune serum raised against Sendai virus was a gift from Dr S. Haahr (Institute of Medical Microbiology, Aarhus University, Aarhus, Denmark). Table 1 shows the 18 h IFN yields harvested from the different cell cultures. Data represent the mean values of five independent experiments. Uninduced and mockinfected cultures failed to produce any detectable amount of IFN (not shown). All cell types were able to produce IFN after induction with Sendai virus. Macrophages were the best producers of IFN but high interferon yields were also produced by trophoblasts and JAR cells. Cultures of JEG-3 cells produced less IFN than those of either trophoblasts or JAR cells. The BeWo cells proved relatively poor producers in comparison to the other cell types tested.
0
w
0
5
10 15 Time after induction (h)
20
Fig. 2. Kinetics of IFN production in cultures of trophoblasts and choriocarcinoma cells induced with Sendai virus. O, Trophoblast; e , JAR; ~7, JEG-3; V, BeWo. Mean + standard deviation is indicated for each point.
The time course of IFN production by three subpopulations of term placental cells is shown in Fig. 1. The kinetics of IFN release was similar in five experiments. Sendai virus-induced IFN was detectable after 4 h in all cultures. IFN production by trophoblasts and macrophages peaked at 6 h and declined thereafter. The peak in the rate of IFN production by fibroblast cultures was delayed as compared to that of trophoblast and macrophage cultures. The maximum yields produced by macrophages were about twofold greater than that of trophoblasts. The maximum titre of IFN produced by fibroblasts was roughly two-thirds of the maximal yield from trophoblasts. No appreciable differences were found in the time course of IFN production by trophoblasts and choriocarcinoma cells (Fig. 2). Results of neutralization studies are shown in Table 2. IFN activities in supernatants from choriocarcinoma cell lines could be completely neutralized by antiserum against human IFN-fl, but not by anti-IFN-~ or antiIFN-~ antiserum. IFNs from trophoblast, macrophage and fibroblast cultures apparently consisted of a mixture of IFN-~ and IFN-fl. Of the total trophoblast IFN activity, 75~ was IFN-fl and 25% was IFN-ct, the macrophage IFN activity was 65% IFN-fl and 35% IFN-~ and the fibroblast IFN activity was 90~ IFN-fl and 10% IFN-~. IFNs from all sources were equally stable to pH 2.0 for 24 h (data not shown). The replication of Sendai virus in the various cells was monitored by titrating samples of cell culture medium for
Short communication
Table 2. Characterization o f Sendai virus-induced I F N using antibodies to human IFNs Residual IFN activity (~) after incubation with antibody to IFN sample
IFN-~t
IFN-fl
IFN 7
Standard ~t Standard fl Standard 7 Trophoblast Macrophage Fibroblast JAR JEG-3 BeWo
0 100 100 75 65 90 100 100 100
100 0 100 25 35 10 0 0 0
100 100 0 100 100 100 100 100 100
HA activity. The virus-specific nature of HA was checked by an HI test. Virus replication reached a maximum of 60 to 80 HA units/106 cells by 24 h in all types of cell cultures tested (data not shown). The results of the present study are the first demonstration that trophoblasts are good producers of IFN upon virus stimulation. No appreciable differences were found in the amounts of IFN detected in cultures of trophoblasts and two choriocarcinoma cell lines (JAR and JEG-3), whereas a third choriocarcinoma cell line, BeWo, studied here responded with much less IFN production than normal trophoblasts. It is not clear whether BeWo cells generally tend to produce less IFN. There was no correlation between IFN yield and replication of Sendai virus in the cell types tested. Both IFN-0t and IFN-fl can be synthesized in normal trophoblasts after induction with Sendal virus, whereas the IFN produced by different malignant trophoblast cell lines upon induction by the same inducer is of the IFN-fl type. The expression of the IFN-0t and IFN-fl genes in different cells is apparently controlled by at least two factors, the cell type and the mode of induction and the results of Lebon et al. (1982) provide evidence for the existence of different mechanisms for IFN-~ and IFN-fl induction. The IFN produced by Sendai virus-induced trophoblasts seems to be distinct from that released from Sendai virus-infected human amniotic membrane (Duc-Goiran et al., 1983) and from that detected in human placental blood (Duc-Goiran et al., 1985). The latter two IFNs consist of ~ and B species as well as three other species neutralized to almost the same extent by IFN-~ and IFN-fl antibodies. Circulating human monocytes produce IFN-~t upon induction with Sendai virus (Saksela et al., 1984). In our study, the IFN produced by placental macrophages was mainly of the fl type. Newcastle disease virus (NDV)-induced mouse peritoneal macrophages are IFN-fl producers (de Maeyer & de MaeyerGuignard, 1986) but, when the macrophages originate from bone marrow precursors in vitro, a significant
3069
fraction of the IFN produced upon induction with NDV is IFN-at (Brehm & Kirchner, 1986). Thus, our observations on the IFN production of tissue macrophages is the first indication in a human system that the stage of differentiation of the macrophage has an influence on the types of IFN produced. Our finding, that IFN-fl is the major component in virus-induced preparations from fibroblasts, agrees well with earlier results (Havell et al., 1978). The demonstration that human trophoblasts produce large amounts of IFN when exposed in vitro to Sendai virus is interesting because the trophoblast layer of the human placenta had been thought to play an important role in defence against viruses. It remains to be determined whether viruses involved in infection of the placenta are IFN inducers in trophoblasts varying in gestational age.
References Bocci, V., PAULESCU, L. & RICCI, M. G. (1985). The physiological interferon response. IV. Production of interferon by the perfused human placenta at term. Proceedings of the Society for Experimental Biology and Medicine 18ti, 137-143. BREHM, G. & KIRCHNER, H. (1986). Analysis of interferons induced in mice in vivo and in macrophages in vitro by Newcastle disease virus and by polyinosinic-polycytidylic acid. Journal oflnterferon Research 6, 21-28. DE MAEYER, E. & DE MAEYER-GUIGNARD, J. (1986). Interferon structural and regulatory genes in mouse. In lnterferons as Cell Growth lnhibitors and Antitumor Factors. UCLA Symposia on Molecular and Cellular Biology, New Series, vol. 50, pp. 435-445. Edited by R. M. Friedman, T. Merigan & T. Sreevalsan. New York: Alan R. Liss. DOUGLAS, G. C. & KING, B. F. (1989). Isolation of pure villous cytotrophoblast from term human placenta using immunomagnetic microspheres. Journal of Immunological Methods 119, 259-268. Duc-GoIRAN, P., ROBERT-GALLIOT, B., CHUDZIO, T. & CrlANY, C. (1983). Unusual human interferons produced by virus-infected amniotic membranes. Proceedings of the National Academy oJ Sciences, U.S.A. 80, 2628-2631. Duc-GOIRAN, P., ROBERT-GALLIOT,B., LOPEZ, J. & CHANY, C. (1985). Unusual apparently constitutive interferons and antagonists in human placental blood. Proceedings of the National Academy oJ Sciences, U.S.A. 82, 5010-5014. HAVELL, E. A., HAYES, T. G. & VILCEK, J. (1978). Synthesis of two distinct interferons by human fibroblasts. Virology 89, 330-334. LEBON, P., COMMOY-CHEVALIER,M. J., ROBERT-GALLIOT,B. & CHANY, C. (1982). Different mechanisms for g and fl interferon induction. Virology 119, 504-507. LEE, S. H. S. (1969). Interferon production by human leukocytes in vitro; some biological characteristics. Applied Microbiology 18, 731-739. SAKSELA,E., VIRTANEN,I., Hovl, T., SECHER, D. S. & CANTELL,K. (1984). Monocyte is the main producer of human leukocyte alpha interferons following Sendal virus induction. Progress in Medical Virology 30, 78-86. T6TH, F. D., JUHL, C., NORSKOV-LAURITSEN,N., MOSBORG-PETERSEN, P. & EBBESEN, P. (1990). Interferon production by cultured human trophoblast induced with double-stranded polyribonucleotide. Journal of Reproductive Immunology 17, 217-227. WILSON, C. B., HAAS, J. E. & WEAVER, W. M. (1983). Isolation, purification and characteristics of mononuclear phagocytes from human placentas. Journal of Immunological Methods 56, 305-317.
(Received 14 May 1990; Accepted 14 August 1990)
