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Study of interferon production during pregnancy in mice and antiviral activity in the placenta
Study of interferon production during pregnancy in mice and antiviral activity in the placenta Kazunori Yamada, M.D., Yoshinobu Shimizu, M.D., Kunihiro Okamura, M.D., Katsuo Kumagai, M.D., and Masakuni Suzuki, M.D. Sendai, japan Although the mortality rate after herpes simplex virus type 2 inoculation was not significantly different between pregnant mice and nonpregnant mice, systemic interferon production was very high during late pregnancy compared with that in nonpregnant mice. Antiviral activity was detected in placentas from all noninfected pregnant mice (80 to 320 U/ml in 20% suspension). The antiviral activity had a broad spectrum and was also effective in the cells of other species; an antiviral effect was shown even if the cells were treated after challenge with a virus. In addition, this activity was not inactivated by anti mouse interferon-neutralizing antisera. The molecular weight of this placental antiviral substance was estimated to be 200,000 to 450,000 daltons by gel filtration, and it was inactivated by heat, acid, and trypSin. Noninterferon antiviral activity (40 to 80 U/ml) was also detected in more than half the sera (61 .5%) of noninfected mice in late pregnancy. (AM J OBSTET GVNECOL 1985;153:335-41.)
Key words: Pregnancy, herpes simplex virus type 2, interferon, placenta, antiviral substance
There have been a number of studies showing that various immunologic reactions are suppressed during pregnancy or suppressed by the sera of pregnant women. 1-3 Pregnancy-specific substances such as human chorionic gonadotropin , human placental lactogen , (Xfetoprotein, pregnancy-specific protein 1, and pregnancy-specific protein 2 and an increase of steroid hormones such as estrogens and progesterone are thought to partially account for the low immunologic responsiveness during pregnancy. Although the low responsiveness is favorable in that it allows the pregnant woman to accept the antigenically foreign fetus, it is also unfavorable since pathogenic organisms may invade the pregnant woman with ease and cause death if the whole maternal immune system is suppressed. On the other hand, in addition to an increase of neutrophils and macro phages in phagocytic and bactericidal activity'" 5 enhancement of the complement system 6 and the antibody response to specific antigens 7 during pregnancy has been reported. The enhancement of these immunologic activities seems to compensate for the suppressed lymphocyte functions. We studied the changes in the interferon response, one of the most important humoral immunologic re-
From the Department of Obstetrics and Gynecology, School of Medicine, and the Department of Microbiology, School of Dentistry, Tohoku University. Received for publication March 14, /985; revised May 30, 1985; accepted June 26, 1985. Reprint requests: K. Yamada, M .D., Department of Obstetrics and Gynecology, Tohoku University, School of Medicine, 1-1 Seiryocho, Sendai 980, japan.
sponses against viral infection, during pregnancy in mice. In addition to metabolic and endocrinologic functions, embryonal tissues including the placenta are reported to have various immunologically active substances. They are the suppressors of the blastogenesis oflymphocytes 8 and the activators ofT-cell-dependent antibody production,9 interferon,' o. 11 and its inhibitor. I. These immunologically active substances are very interesting, considering not only the immunologic relationship between mother and fetus but also the defense mechanism against intrauterine infection of the fetus. From this point of view, we researched the antiviral activity of the murine placenta. Material and methods Material. The 9- to 12-week-old female Balb/c mice were mated with male Balb/c mice overnight, and the presence of a vaginal plug was taken as evidence of successful mating, with that day designated as day 0 of pregnancy. Mice of 4 to 5 days of gestation were designated as being in early pregnancy, and those of 1 I to 12 days of gestation were designated as being in late pregnancy. In some experiments, gestation was divided into three periods: early (0 to 6 days of gestation), middle (7 to 13 days of gestation), and late (14 days of gestation). Vesicular stomatitis virus (Indiana strain), herpes simplex virus type 2 (HSV-2, UW268 strain), poliovirus type 3, rubella virus, and adenovirus type 2 were used for antiviral activity assay including interferon assay. In inoculation studies, 33 nonpregnant mice, seven 335
336
Yamada et al.
October I, 1985 Am J Obstet Gynecol
100 90 80 • 70 II 60 111 50 ... 40 111 > 30 ...:;,> 20 III 10 ~
o
1 2 3 4 5 6 7 8 9 10 11 12 13 14 days post
inoculation
Fig.!. The survival rate of pregnant Balblc mice inoculated with HSV -2. Thirty-three nonpregnant mice (_ _ ). seven mice in early pregnancy (4 to 5 days of gestation, 0---0), and nine mice in late pregnancy (11 to 12 days of gestation, f'>--f'» were inoculated with HSV-2 (2 x 103 plaqueforming units per mouse) by intraperitoneal injection.
mice in early pregnancy, and nine mice in late pregnancy were inoculated with 2 x 10' plaque-forming units of HSV-2 in 200 f.Ll of phosphate-buffered saline by intraperitoneal injection. Continuous mouse fibroblasts (L929, C3H2KC), secondary mouse embryo cells (MEC), continuous human amniotic cells (WISH, FL), secondary human embryonic lung cells (HEL), continuous monkey kidney cells (GMK, CV-l), and continuous hamster kidney cells (BHK-21) were propagated by standard methods in growth medium. The growth medium consisted of Eagle's minimum essential medium containing 10% calf serum (Gibco, Ohio) penicillin (100 U/ml), and streptomycin (100 f.Lgl ml); maintenance medium for antiviral activity assay was similar except that 2% calf serum was used. Placentas were collected from mice on day 18 or 19 of gestation, washed with phosphate-buffered saline to remove blood, and homogenized in phosphate-buffered saline or minimum essential medium. The supernatant resulting from centrifugation at 100,000 x g for 60 minutes was harvested as a cytosol fraction and sterilized by passage through a 0.22 f.L microfilter. Antisera were antimouse a/~-interferon antiserum purchased from Enzo Biochemical, Inc. (New York) and antimouse 'V-interferon antiserum pr~pared by Professor H. M. Johnson (Department of Microbiology, University of Texas, Galveston, Texas). Trypsin was bovine pancreas type V purchased from Sigma Chemical Co. (St. Louis, Missouri). Interferon assay. The interferon assay was a conventional plaque-inhibition assay with L929 cells used as the indicator cells and vesicular stomatitis virus used
as the challenging virus. In our assay system, 1 U was equivalent to 1.5 IU (compared with National Institutes of Health standard interferon). Species specificity. Confluent cell cultures were incubated with 2% (w/v) placental extract for 24 hours at 37° C with 5% carbon dioxide. After the supernatants were removed, the cell cultures were challenged with vesicular stomatitis virus for 60 minutes and the incubation was continued until cytopathic effect was visible in control cells (approximately 24 hours). Cytopathic effect was expressed as follows: -. cytopathic effect not observed; ±, cytopathic effect suspected; +, obvious cytopathic effect observed in some . part; + +, cytopathic effect observed in almost all of culture; + + +, all cells lost owing to cytopathic effect. Virus spectrum. Monolayer cell cultures in capped test tubes were incubated with 2% (w/v) placental extract for 24 hours at 37° C and challenged with viruses for 90 minutes. Washed with serum-free minimum essential medium three times, cell cultures were incubated with 1 ml of minimum essential medium or minimum essential medium containing 2% (w/v) placental extract until cytopathic effect was visible in control cells. In the rubella virus assay, serum-free minimum essential medium was used in all procedures. Expressions of cytopathic effect were the same as those in the species specificity assay. Mode of action Experiment with vesicular stomatitis virus and L929. Confluent monolayer cultures of L929 were incubated with minimum essential medium or minimum essential medium containing 2% (w/v) placental extract for 24 hours at 37° C in 5% carbon dioxide. After removal of the
Interferon production during pregnancy in mice
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•• •• <40 lGt.m=~.a.1I 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 days post inoculation Fig. 2. Systemic interferon (IFN) production by pregnant Balblc mice inoculated with HSV-2. Nonpregnant mice (e), mice in early pregnancy (0), and mice in late pregnancy (t.) were inoculated with HSV-2 (2 x 10' pfu per mouse) by intraperitoneal iqjection, and serum interferon titers were assayed by a 50% plaque-reduction assay with the use of L929 and vesicular stomatitis virus.
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Fig. 3. Interferon-like antiviral activity in sera of noninoculated pregnant mice. Pregnant Balblc mice 9 to 12 weeks old were put to death on day 3 (early), 10 (middle), or 16 to 19 (late) of gestation. The sera were assayed for antiviral activity by a 50% plaque-reduction assay with the use of L929 and vesicular stomatitis virus.
incubating medium, vesicular stomatitis virus in minimum essential medium or in minimum essential medium containing 2% (w/v) placental extract was added to the cell cultures and incubated for 90 minutes for adsorption. The supernatants were removed and minimum essential medium or minimum essential medium containing 2% (w/v) placental extract was added to the cultures and the incubation was continued until cytopathic effect was visible in control cells (approximately 24 hours). The culture media were harvested for virus yield assay and the plaques were counted. After serial tenfold dilutions of the harvested culture media, 1 ml was added to monolayer L929 cells. Cell cultures were incubated until cytopathic effect was visible (approximately 24 hours). Virus yields were calculated as a re-
ciprocal of the highest dilution of the supernatants that caused a cytopathic effect on the cells. Experiment with HSV-2 and WISH cells. Confluent monolayer cultures of WISH cells in capped test tubes were incubated with minimum essential medium or minimum essential medium containing 2% (w/v) placental extract for 24 hours at 37 0 C. Then WISH cells were challenged with HSV-2 for 60 minutes. Infected cells were cultured with minimum essential medium or minimum essential medium containing 2% (w/v) placental extract until cytopathic effect was visible in control cells (approximately 72 hours). When cytopathic effect was visible in control cells, cells were frozen and thawed three times rapidly. After serial tenfold dilutions of the supernatant ofa centrifugation at 1500 x g
338 Yamada et al.
October I, 1985 Am J Obstet Gynecol
Table I. Neutralizing test of antiviral activity in sera of noninoculated pregnant mice by anti mouse interferon antisera
Table III. Neutralizing test of placental antiviral substance by antimouse interferon antisera
Treatment Minimum essential medium (Ulml)
Sample
Pregnant mouse serum Mouse ~-interferon Mouse 'I-interferon
Antimouse
0.1 {3-interferon antiserum (Ulml)
Treatment Antimouse 'I-interferon antiserum (Ulml)
32
16
32
1024 32
<4 ND
ND <4
The samples were treated with antisera for 90 minutes at 37° C. The antiviral activity was measured by interferon assay after treatment. ND = Not done.
Treatment
Titer (U/ml)
256
16
Placental extract Experiment 1 Experiment 2 Mouse ~-interferon (experiment I) Mouse 'I-interferon (experiment 2)
Antimouse antiserum (Ulml)
Antimouse 'I-interferon antiserum (Ulml)
64 128 128
32 ND <4
ND 64 ND
128
ND
<4
0.1 {3-interferon
The samples were treated with antiserum for 90 minutes at 37° C. The antiviral activity was assayed by interferon assay after treatment. ND = Not done.
Table II. Stability of placental antiviral substance in various conditions
None
Sample
Minimum essential medium (Ulml)
56° C, 30 min
Trypsin*
4
16
The figures show antiviral activity in units per milliliter assayed by 50% plaque-reduction assay with the use of L929 and vesicular stomatitis virus after treatment. *For 30 minutes at 37° C in 0.2% trypsin.
for 30 minutes, the supernatant was added to monolayer cultures of BHK cells. The cells were incubated until cytopathic effect was visible (approximately 96 hours) and virus yields were calculated as a reciprocal of the highest dilution of the supernatant that caused cytopathic effect on the BHK cells. Gel filtration. A total of 5 ml of 40% (w/v) placental extract in phosphate-buffered saline was applied to a 2.6 by 60 cm column of Sephacryl S-300 (Pharmacia, Vppsala, Sweden). The sample was eluted with phosphate-buffered saline at a flow rate of 5.7 cm/hr. Fractions of 3 mt were collected and assayed for their antiviral activity by the interferon assay. In order to estimate the molecular weight of placental antiviral substance, marker proteins, that is, egg albumin (molecular weight 45,000), catalase (molecular weight 240,000), and ferritin (molecular weight 450,000), were used.
Results Susceptibility of pregnant mice to HSV-2 infection and systemic interferon production. Pregnancy seemed to increase the susceptibility of mice to HSV-2 infection, that is, all pregnant mice died by 13 days after virus inoculation, while 18.2% (six of 33) of nonpregnant mice survived more than 14 days after virus
Table IV. Species specificity of placental antiviral substance Cytopathic effect Cell
Mouse L929 C3H2KC MEC Human WISH FL HEL Monkey GMK CV-I Hamster, BHK-21
Placental extract
Control
+++ + + ++ +++
+++ +++ +++
+
++ ++
+++
+++
The results are expressed as extent of cytopathic effect formed on the cells of various species that were treated with 2% (w/v) placental extract before vesicular stomatitis virus challenge (see text for explanation).
inoculation (Fig. 1). However, the differences in mortality rates between the three groups were not statistically significant. The interferon titers in sera of HSV-2-inoculated nonpregnant mice were 40 to 160 Vlml on the fourth day after inoculation. In mice in early pregnancy, the postinoculation interferon titers in sera were 40 to 320 Vlml from day 2 to day 4. In mice in late pregnancy, very high systemic interferon production was observed; the titers of interferon in sera were 40 to 640 Vlml from day 1 to day 5 (Fig. 2). Antiviral activity (40 to 80 Vlml) was detected in the sera of about half the pregnant mice before virus inoculation, and the incidence of this antiviral activity increased as pregnancy advanced. It was detected in
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Table V. Virus spectrum of placental antiviral substance Cytopathic effect
I postchallenge Prechallenge and I No treatment treatment
BHK-21 (hamster)
FL (huroon)
Virus
Prechallenge treatment
Vesicular stomatitis virus Polio virus Rubella virus HSV-2 Adenovirus
Prechallenge treatment
+ + + + +
± ±
I postchallenge Prechallenge and I treatment No treatment
+++
+
+++
± ±
±
+ + +
+
The results are expressed as extent of cytopathic effect of various viruses formed on host cells that were treated with 2%
(w/v) placental extract either prior to or both before and after virus challenge.
Table VI. Stage of action of placental antiviral substance Vesicular storootitis virus, L929 Plaque Stage of action
-24 hr Time 0 90 min -24 hr 90 min None
time 0 90 min 24 hr time 0 24 hr
No.
28 252 104 0
I
Inhibition (%)
Virus yield
Log'o inhibition virus yield
HSV-2, WISH: Virus yield
93.2 39.0 74.8 100
10' 10' 10' 102
2.0 1.0 1.0 3.0
>10' ND 10' 10
413
10'
> 10'
In the plaque-inhibition assay, the inhibition rate was calculated with the numbers of plaques formed by vesicular stomatitis virus on L929 cells treated with 2% (w/v) placental extract in various stages. In the virus yield-inhibition assay, the cells were treated with 2% (w/v) placental extract in various stages of virus replication and serial tenfold dilutions of the harvested culture media were added to assay cells. Virus yield was calculated as the highest dilution that caused cytopathic effect. ND = Not done.
61.5% (eight of 13) of the mice in late pregnancy (Fig. 3). This antiviral activity was not inactivated by antimouse u/l3-interferon antiserum or antimouse -y-interferon antiserum (Table I) . Characterization of placental antiviral substance. An antiviral activity (80 to 320 V im I) was detected in a 20% (w/v) suspension of placentas of noninfected mice by the interferon assay. Placental antiviral substance was inactivated by acid (pH 2, 24 hours), heat (56 0 C, 30 minutes), and trypsin treatment (Table II). The placental antiviral substance was not inactivated by antimouse u/l3-interferon-neutralizing antiserum or antimouse -y-interferon-neutralizing antiserum (Table III) . The activity of placental antiviral substance on vesicular stomatitis virus growth was evident in all mouse cells examined (L929, C3H2KC, and MEC) and also evident in the human FL cell, less evident in the human WISH cell, and not at all evident in the HEL cell. Activity was also evident in monkey cells CV-I and GMK but not in hamster cells (BHK) (Table IV). These results indicate the low species specificity of placental antiviral substance. Placental antiviral substance was effective against both the ribonucleic acid viruses and deoxyri-
.bonucleic acid viruses examined. In the experiment with FL used as a host cell, it was effective against ribonucleic acid viruses, that is, vesicular stomatitis virus, poliovirus, and rubella virus, and also effective against deoxyribonucleic acid viruses, that is, HSV-2 and adenovirus, with prechallenge treatment. In the experiment with the BHK cell used as the host cell, although placental antiviral substance was effective only against rubella virus and HSV -2 when prechallenge treatment alone was done, it was effective against vesicular stomatitis virus and adenovirus by addition of the postchallenge treatment (Table V). In the experiment to determine the stage of action for vesicular stomatitis virus and the L929 cell, placental antiviral substance was most effective when both prechallenge and postchallenge treatment was done; prechallenge treatment only was next in both the plaquereduction assay and the virus yield-inhibition assay. In the virus yield-inhibition assay with the use of HSV-2 and the WISH cell, although placental antiviral substance was most effective when both prechallenge and postchallenge treatment was done, postchallenge treatment only was next, and prechallenge treatment only was ineffective (Table VI) .
340
Yamada et al.
October 1, 1985 Am J Obstet Gynecol
blue ferritin catalase dextran (450000) (240000) 2.0
~
l
~
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~ I
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8 oIII
0
0
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0 40
50
60
70 80 fraction
90 100 number
110
120
130
Fig. 4. Gel filtration of murine placental extract with the use of Sephacryl S-300. An amount of 5 ml of 40% (w/v) placental extract was applied to a 2.6 by 60 cm column, eluted with phosphatebuffered saline at a flow rate of 5.7 cm/hr, and fractions of 3 ml were collected and measured by interferon assay.
The molecular weight of placental antiviral substance was estimated to be 200,000 to 450,000 daltons by gel filtration with Sephacryl S-300 (Fig. 4).
Comment The changes in immunologic responses during pregnancy can be divided into two types, suppressive and enhancing responses. The suppressions are of mixed leukocyte reaction,' natural killer cell activity; and blastogenesis oflymphocytes by lectins in vitro.' On the other hand, the enhancements are of the phagocytic and bactericidal activities of neutrophils and macrophages:' 5 the antibody response to specific antigens,6 the complement system,7 the interferon response after virus inoculation, and antiviral activity in serum without any inducing stimuli. The suppressive responses are genetically highly developed immunologic responses in which there is individual recognition of "self" and "notself"; the enhancing responses are genetically primitive nonspecific immunities except for the antibody response. It seems to be very reasonable that the specific responses are suppressed during pregnancy so that the pregnant woman can accept the antigenically allogeneic fetus. The weakened specific immunity is compensated by enhancement of nonspecific immunity. Although the mechanism governing the high systemic interferon response remains to be investigated, the increase in protein synthesis in cells that results from the raised serum estrogen level during pregnancy might partially account for it. In the present work we detected antiviral activity in more than half the sera (61.5%) of mice in late pregnancy without any induction. It is unlikely that the antiviral activity in serum is the result of interferon pro-
duced by a latent viral infection activated during pregnancy because it was not neutralized by anti-interferon sera. The origin ofthe antiviral activity is not clear, and the possibility also cannot be discarded that it is the same substance as placental antiviral substance. In humans, our colleague Hamazaki '3 has reported that the interferon-like antiviral activity was detected in the sera of pregnant women. It was detected in 59% (27 of 46) of pregnant women in the third trimester. It is not clear why some pregnant women had antiviral activity in the sera and others did not or why it appeared with about the same incidence in syngeneically mated mice and in allogeneically coupled humans. However, the antiviral activity in serum is probably related to pregnancy for it was detected in none of the sera of nonpregnant women and the incidence increased as pregnancy advanced in both the mouse and the human. With reference to the antiviral substance in the placenta, Fowler et al. lO have reported that the antiviral substance detected in the murine placenta was interferon, but the results of our experiments suggest that placental antiviral substance is different from interferon,judging from its unusual antigenicity, low species specificity, unusual stage at action, and high molecular weight. Regarding antiviral substances related to fetoplacental tissues, the "virus inhibitor" spontaneously produced by many types of cells, including mouse embryo cells in culture, has also been reported. 14 Although the "virus inhibitor" is similar to placental antiviral substance in terms of low species specificity, its molecular weight was estimated to be no higher than that of a middle-sized protein while that of placental antiviral substance was estimated as 200,000 to 450,000 daltons and the inhibition of the "virus inhibitor" required the
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simultaneous presence of inhibitor, virus, and cells, while the inhibition of placental antiviral substance did not. Placental antiviral substance is interesting in view of its high molecular weight, for Duc-Goiran et al. 15 reported on the high-molecular weight interferons produced by human amniotic membrane with the induction of Sendai virus. They showed that the highmolecular weight interferons did not have strict species specificity; some species of these high-molecular weight interferons were effective in bovine MDBK cells as well as in human cells, while other species were effective in only human cells. Although placental antiviral substance has not been clearly characterized by the research done so far, the available evidence suggests the possibility that it may be an antigenically immature, primitive interferon. It is necessary to further purify this substance and prepare antiserum raised to placental antiviral substance in order to clarify whether it is synthesized spontaneously by fetoplacental tissues including trophoblasts, like human chorionic gonadotropin and steroid hormones, or whether it is produced by immunologically competent cells with some inducing stimuli. Further study is also needed to reveal how placental antiviral substance might work to prevent intrauterine viral infection of the fetus. We are grateful to Miss Midori Takanashi for preparation of the figures. REFERENCES I. Kasakura S. A factor in maternal plasma during pregnancy that suppresses the reactivity of mixed leukocyte cultures. j Immunol 1971; 107: 1296. 2. Okamura K, Furukawa K, Nakakuki M, Yamada K, Suzuki M. Natural killer cell activity during pregnancy. AM j OBSTET GYNECOL 1984; 149:396.
Interferon production during pregnancy in mice
341
3. Finn R, St Hill CA, Govan Aj, Ralfs IG, Gurney Fj, Denye V. Immunological responses in pregnancy and survival of fetal homograft. Br Medj 1972;15:150. 4. Mitchell GW jr, McRipley Rj, Selvaraj Rj, Sbarra AJ. The role of the phagocyte in host-parasite interactions. IV. The phagocytic activity of leukocytes in pregnancy and its relationship to urinary tract infection. AM j OBSTET GyNECOL 1966;96:687. 5. Mitchell GW jr, jacobs AA, Haddad V, Paul BB, Strauss RR, and Sbarra AJ. The role of the phagocyte in hostparasite interactions. XXV. Metabolic and bactericidal activities ofleukocytes from pregnant women. AM j OBSTET GYNECOL 1970; 108:805. 6. Tedder RS, Nelson M, Eisen V. Effects on serum complement of normal and pre-eclamptic pregnancy and of oral contraceptives. Br j Pathol 1975;56:389. 7. Fabris N, Serri F. Immunological reactivity during pregnancy in the mouse. In: Cantaro A, Carreti N, eds. Immunology in obstetrics and gynecology. New York: Elsevier, 1974:183. 8. Kouttab NM, Fowler AK, Strickland jE, Hellman A. Suppression of in vitro lymphocyte stimulation in mice by uterine and placental extracts. j Immunol1976; 177: 1644. 9. Barg M, Burton RC, Smith jA, Luckenbach GA, Decker j, Mitchell GF. Effects of placental tissue on immunological responses. Clin Exp Immunol 1978;34:441. 10. Fowler AK, Reed CD, Giron DJ. Identification of an interferon in murine placentas. Nature 1980;286:266. II. Lebon P, Girard S, Thepot F, Chany C. The presence of ex-interferon in human amniotic fluid. j Gen Virol 1982;59:393. 12. Cembrzynska-Nowak M. Physicochemical characteristics of IME-inhibitor of interferon activity from mouse embryo tissues. Arch Immunol Ther Exp 1977;25:663. 13. Hamazaki Y. Interferon-like substance in pregnancy sera. Acta Obstet Gynaecoljpn 1983;35:621. 14. Baron S, McKerlie L. Broadly active inhibitor of viruses spontaneously produced by many cell type culture. Infect Immun 1981;32:449. 15. Duc-Goiran P, Robert-Gailliot B, Chudzio T, Chany C. Unusual human interferons producted by virus-infected amniotic membranes. Proc Nat! Acad Sci USA 1983; 80:2628.
Key words: Pregnancy, herpes simplex virus type 2, interferon, placenta, antiviral substance
There have been a number of studies showing that various immunologic reactions are suppressed during pregnancy or suppressed by the sera of pregnant women. 1-3 Pregnancy-specific substances such as human chorionic gonadotropin , human placental lactogen , (Xfetoprotein, pregnancy-specific protein 1, and pregnancy-specific protein 2 and an increase of steroid hormones such as estrogens and progesterone are thought to partially account for the low immunologic responsiveness during pregnancy. Although the low responsiveness is favorable in that it allows the pregnant woman to accept the antigenically foreign fetus, it is also unfavorable since pathogenic organisms may invade the pregnant woman with ease and cause death if the whole maternal immune system is suppressed. On the other hand, in addition to an increase of neutrophils and macro phages in phagocytic and bactericidal activity'" 5 enhancement of the complement system 6 and the antibody response to specific antigens 7 during pregnancy has been reported. The enhancement of these immunologic activities seems to compensate for the suppressed lymphocyte functions. We studied the changes in the interferon response, one of the most important humoral immunologic re-
From the Department of Obstetrics and Gynecology, School of Medicine, and the Department of Microbiology, School of Dentistry, Tohoku University. Received for publication March 14, /985; revised May 30, 1985; accepted June 26, 1985. Reprint requests: K. Yamada, M .D., Department of Obstetrics and Gynecology, Tohoku University, School of Medicine, 1-1 Seiryocho, Sendai 980, japan.
sponses against viral infection, during pregnancy in mice. In addition to metabolic and endocrinologic functions, embryonal tissues including the placenta are reported to have various immunologically active substances. They are the suppressors of the blastogenesis oflymphocytes 8 and the activators ofT-cell-dependent antibody production,9 interferon,' o. 11 and its inhibitor. I. These immunologically active substances are very interesting, considering not only the immunologic relationship between mother and fetus but also the defense mechanism against intrauterine infection of the fetus. From this point of view, we researched the antiviral activity of the murine placenta. Material and methods Material. The 9- to 12-week-old female Balb/c mice were mated with male Balb/c mice overnight, and the presence of a vaginal plug was taken as evidence of successful mating, with that day designated as day 0 of pregnancy. Mice of 4 to 5 days of gestation were designated as being in early pregnancy, and those of 1 I to 12 days of gestation were designated as being in late pregnancy. In some experiments, gestation was divided into three periods: early (0 to 6 days of gestation), middle (7 to 13 days of gestation), and late (14 days of gestation). Vesicular stomatitis virus (Indiana strain), herpes simplex virus type 2 (HSV-2, UW268 strain), poliovirus type 3, rubella virus, and adenovirus type 2 were used for antiviral activity assay including interferon assay. In inoculation studies, 33 nonpregnant mice, seven 335
336
Yamada et al.
October I, 1985 Am J Obstet Gynecol
100 90 80 • 70 II 60 111 50 ... 40 111 > 30 ...:;,> 20 III 10 ~
o
1 2 3 4 5 6 7 8 9 10 11 12 13 14 days post
inoculation
Fig.!. The survival rate of pregnant Balblc mice inoculated with HSV -2. Thirty-three nonpregnant mice (_ _ ). seven mice in early pregnancy (4 to 5 days of gestation, 0---0), and nine mice in late pregnancy (11 to 12 days of gestation, f'>--f'» were inoculated with HSV-2 (2 x 103 plaqueforming units per mouse) by intraperitoneal injection.
mice in early pregnancy, and nine mice in late pregnancy were inoculated with 2 x 10' plaque-forming units of HSV-2 in 200 f.Ll of phosphate-buffered saline by intraperitoneal injection. Continuous mouse fibroblasts (L929, C3H2KC), secondary mouse embryo cells (MEC), continuous human amniotic cells (WISH, FL), secondary human embryonic lung cells (HEL), continuous monkey kidney cells (GMK, CV-l), and continuous hamster kidney cells (BHK-21) were propagated by standard methods in growth medium. The growth medium consisted of Eagle's minimum essential medium containing 10% calf serum (Gibco, Ohio) penicillin (100 U/ml), and streptomycin (100 f.Lgl ml); maintenance medium for antiviral activity assay was similar except that 2% calf serum was used. Placentas were collected from mice on day 18 or 19 of gestation, washed with phosphate-buffered saline to remove blood, and homogenized in phosphate-buffered saline or minimum essential medium. The supernatant resulting from centrifugation at 100,000 x g for 60 minutes was harvested as a cytosol fraction and sterilized by passage through a 0.22 f.L microfilter. Antisera were antimouse a/~-interferon antiserum purchased from Enzo Biochemical, Inc. (New York) and antimouse 'V-interferon antiserum pr~pared by Professor H. M. Johnson (Department of Microbiology, University of Texas, Galveston, Texas). Trypsin was bovine pancreas type V purchased from Sigma Chemical Co. (St. Louis, Missouri). Interferon assay. The interferon assay was a conventional plaque-inhibition assay with L929 cells used as the indicator cells and vesicular stomatitis virus used
as the challenging virus. In our assay system, 1 U was equivalent to 1.5 IU (compared with National Institutes of Health standard interferon). Species specificity. Confluent cell cultures were incubated with 2% (w/v) placental extract for 24 hours at 37° C with 5% carbon dioxide. After the supernatants were removed, the cell cultures were challenged with vesicular stomatitis virus for 60 minutes and the incubation was continued until cytopathic effect was visible in control cells (approximately 24 hours). Cytopathic effect was expressed as follows: -. cytopathic effect not observed; ±, cytopathic effect suspected; +, obvious cytopathic effect observed in some . part; + +, cytopathic effect observed in almost all of culture; + + +, all cells lost owing to cytopathic effect. Virus spectrum. Monolayer cell cultures in capped test tubes were incubated with 2% (w/v) placental extract for 24 hours at 37° C and challenged with viruses for 90 minutes. Washed with serum-free minimum essential medium three times, cell cultures were incubated with 1 ml of minimum essential medium or minimum essential medium containing 2% (w/v) placental extract until cytopathic effect was visible in control cells. In the rubella virus assay, serum-free minimum essential medium was used in all procedures. Expressions of cytopathic effect were the same as those in the species specificity assay. Mode of action Experiment with vesicular stomatitis virus and L929. Confluent monolayer cultures of L929 were incubated with minimum essential medium or minimum essential medium containing 2% (w/v) placental extract for 24 hours at 37° C in 5% carbon dioxide. After removal of the
Interferon production during pregnancy in mice
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• •• 0
A
•• •• <40 lGt.m=~.a.1I 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 days post inoculation Fig. 2. Systemic interferon (IFN) production by pregnant Balblc mice inoculated with HSV-2. Nonpregnant mice (e), mice in early pregnancy (0), and mice in late pregnancy (t.) were inoculated with HSV-2 (2 x 10' pfu per mouse) by intraperitoneal iqjection, and serum interferon titers were assayed by a 50% plaque-reduction assay with the use of L929 and vesicular stomatitis virus.
E
::::>
>.
> u
ro
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-
160
40
I
c
ro <40
•
••
80
..
early ( 3days)
•
IU
• middle
ell
(10days)
la te (16-19days)
stage
Fig. 3. Interferon-like antiviral activity in sera of noninoculated pregnant mice. Pregnant Balblc mice 9 to 12 weeks old were put to death on day 3 (early), 10 (middle), or 16 to 19 (late) of gestation. The sera were assayed for antiviral activity by a 50% plaque-reduction assay with the use of L929 and vesicular stomatitis virus.
incubating medium, vesicular stomatitis virus in minimum essential medium or in minimum essential medium containing 2% (w/v) placental extract was added to the cell cultures and incubated for 90 minutes for adsorption. The supernatants were removed and minimum essential medium or minimum essential medium containing 2% (w/v) placental extract was added to the cultures and the incubation was continued until cytopathic effect was visible in control cells (approximately 24 hours). The culture media were harvested for virus yield assay and the plaques were counted. After serial tenfold dilutions of the harvested culture media, 1 ml was added to monolayer L929 cells. Cell cultures were incubated until cytopathic effect was visible (approximately 24 hours). Virus yields were calculated as a re-
ciprocal of the highest dilution of the supernatants that caused a cytopathic effect on the cells. Experiment with HSV-2 and WISH cells. Confluent monolayer cultures of WISH cells in capped test tubes were incubated with minimum essential medium or minimum essential medium containing 2% (w/v) placental extract for 24 hours at 37 0 C. Then WISH cells were challenged with HSV-2 for 60 minutes. Infected cells were cultured with minimum essential medium or minimum essential medium containing 2% (w/v) placental extract until cytopathic effect was visible in control cells (approximately 72 hours). When cytopathic effect was visible in control cells, cells were frozen and thawed three times rapidly. After serial tenfold dilutions of the supernatant ofa centrifugation at 1500 x g
338 Yamada et al.
October I, 1985 Am J Obstet Gynecol
Table I. Neutralizing test of antiviral activity in sera of noninoculated pregnant mice by anti mouse interferon antisera
Table III. Neutralizing test of placental antiviral substance by antimouse interferon antisera
Treatment Minimum essential medium (Ulml)
Sample
Pregnant mouse serum Mouse ~-interferon Mouse 'I-interferon
Antimouse
0.1 {3-interferon antiserum (Ulml)
Treatment Antimouse 'I-interferon antiserum (Ulml)
32
16
32
1024 32
<4 ND
ND <4
The samples were treated with antisera for 90 minutes at 37° C. The antiviral activity was measured by interferon assay after treatment. ND = Not done.
Treatment
Titer (U/ml)
256
16
Placental extract Experiment 1 Experiment 2 Mouse ~-interferon (experiment I) Mouse 'I-interferon (experiment 2)
Antimouse antiserum (Ulml)
Antimouse 'I-interferon antiserum (Ulml)
64 128 128
32 ND <4
ND 64 ND
128
ND
<4
0.1 {3-interferon
The samples were treated with antiserum for 90 minutes at 37° C. The antiviral activity was assayed by interferon assay after treatment. ND = Not done.
Table II. Stability of placental antiviral substance in various conditions
None
Sample
Minimum essential medium (Ulml)
56° C, 30 min
Trypsin*
4
16
The figures show antiviral activity in units per milliliter assayed by 50% plaque-reduction assay with the use of L929 and vesicular stomatitis virus after treatment. *For 30 minutes at 37° C in 0.2% trypsin.
for 30 minutes, the supernatant was added to monolayer cultures of BHK cells. The cells were incubated until cytopathic effect was visible (approximately 96 hours) and virus yields were calculated as a reciprocal of the highest dilution of the supernatant that caused cytopathic effect on the BHK cells. Gel filtration. A total of 5 ml of 40% (w/v) placental extract in phosphate-buffered saline was applied to a 2.6 by 60 cm column of Sephacryl S-300 (Pharmacia, Vppsala, Sweden). The sample was eluted with phosphate-buffered saline at a flow rate of 5.7 cm/hr. Fractions of 3 mt were collected and assayed for their antiviral activity by the interferon assay. In order to estimate the molecular weight of placental antiviral substance, marker proteins, that is, egg albumin (molecular weight 45,000), catalase (molecular weight 240,000), and ferritin (molecular weight 450,000), were used.
Results Susceptibility of pregnant mice to HSV-2 infection and systemic interferon production. Pregnancy seemed to increase the susceptibility of mice to HSV-2 infection, that is, all pregnant mice died by 13 days after virus inoculation, while 18.2% (six of 33) of nonpregnant mice survived more than 14 days after virus
Table IV. Species specificity of placental antiviral substance Cytopathic effect Cell
Mouse L929 C3H2KC MEC Human WISH FL HEL Monkey GMK CV-I Hamster, BHK-21
Placental extract
Control
+++ + + ++ +++
+++ +++ +++
+
++ ++
+++
+++
The results are expressed as extent of cytopathic effect formed on the cells of various species that were treated with 2% (w/v) placental extract before vesicular stomatitis virus challenge (see text for explanation).
inoculation (Fig. 1). However, the differences in mortality rates between the three groups were not statistically significant. The interferon titers in sera of HSV-2-inoculated nonpregnant mice were 40 to 160 Vlml on the fourth day after inoculation. In mice in early pregnancy, the postinoculation interferon titers in sera were 40 to 320 Vlml from day 2 to day 4. In mice in late pregnancy, very high systemic interferon production was observed; the titers of interferon in sera were 40 to 640 Vlml from day 1 to day 5 (Fig. 2). Antiviral activity (40 to 80 Vlml) was detected in the sera of about half the pregnant mice before virus inoculation, and the incidence of this antiviral activity increased as pregnancy advanced. It was detected in
Interferon production during pregnancy in mice
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339
Table V. Virus spectrum of placental antiviral substance Cytopathic effect
I postchallenge Prechallenge and I No treatment treatment
BHK-21 (hamster)
FL (huroon)
Virus
Prechallenge treatment
Vesicular stomatitis virus Polio virus Rubella virus HSV-2 Adenovirus
Prechallenge treatment
+ + + + +
± ±
I postchallenge Prechallenge and I treatment No treatment
+++
+
+++
± ±
±
+ + +
+
The results are expressed as extent of cytopathic effect of various viruses formed on host cells that were treated with 2%
(w/v) placental extract either prior to or both before and after virus challenge.
Table VI. Stage of action of placental antiviral substance Vesicular storootitis virus, L929 Plaque Stage of action
-24 hr Time 0 90 min -24 hr 90 min None
time 0 90 min 24 hr time 0 24 hr
No.
28 252 104 0
I
Inhibition (%)
Virus yield
Log'o inhibition virus yield
HSV-2, WISH: Virus yield
93.2 39.0 74.8 100
10' 10' 10' 102
2.0 1.0 1.0 3.0
>10' ND 10' 10
413
10'
> 10'
In the plaque-inhibition assay, the inhibition rate was calculated with the numbers of plaques formed by vesicular stomatitis virus on L929 cells treated with 2% (w/v) placental extract in various stages. In the virus yield-inhibition assay, the cells were treated with 2% (w/v) placental extract in various stages of virus replication and serial tenfold dilutions of the harvested culture media were added to assay cells. Virus yield was calculated as the highest dilution that caused cytopathic effect. ND = Not done.
61.5% (eight of 13) of the mice in late pregnancy (Fig. 3). This antiviral activity was not inactivated by antimouse u/l3-interferon antiserum or antimouse -y-interferon antiserum (Table I) . Characterization of placental antiviral substance. An antiviral activity (80 to 320 V im I) was detected in a 20% (w/v) suspension of placentas of noninfected mice by the interferon assay. Placental antiviral substance was inactivated by acid (pH 2, 24 hours), heat (56 0 C, 30 minutes), and trypsin treatment (Table II). The placental antiviral substance was not inactivated by antimouse u/l3-interferon-neutralizing antiserum or antimouse -y-interferon-neutralizing antiserum (Table III) . The activity of placental antiviral substance on vesicular stomatitis virus growth was evident in all mouse cells examined (L929, C3H2KC, and MEC) and also evident in the human FL cell, less evident in the human WISH cell, and not at all evident in the HEL cell. Activity was also evident in monkey cells CV-I and GMK but not in hamster cells (BHK) (Table IV). These results indicate the low species specificity of placental antiviral substance. Placental antiviral substance was effective against both the ribonucleic acid viruses and deoxyri-
.bonucleic acid viruses examined. In the experiment with FL used as a host cell, it was effective against ribonucleic acid viruses, that is, vesicular stomatitis virus, poliovirus, and rubella virus, and also effective against deoxyribonucleic acid viruses, that is, HSV-2 and adenovirus, with prechallenge treatment. In the experiment with the BHK cell used as the host cell, although placental antiviral substance was effective only against rubella virus and HSV -2 when prechallenge treatment alone was done, it was effective against vesicular stomatitis virus and adenovirus by addition of the postchallenge treatment (Table V). In the experiment to determine the stage of action for vesicular stomatitis virus and the L929 cell, placental antiviral substance was most effective when both prechallenge and postchallenge treatment was done; prechallenge treatment only was next in both the plaquereduction assay and the virus yield-inhibition assay. In the virus yield-inhibition assay with the use of HSV-2 and the WISH cell, although placental antiviral substance was most effective when both prechallenge and postchallenge treatment was done, postchallenge treatment only was next, and prechallenge treatment only was ineffective (Table VI) .
340
Yamada et al.
October 1, 1985 Am J Obstet Gynecol
blue ferritin catalase dextran (450000) (240000) 2.0
~
l
~
egg albumin (45000)
J
•
32
1.5
~ I
<
0
16 :;.
co
N
III
III
1.0
8 oIII
0
0
0.5
<
4 ;:;: '<
0 40
50
60
70 80 fraction
90 100 number
110
120
130
Fig. 4. Gel filtration of murine placental extract with the use of Sephacryl S-300. An amount of 5 ml of 40% (w/v) placental extract was applied to a 2.6 by 60 cm column, eluted with phosphatebuffered saline at a flow rate of 5.7 cm/hr, and fractions of 3 ml were collected and measured by interferon assay.
The molecular weight of placental antiviral substance was estimated to be 200,000 to 450,000 daltons by gel filtration with Sephacryl S-300 (Fig. 4).
Comment The changes in immunologic responses during pregnancy can be divided into two types, suppressive and enhancing responses. The suppressions are of mixed leukocyte reaction,' natural killer cell activity; and blastogenesis oflymphocytes by lectins in vitro.' On the other hand, the enhancements are of the phagocytic and bactericidal activities of neutrophils and macrophages:' 5 the antibody response to specific antigens,6 the complement system,7 the interferon response after virus inoculation, and antiviral activity in serum without any inducing stimuli. The suppressive responses are genetically highly developed immunologic responses in which there is individual recognition of "self" and "notself"; the enhancing responses are genetically primitive nonspecific immunities except for the antibody response. It seems to be very reasonable that the specific responses are suppressed during pregnancy so that the pregnant woman can accept the antigenically allogeneic fetus. The weakened specific immunity is compensated by enhancement of nonspecific immunity. Although the mechanism governing the high systemic interferon response remains to be investigated, the increase in protein synthesis in cells that results from the raised serum estrogen level during pregnancy might partially account for it. In the present work we detected antiviral activity in more than half the sera (61.5%) of mice in late pregnancy without any induction. It is unlikely that the antiviral activity in serum is the result of interferon pro-
duced by a latent viral infection activated during pregnancy because it was not neutralized by anti-interferon sera. The origin ofthe antiviral activity is not clear, and the possibility also cannot be discarded that it is the same substance as placental antiviral substance. In humans, our colleague Hamazaki '3 has reported that the interferon-like antiviral activity was detected in the sera of pregnant women. It was detected in 59% (27 of 46) of pregnant women in the third trimester. It is not clear why some pregnant women had antiviral activity in the sera and others did not or why it appeared with about the same incidence in syngeneically mated mice and in allogeneically coupled humans. However, the antiviral activity in serum is probably related to pregnancy for it was detected in none of the sera of nonpregnant women and the incidence increased as pregnancy advanced in both the mouse and the human. With reference to the antiviral substance in the placenta, Fowler et al. lO have reported that the antiviral substance detected in the murine placenta was interferon, but the results of our experiments suggest that placental antiviral substance is different from interferon,judging from its unusual antigenicity, low species specificity, unusual stage at action, and high molecular weight. Regarding antiviral substances related to fetoplacental tissues, the "virus inhibitor" spontaneously produced by many types of cells, including mouse embryo cells in culture, has also been reported. 14 Although the "virus inhibitor" is similar to placental antiviral substance in terms of low species specificity, its molecular weight was estimated to be no higher than that of a middle-sized protein while that of placental antiviral substance was estimated as 200,000 to 450,000 daltons and the inhibition of the "virus inhibitor" required the
Volume 153 Number 3
simultaneous presence of inhibitor, virus, and cells, while the inhibition of placental antiviral substance did not. Placental antiviral substance is interesting in view of its high molecular weight, for Duc-Goiran et al. 15 reported on the high-molecular weight interferons produced by human amniotic membrane with the induction of Sendai virus. They showed that the highmolecular weight interferons did not have strict species specificity; some species of these high-molecular weight interferons were effective in bovine MDBK cells as well as in human cells, while other species were effective in only human cells. Although placental antiviral substance has not been clearly characterized by the research done so far, the available evidence suggests the possibility that it may be an antigenically immature, primitive interferon. It is necessary to further purify this substance and prepare antiserum raised to placental antiviral substance in order to clarify whether it is synthesized spontaneously by fetoplacental tissues including trophoblasts, like human chorionic gonadotropin and steroid hormones, or whether it is produced by immunologically competent cells with some inducing stimuli. Further study is also needed to reveal how placental antiviral substance might work to prevent intrauterine viral infection of the fetus. We are grateful to Miss Midori Takanashi for preparation of the figures. REFERENCES I. Kasakura S. A factor in maternal plasma during pregnancy that suppresses the reactivity of mixed leukocyte cultures. j Immunol 1971; 107: 1296. 2. Okamura K, Furukawa K, Nakakuki M, Yamada K, Suzuki M. Natural killer cell activity during pregnancy. AM j OBSTET GYNECOL 1984; 149:396.
Interferon production during pregnancy in mice
341
3. Finn R, St Hill CA, Govan Aj, Ralfs IG, Gurney Fj, Denye V. Immunological responses in pregnancy and survival of fetal homograft. Br Medj 1972;15:150. 4. Mitchell GW jr, McRipley Rj, Selvaraj Rj, Sbarra AJ. The role of the phagocyte in host-parasite interactions. IV. The phagocytic activity of leukocytes in pregnancy and its relationship to urinary tract infection. AM j OBSTET GyNECOL 1966;96:687. 5. Mitchell GW jr, jacobs AA, Haddad V, Paul BB, Strauss RR, and Sbarra AJ. The role of the phagocyte in hostparasite interactions. XXV. Metabolic and bactericidal activities ofleukocytes from pregnant women. AM j OBSTET GYNECOL 1970; 108:805. 6. Tedder RS, Nelson M, Eisen V. Effects on serum complement of normal and pre-eclamptic pregnancy and of oral contraceptives. Br j Pathol 1975;56:389. 7. Fabris N, Serri F. Immunological reactivity during pregnancy in the mouse. In: Cantaro A, Carreti N, eds. Immunology in obstetrics and gynecology. New York: Elsevier, 1974:183. 8. Kouttab NM, Fowler AK, Strickland jE, Hellman A. Suppression of in vitro lymphocyte stimulation in mice by uterine and placental extracts. j Immunol1976; 177: 1644. 9. Barg M, Burton RC, Smith jA, Luckenbach GA, Decker j, Mitchell GF. Effects of placental tissue on immunological responses. Clin Exp Immunol 1978;34:441. 10. Fowler AK, Reed CD, Giron DJ. Identification of an interferon in murine placentas. Nature 1980;286:266. II. Lebon P, Girard S, Thepot F, Chany C. The presence of ex-interferon in human amniotic fluid. j Gen Virol 1982;59:393. 12. Cembrzynska-Nowak M. Physicochemical characteristics of IME-inhibitor of interferon activity from mouse embryo tissues. Arch Immunol Ther Exp 1977;25:663. 13. Hamazaki Y. Interferon-like substance in pregnancy sera. Acta Obstet Gynaecoljpn 1983;35:621. 14. Baron S, McKerlie L. Broadly active inhibitor of viruses spontaneously produced by many cell type culture. Infect Immun 1981;32:449. 15. Duc-Goiran P, Robert-Gailliot B, Chudzio T, Chany C. Unusual human interferons producted by virus-infected amniotic membranes. Proc Nat! Acad Sci USA 1983; 80:2628.