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Obstetrics & Gynecology 1999;94:279-283
© 1999 by The American College of Obstetricians and Gynecologists
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ORIGINAL RESEARCH

Apoptosis in Placentas From Human T-Lymphotropic Virus Type I–Seropositive Pregnant Women: A Possible Defense Mechanism Against Transmission From Mother to Fetus

TOSHINORI FUJINO, MD, ICHIRO IWAMOTO, MD, HIROFUMI OTSUKA, MD, TOSHIRO IKEDA, MD, SHUNJI TAKESAKO, MD and YUKIHIRO NAGATA, MD

From the School of Health Sciences and the Department of Obstetrics and Gynecology, Faculty of Medicine, Kagoshima University, Kagoshima, Japan.

Address reprint requests to: Toshinori Fujino, MD School of Health Sciences Kagoshima University 8-35-1 Sakuragaoka Kagoshima, 890-8506 Japan E-mail: toshinet{at}health.nop.kagoshima-u-ac-jp


   Abstract
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Abstract
Materials and Methods
 Results
 Discussion
 References
 
Objective: The mechanism by which the placenta serves as the barrier against mother-to-fetus transmission of microorganisms remains to be elucidated. Programmed cell death, apoptosis, is considered a cellular defense mechanism against infection. The hypothesis of this study is that apoptosis of human T-lymphotropic virus type I (HTLV-I)–infected placental villous cells is involved in the defense mechanism against mother-to-fetus transmission of HTLV-I.

Methods: Apoptosis was compared in term placentas from eight HTLV-I–seropositive pregnant women and eight HTLV-I–seronegative pregnant women by the terminal deoxynucleotidyl transferase-mediated deoxyuridine nick end-labeling method. In addition, an in vitro cocultivation with an HTLV-I–infected lymphocyte cell line (MT-2 cells) was performed to examine whether placental villous cells were infected with HTLV-I and apoptosis was induced.

Results: The incidence of apoptosis-positive cells (nuclei) in placentas from the HTLV-I–seropositive pregnant women was higher than in the HTLV-I–seronegative pregnant women (P < .02). Cocultivation with MT-2 cells showed that trophoblast cells were able to be infected with HTLV-I and that apoptosis was induced in the placental villous cells.

Conclusion: HTLV-I infection induces apoptosis in the placenta. We speculate that apoptosis may be involved in the defense mechanism of the placenta against mother-to-fetus transmission of HTLV-I.

The barrier system at the materno-fetal interface against mother-to-fetus viral transmission is considered to consist mainly of two factors: maternal neutralizing antibodies, and the infection-preventive barrier system in the placenta.1 It is well demonstrated that the maternal neutralizing antibodies have preventive activities against mother-to-fetus transmission of viruses of many types. If the virus infects the placenta despite maternal neutralizing antibodies, the placenta itself has to exert its defense mechanism to protect the fetus from infection. However, almost nothing has been demonstrated about the mechanism of the placental barrier system except that macrophages in the placenta, called Hofbauer cells, possessing phagocytic capacity,2 may play roles in the barrier system.

Human T-lymphotropic virus type I (HTLV-I),3 a causative agent of adult T-cell leukemia, is known to be transmitted vertically from mothers to children, and the main route of mother-to-child transmission is postnatal breast-feeding.4 Intrauterine HTLV-I transmission is reported to be rare.5 Of 115 cord blood samples from HTLV-I–seropositive pregnant women, none was positive for HTLV-I antigen, and only five cases (4.3%) were positive for HTLV-I proviral genome.6 On the other hand, HTLV-I can infect the placenta. We reported previously, by testing HTLV-I antigen and HTLV-I proviral genome in cultured placental villous cells, that two of nine placentas from HTLV-I–seropositive mothers were infected with HTLV-I ( Fujino T, Fujiyoshi T, Yashiki S, Sonoda S, Otsuka H, Nagata Y. HTLV-I transmission from mother to fetus via placenta. Lancet 1992;340:1157). This difference in HTLV-I infection rates between placentas and fetuses suggests that the infection-preventive barrier system in the placenta operates to prevent fetal infection when the placenta is infected with HTLV-I despite maternal neutralizing antibodies.

Programmed cell death, or apoptosis, is a process whereby developmental or environmental stimuli activate a genetic program to cause the death and efficient disposal of a cell. If apoptosis is induced in virus-infected cells, and they are cleared rapidly without further proliferation and spread of pathogens, this can be regarded as a defense mechanism. Reports have been accumulating that suggest that apoptosis is involved in defense mechanism against infection.7–9 Recently, Fratazzi et al10 gave evidence that programmed cell death of Mycobacterium avium–infected human macrophages was an important defense mechanism, preventing the spread of infection by sequestering the mycobacteria and contributing to their demise by activation of newly recruited uninfected macrophages.

We hypothesized that apoptosis of HTLV-I–infected placental villous cells might be involved in the defense mechanism of the placenta against mother-to-fetus transmission of HTLV-I. To raise the hypothesis in the present study, we examined apoptosis in placentas from HTLV-I–seropositive pregnant women. Placental sections were tested to compare the incidence of apoptosis-positive cells between HTLV-I–seropositive and HTLV-I–seronegative pregnant women. In addition, an in vitro experiment was performed to examine whether placental villous cells were infected with HTLV-I and apoptosis was induced in the placental villous cells by cocultivation with an HTLV-I–infected cell line.


   Materials and Methods
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Abstract
 Materials and Methods
Results
 Discussion
 References
 
Term placentas were obtained from eight HTLV-I–seropositive11 and eight HTLV-I–seronegative Japanese pregnant women without any obstetric complications after normal vaginal delivery at Kagoshima University Hospital between January 1997 and January 1998. Informed consent was obtained. All the pregnant women were human immunodeficiency virus–seronegative. None of the pregnant women had ruptured membranes for longer than 24 hours. Maternal ages, gestational weeks at delivery, placenta weight, and birth weight of infants were matched between the HTLV-I–seropositive and –seronegative mothers (Table 1Go). All infants born were classified appropriately for dates.


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  		Table 1. Maternal, Placental, and Infant Characteristics
 
Placental tissue was obtained from the central cotyledon, cut into small pieces, and fixed in buffered formalin. The specimens were sampled at random and formed into three different paraffin-embedded blocks; 4-µm sections were cut and mounted on microscope slides. Five sections were obtained randomly from the three different blocks of tissue. The placental sections were tested for apoptosis.

Apoptosis in the placentas was examined by the terminal deoxynucleotidyl transferase–mediated deoxyuridine nick end-labeling method (TUNEL)12 using a commercial kit (ApopTag Plus Peroxidase; Oncor, Gaithersberg, MD) according to the instruction manual of the manufacturer. A negative control (without terminal deoxynucleotidyl transferase) and a positive control (specimen supplied in the commercial kit) also were run. Placental sections were examined under light microscopy. Apotosis-positive cells (nuclei) were detected easily because they labeled brown, compared with apoptosis-negative nuclei that were green (methylgreen counterstain). Apoptosis-positive nuclei in the placental villi, but not nuclei in the intervillous space, were counted. For each placenta, 500 microscopic fields were examined (100 fields from each section at a magnification of x200). Approximately 10,000–15,000 nuclei were counted for each placenta. The number of apoptosis-positive nuclei per 1000 nuclei examined was calculated for each placenta. All counts were performed by a single blinded observer. Intraobserver error was less than 5%.

To examine whether placental villous cells could be infected with HTLV-I, and whether apoptosis was induced in the placental villous cells by cocultivation with HTLV-I–infected cells, the following in vitro experiment was performed. Placental villous cell suspensions of term placentas from HTLV-I–seronegative pregnant women were obtained by the methods of Kliman et al13 and Douglas and King14 with slight modifications. In brief, placental villous tissue was digested by collagenase/disperse (Boehringer Mannheim GmbH Biochemica, Mannheim, Germany) (1.7 mg/mL) and trypsin. After Percoll gradient centrifugation, the cells at density between 1.035 and 1.074 were collected. The cells positioned between these densities consisted of trophoblast cells, fibroblasts, macrophages, and endothelial cells. The presence of each type of cell was confirmed by immunostaining. The placental villous cells that included all these cell types were plated out (3 x 105/mL) on coverslips (25 mm in diameter, 1 x 105 cells/coverslip) in dishes and cultured in the RPMI 1640 medium supplemented with 10% fetal calf serum for 2 days.

After removing dead cells, the placental villous cells were cocultivated with MT-2 cells15 (an HTLV-I–infected cell line, 1 x 105/coverslip) in the RPMI 1640 medium supplemented with 10% fetal calf serum for 48 hours. Human T-lymphotropic virus type I infection and apoptosis of the placental villous cells were examined at 9, 18, 24, 32, and 48 hours of cocultivation. Control cultures were run without MT-2 cells or with K 562 cells (an erythroleukemia cell line, 1 x 105/cover-slip). After each incubation period, the coverslips were washed throughly with phosphate-buffered saline seven times to remove MT-2 cells or K 562 cells completely, and the placental villous cells on the coverslips were fixed with cold methanol for 20 minutes. The experiment of the cocultivation with MT-2 cells or K-562 cells and placental villous cells cultured alone was repeated five times using different placentas from HTLV-I–seronegative pregnant women.

The presence of HTLV-I–infected trophoblast cells was examined by double immunostaining using two monoclonal antibodies: GIN-14,16 against HTLV-I p19 protein, and CAM 5.2,17 which is reactive with cytokeratin. Immunostaining for negative control was run using an irrelevant monoclonal antibody. Three randomly selected coverslips were tested for double immunostaining for each incubation time in each incubation experiment.

Apoptosis of the placental villous cells was examined by the same TUNEL method described for the placental sections. Three coverslips selected at random were tested for each incubation time again in each experiment. The number of apoptosis-positive placental villous cells (nuclei) was counted per 10,000 cells. All counts were made by the single-blinded observer. Comparison was made between the cocultivation with MT-2 cells and placental villous cells alone.

To examine the presence of apoptosis in the HTLV-I–infected placental villous cells, double staining was performed; after immunostaining using GIN-14 (to test HTLV-I infection), apoptosis of the HTLV-I–infected placental villous cells (GIN-14–reactive placental villous cells) was tested by the TUNEL method. Twenty HTLV-I–infected placental villous cells were tested for apoptosis.

Statistical analysis was carried out on a Power Macintosh (Apple Computer, Inc., Cupertino, CA) personal computer with the statistics software package Statview 4.2 (Abacus Concepts, Berkeley, CA). Because all data were considered nonparametric, the Mann-Whitney U test was used. Statistical significance was set at P < .05.


   Results
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Abstract
 Materials and Methods
 Results
Discussion
 References
 
Apoptosis was detected in the placental sections from both the HTLV-I–seropositive and –seronegative pregnant women. Apoptosis was observed near fibrin deposition (Figure 1Go), in the syncytiotrophoblast layer, and in stromal cells in the placental villi (Figure 2Go). Most apoptosis-positive nuclei were observed at the outer surface of the villi. When the number of apoptosis-positive nuclei per 1000 nuclei of the placentas was compared, apoptosis-positive nuclei in the placentas from the HTLV-I–seropositive pregnant women were significantly higher than in those from the HTLV-I–seronegative pregnant women (P < .02, Mann-Whitney U test) (Figure 3Go). In the placentas from HTLV-I–seropositive pregnant women, most apoptosis-positive nuclei were detected in cluster. None of the placentas tested for apoptosis had obvious chorioamnionitis or severe infiltration of polymorphonuclear leukocytes in villi or intervillous space.



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  		Figure 1. Apoptosis-positive nuclei (arrow) near fibrin deposition (original magnification: x200).

 


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  		Figure 2. Apoptosis-positive nuclei in the syncytiotrophoblast (large arrow) and stroma (small arrow) (original magnification: x200).

 


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  		Figure 3. Comparison of the incidence of apoptosis-positive nuclei. The medians are shown by horizontal bars. HTLV-I = human T-lymphotropic virus type I.

 
By cocultivation of placental villous cells with MT-2 cells (HTLV-I–infected cell line), GIN-14– and CAM 5.2–double-reactive cells, that is, HTLV-I–infected and cytokeratin-positive cells, were found in samples of cocultivation of 24, 32, and 48 hours, but not in 9- or 18-hour incubation time (Figure 4Go). GIN-14–reactive cells were stained brown, and CAM 5.2–reactive cells were stained gray-purple. Because it had been tested that trophoblast cells, but not MT-2 cells, macrophages, fibroblasts, or endothelial cells in villi reacted with CAM 5.2, the GIN-14– and CAM 5.2–double-reactive cells were considered to be HTLV-I–infected trophoblast cells. Therefore, it was shown that HTLV-I was able to infect trophoblast cells. However, the number of the HTLV-I–infected trophoblast cells was very small (one in 10,000 placental villous cells).



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  		Figure 4. A CAM 5.2– and GIN-14–double-reactive cell (an HTLV-I–infected trophoblast cell) (arrow) (original magnification: x200).

 
Apoptosis-positive placental villous cells (nuclei) (Figure 5Go) were detected in cultures both with MT-2 cells and without MT-2 cells. However, the number of apoptosis-positive placental villous cells (nuclei) per 10,000 cells (nuclei) that were cocultivated with MT-2 cells was higher than the cultures of placental villous cells alone; the difference reached statistical significance in the cocultivation of 24 hours and 48 hours (P < .02, Mann-Whitney U test) (Figure 6Go). The number of apoptosis-positive placental villous cells (nuclei) cocultivated with K 562 cells was comparable to the placental villous cells cultured alone.



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  		Figure 5. An apoptosis-positive placental villous cell (nucleus) (arrow) (original magnification: x200).

 


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  		Figure 6. Comparison of the incidence of apoptosis-positive placental villous cells. The medians are shown by the horizontal bars. HTLV-I = human T-lymphotropic virus type I.

 
When the 20 GIN-14–reactive (HTLV-I–infected) placental villous cells were tested for apoptosis by double-staining (immunostaining and TUNEL method), no double-positive (HTLV-I–infected and apoptosis-positive) placental villous cells were found.


   Discussion
Top
Abstract
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we compared the incidence of apoptosis in placentas between HTLV-I–seropositive and HTLV-I–seronegative pregnant women by examining placental sections. Apoptosis was detected in the placentas from both HTLV-I–seropositive and –seronegative pregnant women. The incidence of apoptosis in the placentas from HTLV-I–seronegative pregnant women was comparable to those that were reported previously.18 The number of apoptosis-positive cells (nuclei) in the placentas from the HTLV-I–seropositive pregnant women was significantly higher than that from the HTLV-I–seronegative pregnant women.

In the in vitro experiment of the present study, in which placental villous cells were cocultivated with an HTLV-I–infected cell line (MT-2 cells), it was found that trophoblast cells were able to be infected by HTLV-I and that apoptosis was really induced in the placental villous cells. Apoptosis is induced by cytokines.8 To examine whether the apoptosis that was induced in the placental villous cells by cocultivating with MT-2 cells was due to cytokines that were released in the culture medium, placental villous cells were cocultivated with K 562 cells, an erythroleukemia cell line, instead of MT-2 cells. Apoptosis in the placental villous cells was not induced by cocultivation with K 562 cells, suggesting that it was contact with HTLV-I–infected cells (MT-2 cells) and subsequent infection with HTLV-I, not cytokines alone, that induced apoptosis of the placental villous cells.

In the in vitro experiment of cocultivation of placental villous cells with MT-2 cells, the number of HTLV-I–infected trophoblast cells (trophoblast cells expressing HTLV-I antigen on their cell surface) was very small, and placental villous cells that were double-positive for HTLV-I antigen and apoptosis were not detected. It may be that if trophoblast cells are infected with HTLV-I, apoptosis will occur in most of the infected cells and they will be quickly cleared before they produce HTLV-I antigen and express it on their cell surface. Among the HTLV-I–infected trophoblast cells, only those that have escaped from apoptosis express HTLV-I antigen on their cell surface.

Thus, this study showed that HTLV-I infection induces apoptosis in placental villous cells. We speculate that apoptosis may be involved in the placental barrier system against mother-to-fetus transmission of HTLV-I. Although apoptosis may be a coincidental phenomenon, this approach may be an important step to elucidate the defense mechanism of the placenta.


   Footnotes
 
Supported by Grant-in-Aid for Scientific Research 05671383 from the Ministry of Education, Science and Culture, Japan.

PII S0029-7844(99)00322-1

Received September 28, 1998. Received in revised form January 25, 1999. Accepted February 3, 1999.


   References
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Abstract
 Materials and Methods
 Results
 Discussion
 References
 
1. Fox H. The placenta and infection. In: Redman CWG, Sargent IL, Starky PM, eds. The human placenta. Oxford: Blackwell Scientific Publications, 1993:313–33.

2. Wood GW. Mononuclear phagocytes in the human placenta. Placenta 1980;1:113–23.[Medline]

3. Hinuma Y, Nagata K, Hanaoka M, Nakai M, Matsumoto T, Kinoshita K, et al. Adult T cell leukemia: Antigen in an ATL cell line and detection of antibodies to the antigen in human sera. Proc Natl Acad Sci U S A 1981;78:6476–80.[Abstract/Free Full Text]

4. Hino S, Yamaguchi K, Katamine S, Sugiyama H, Amagasaki T, Kinoshita K, et al. Mother-to-child transmission of human T-cell leukemia virus type I. Gann (Jpn J Cancer Res) 1985;76:474–80.

5. Kawase K, Katamine S, Moriuchi R, Miyamoto T, Kubota K, Igarashi H, et al. Maternal transmission of HTLV-I other than through breast milk: Discrepancy between the polymerase chain reaction positivity of cord blood samples for HTLV-I and the subsequent seropositivity of individuals. Jpn J Cancer Res 1992;83: 968–77.[Medline]

6. Saito S, Ichijo M. Detection of HTLV-I sequence in infants born to HTLV-I carrier mothers by polymerase chain reaction. Gann (Jpn J Cancer Res) 1992;39:175–85.

7. Steller H. Mechanisms and genes of cellular suicide. Science 1995;267:1445–9.[Abstract/Free Full Text]

8. Levine B, Huang Q, Isaacs JT, Reed JC, Griffin DE, Hardwick JM. Conversion of lytic to persistent alphavirus infection by the bcl-2 cellular oncogene. Nature 1993;361:739–42.[Medline]

9. Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science 1995;267:1456–62.[Abstract/Free Full Text]

10. Fratazzi C, Arbeit RD, Carini C, Remold HG. Programmed cell death of Mycobacterium avium serovar 4-infected human macrophages prevents the mycobacteria from spreading and induces mycobacterial growth inhibition by freshly added, uninfected macrophages. J Immunol 1997;158:4320–7.[Abstract]

11. Fujiyama C, Fujiyoshi T, Matsumoto D, Tamashiro H, Sonoda S. Evaluation of commercial HTLV-I test kits by a standard HTLV-I serum panel. Bull World Health Organ 1995;73:515–21.[Medline]

12. Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labelling of nuclear DNA fragmentation. J Cell Biol 1992;119:493–501.[Abstract/Free Full Text]

13. Kliman HJ, Nestler JE, Sermasi E, Sanger JM, Strauss III JF. Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae. Endocrinology 1986; 118:1567–82.[Abstract]

14. Douglas GC, King BF. Isolation of pure villous cytotrophoblast from term human placenta using immunomagnetic microspheres. J Immunol Methods 1989;119:259–68.[Medline]

15. Miyoshi Y, Kubonishi I, Yoshimoto S, Shiraishi Y. A T-cell line derived from normal human cord leukocytes by co-culturing with human leukemic T-cells. Gann (Jpn J Cancer Res) 1981;72:978–91.

16. Tanaka Y, Koyanagi Y, Chosa T, Yamamoto N, Hinuma Y. Monoclonal antibody reactive with both p28 and p19 of adult T cell leukemia virus-specific polypeptides. Gann (Jpn J Cancer Res) 1983;74:327–30.

17. Makin C, Borow L, Bodmer W. Monoclonal antibody to cytokeratin for use in routine histopathology. J Clin Pathol 1984;37:975–83.[Abstract/Free Full Text]

18. Smith SC, Baker PN, Symonds EM. Placental apoptosis in normal human pregnancy. Am J Obstet Gynecol 1997;177:57–65.[Medline]





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