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Biology of Reproduction 62, 178-185 (2000)
©Copyright 2000 Society for the Study of Reproduction, Inc.

Article

Induction of Apoptosis in Placentas of Pregnant Mice Exposedto Lipopolysaccharides: Possible Involvement of Fas/Fas Ligand System1

Kuniaki Ejimaa,b, Takehiko Koji2,c, Daisuke Tsurutad, Hiroki Nanria, Masamichi Kashimurab, and Masaharu Ikedaa

a Departments of Health Development and b Obstetrics and Gynecology, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan c Department of Histology and Cell Biology, Nagasaki University School of Medicine, Nagasaki 852-8523, Japan d Department of Dermatology, Osaka City University Medical School, Osaka 545-8585, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To explore the pathogenesis in placental dysfunction and abruptio placentae, we analyzed the occurrence of placental cell apoptosis and the role of Fas and Fas ligand (L) in that process in an inflammatory placental dysfunction model of pregnant mice, using lipopolysaccharides (LPS). In the present study, Day 13 pregnant mice were injected i.p. with LPS (50 µg/kg) or saline as a control, and the placentas were isolated at various time points after the injection. Analysis of the isolated DNA in agarose-gel electrophoresis revealed a typical ladder pattern of bands consisting of 180–200 base pairs (bp), which is regarded as a hallmark of apoptosis. The intensity of the bands increased time-dependently, reaching a maximum level at 12 h after LPS injection. In accord with the biochemical data, histochemical analysis using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) revealed that nuclei positive for double-stranded DNA breaks were found in decidua, diploid trophoblasts in the basal zone, and spongiotrophoblasts. The number of positive nuclei was maximized at 12 h after LPS injection. As a next step, we investigated the possible involvement of Fas and Fas L in the induction of apoptosis of the placental cells after LPS injection. Western blot analysis indicated that LPS increased the expression of Fas and Fas L in the placenta by about 4-fold at 12 h and 18 h, respectively, after injection. The cells expressing Fas and Fas L were identified, using immunohistochemistry and nonradioactive in situ hybridization, as decidua, diploid trophoblasts in the basal zone, and spongiotrophoblasts. Furthermore, when the expression of 4-hydroxy-2-nonenal (HNE)-modified proteins was assessed to evaluate the relation of oxidative stress elicited by LPS to the induction of apoptosis, once again decidua, diploid trophoblasts in the basal zone, and spongiotrophoblasts were positive. Therefore, the placental dysfunction by LPS may be brought about by the Fas-mediated apoptosis of various placental cells in a paracrine/autocrine fashion, possibly under the influence of oxidative stress.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Understanding the pathogenesis of placental dysfunction and abruptio placentae in the second trimester with inflammatory and preeclampsia is essential for saving fetuses. Although their pathophysiology is thought to be associated with an active cytokine network [1,2], our information on the precise mechanisms underlying the placental abnormality is only limited. To gain better insight into this issue, we analyzed a mouse model of inflammatory placental dysfunction, in which pregnant mice were administered LPS.

Recently, in a variety of tissue failures including tissue destruction, much attention has been paid to the study of the role of apoptosis, which has been described as active cell death from necrosis of a passive nature. A hallmark of apoptosis is the presence of apoptotic cell death that can be identified practically by a ladder formation of bands consisting of 180- to 200-bp multiples in agarose-gel electrophoresis of DNA [3]. Also, a histochemical approach such as terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) has turned out to be a convenient way to detect apoptotic nuclei with double-stranded DNA breaks [4]. However, the most fascinating aspect of apoptosis may be the requirement for a de novo activation of certain genes [5,6].

Fas (Apo-1, CD95) is a 45-kDa transmembranous glycoprotein that mediates apoptosis [7]. The expression of Fas has been detected in several organs such as liver [8], lung [8], ovary [9], vagina [10], testis [11], colon [12], and thyroid [13,14]. Fas L has been found in cytotoxic T-cell hybridoma as a natural ligand for Fas, and now the Fas/Fas L system is widely accepted as a major molecular mechanism to induce apoptosis in various normal [8] and pathological cells [12–15]. Although the Fas-Fas L system has recently also been thought to be involved in the induction of apoptosis in placenta [16–21], the relationship between the Fas-Fas L system and oxidative stress in placenta is still unknown.

On the other hand, oxidative stress, which generates reactive oxygen species (ROS), has been postulated to be involved in the pathophysiology of some complications such as infectious disease and preeclampsia during pregnancy, occasionally leading to placental dysfunction or abruptio placentae [22–27]. Although LPS induces the expression of a variety of cytokines [28,29] as well as the production of ROS [30], it has not been addressed whether LPS can induce the changes in the expression of Fas and Fas ligand in placenta. Interestingly, it is known that ROS itself is involved directly or indirectly in the induction of apoptosis [31–33].

Therefore, in the present study with an inflammatory placental dysfunction model of pregnant mice, we first investigated the occurrence of apoptotic cells temporarily after LPS injection by means of DNA agarose-gel electrophoresis and TUNEL. As the next step, the expression of Fas and Fas L was assessed by Western blotting, immunohistochemistry, and nonradioactive in situ hybridization with thymine-thymine (T-T) dimerized oligonucleotide probes. Furthermore, the sites of ROS generation were localized by immunostaining of HNE-modified proteins, in which HNE is a toxic aldehyde and an end product of unsaturated fatty acids subjected to oxidative stress.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

A mouse monoclonal antibody (IgG1) against thymine-thymine dimer (M anti T-T) was obtained from Kyowa Medex (Tokyo, Japan). Horseradish peroxidase (HRP)-labeled goat anti-mouse IgG was purchased from Medical Biological Laboratory (Nagoya, Japan). The following reagents were purchased: formamide from Nacarai Chemical Co. (Kyoto, Japan); DAB/4-HCl from Dojindo Chemical Co. (Kumamoto, Japan); proteinase K and WB DNA extractor kit from Wako Pure Chemical Industries (Osaka, Japan); LPS (from Escherichia coli; serotype 011: B4), bovine serum albumin (BSA), salmon testis DNA (phenol-chloroform extract), yeast tRNA (type X-S), dextran sulfate (Mr = 500 000), Brij 35, Triton X-100, heparin, normal mouse IgG, and normal goat IgG from Sigma Chemical Co. (St. Louis, MO); and anti-HNE monoclonal antibody from NOF Co. (Tokyo, Japan). Nitrocellulose filters were purchased from Schleicher and Schuell (Keene, NH). Polyvinylidene difluoride membranes were purchased from Immobilon, Millipore Corp. (Bedford, MA). Terminal deoxynucleotidyl transferase (TdT) and biotinylated 16-dUTP were purchased from Boehringer Mannheim (Mannheim, Germany). All other chemicals and biochemicals used were of analytical and molecular biological grade.

Tissue Preparation

Pregnant 13-day Slc:ICR mice weighing 30–50 g were purchased from Ohtsubo Experimental Animal Co. (Isahaya, Japan). For in vivo treatment with LPS, mice were i.p. injected with saline as a control (n = 3) or LPS from E. coli (50 µg/kg), which is reported to induce trophoblastic apoptosis and increased the serum level of various cytokines in mice [21]. The mice were sacrificed at 6, 12, and 18 h after the injection. For biochemical analysis, placentas were removed, quickly frozen in liquid nitrogen, and stored at -80°C until used. All the other specimens were briefly perfused with 4% paraformaldehyde in PBS (pH 7.4) and removed. After the tissues were cut into small pieces, they were further fixed in the same fixative overnight at room temperature (RT). Then the tissues were embedded in paraffin under standard conditions. Five-micrometer sections were cut and placed onto silane-coated glass slides. Unless otherwise specified, all procedures throughout the present study were conducted at RT.

Oligo-DNA Probes

To synthesize sense and antisense oligo-DNAs probes, parts of mRNA cording for mouse Fas [8] were selected as followed: 5'-ATAGATGAGATCATGCATGACAGCATCCAAGACACAGCTGAGCAG-3' (nucleotides 800–844) or 5'-AAGAGAGGAGAGAGCCTGCCACCCATGATGGAA-3' (nucleotides 1057–1089).

Furthermore, parts of mRNA coding for mouse Fas L [34] to synthesize oligo-DNAs probes were also selected as followed: 5'-GACCGCCACCTCCACCACCACCTGTGTCACCACTACCACCGCCAT-3' (nucleotides 311–355) or 5'-TGCACTACTGGACAGATATGGGCCCACAGCAGCTACCTGGGGGCA-3' (nucleotides 871–915). We conducted a computer-assisted search (GenBank release 103) of the oligo-DNA sequences, and no significant homology with any other published sequence was found.

In Situ Hybridization

The protocol for nonradioactive in situ hybridization has been detailed previously [35,36]. To confirm the specificity of Fas and Fas L mRNA signals, several types of control experiments were conducted. As a positive control to assess the extent of tissue RNA retention, complementary oligo-DNA to 28S ribosomal RNA was used [37]. A sense probe was used as a negative control in every run. As another negative control, some sections were digested with RNase-A (100 µg/ml, 37°C, 1 h) before the postfixation step. Furthermore, some sections were hybridized with Fas or Fas L antisense probes in the presence of an excess amount (50-fold) of the unlabeled corresponding antisense oligo-DNA to validate the sequence specificity of the signals.

Preparation of Polyclonal Antibodies

To generate antibodies, a synthetic oligopeptide sequence, P4, corresponding to the extracellular domain (P4; amino acids 292–306) of mouse Fas [38] was selected. To generate anti-Fas L antibody, a synthetic peptide corresponding to the intracellular domain (P5; amino acids 41–55) of rat Fas ligand [39] was selected. Antisera against Fas and Fas L synthetic oligopeptides were obtained according to the method described previously [9].

Immunoblot Analysis

Placentas were homogenized in lysis buffer (5 mM phosphate buffer, pH 7.2, containing 0.1% Triton X-100, 1 mM phenylmethylsulfonylfluoride, 1 µg/ml leupeptin, and 1 µg/ml chymostatin). The protein content of the preparation was determined by the method of Bradford [40], using BSA as a standard. Sample lysates were mixed with the loading buffer (final concentration, 62.5 mM 1, 4-dithiothreitol, 5% SDS, and 10% glycerol), boiled for 5 min, separated by SDS-PAGE with a 10–20% gel gradient according to Laemmli's method [41], and electrophoretically transferred onto polyvinylidene difluoride membranes. The membranes were blocked for 1 h in PBS with 5% skim milk, and incubated for 2 h with 1:800 rabbit anti-Fas (P4) and anti-Fas L (P5) sera, and then for 1 h with HRP-conjugated goat anti-rabbit IgG F(ab')2 as the second antibody. Bands were visualized with H2O2, 3,3'-diaminobenzidine/4-HCl (DAB; Wako Pure Chemicals, Osaka, Japan), Co2+, and Ni+ according to the method of Adams [42]. The values of fold-increases were determined by densitometric analysis of the stained bands from three independent experiments.

Immunohistochemistry

Fas, Fas L, and HNE-modified proteins were localized using the polyclonal antibodies described above. Immunohistochemistry was performed as reported previously [43]. As a control, some of the sections were subjected to reaction with normal rabbit serum in place of the specific antibody, and some were incubated with primary antibody in the presence of an excess amount (500 times the molar ratio) of the synthetic peptide, which was used to raise the antibody.

TUNEL Staining

To analyze DNA fragmentation in histological sections, TUNEL was carried out according to the method of Gavrieli et al. [4] with a slight modification [11], as described below. Briefly, nuclei of tissue sections were stripped of proteins by incubation with 5 µg/ml proteinase K for 15 min at 37°C, and the slides were then washed in PBS three times for 5 min each time. The sections were rinsed once with distilled deionized water and covered with TdT buffer (pH 6.6) alone for 30 min. Then, the TdT buffer containing 0.1 mM dithiothreitol, 1.5 mM cobalt chloride, 0.2 U/µl TdT, 20 µM dATP, and 10 µM biotinylated 16-dUTP were added to the sections, and the slides were incubated in a humidified atmosphere at 37°C for 60 min. The reaction was terminated by transferring the slides to 50 mM Tris-HCl, pH 7.4, for 15 min and then rinsing with PBS. Endogenous peroxidase was inactivated by immersing the sections in 0.3% H2O2 in methanol for 15 min and then washing three times with PBS for 5 min each time. After incubation with 5% BSA in PBS for 30 min to block nonspecific binding, the sections were incubated with HRP-goat anti-biotin (1:100) diluted with 5% BSA in PBS for 2 h and washed in PBS. The sites of HRP were visualized according to previous methods [42]. As all nuclei became a faint gray under the experimental conditions, only nuclei with strong black staining were regarded as TUNEL-positive. As a control, some of the sections were reacted with TTP in place of biotinylated 16-dUTP.

Electrophoretic Analysis of Extracted DNA

Total DNA was extracted from the frozen tissue samples using the WB DNA extractor kit. The concentration of DNA was measured spectrophotometrically at 260 nm. Aliquots of DNA samples (20 µg/lane) were separated on a 2% agarose gel and stained with ethidium bromide.

Statistical Analysis

TUNEL-positive cells were counted as apoptotic cells during examination of 1000 placental cells in each case, and the frequency was finally expressed as the number of apoptotic placental cells per 1000 placental cells (mean ± SD). Data were analyzed for significant differences using ANOVA. A P value of less than 0.05 denoted the presence of a statistically significant difference.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Electrophoretic Analysis of Extracted DNA

As a biochemical hallmark of apoptosis, the finding of a DNA ladder with multiple 180- to 200-bp bands in agarose gel-electrophoresis of DNA often has been examined [3]. To obtain biochemical evidence of the involvement of apoptosis in the dysfunction of LPS-treated placenta, we analyzed the extracted DNA for the presence or absence of the band laddering. As shown in Figure 1, a faint but visible ladder appeared at 6 h after LPS injection, and the intensity of the laddering bands reached a maximum level at 12 h after injection (Fig. 1).



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  		FIG. 1. Electrophoretic analysis of DNA extracted from placenta after LPS injection; 20 µg of DNA was applied to each lane. Lane 1, saline as control; lane 2, 6 h after LPS injection; lane 3, 12 h after LPS injection; lane 4, 18 h after LPS injection. M, Molecular marker.

Analysis of Apoptotic Cells by TUNEL Staining

To assess the temporal and spatial distribution of apoptotic cells with DNA strand breaks, we performed TUNEL staining using paraffin-embedded placental sections. As shown in Figure 2, a and b, there were only a few positive cells in the normal placenta. Six hours after LPS injection, however, TUNEL-positive cells were detected that were localized to the areas of decidua, diploid trophoblasts in the basal zone, and spongiotrophoblasts. The number of TUNEL-positive cells increased time-dependently in all those areas after LPS injection (Fig. 2, c–h) and the total number of TUNEL-positive cells reached the highest value in the placenta 12 h after LPS injection (Fig. 3). When normal placental sections were incubated with the reaction mixture omitting biotinylated 16-dUTP, no positive nuclei were found (data not shown). In this experiment, we confirmed that the number of TUNEL-positive cells after LPS injection increase in tandem with the appearance of the DNA ladder.



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  		FIG. 2. TUNEL staining in placentas after LPS treatment. a,b) Sections of normal mouse placenta without LPS exposure; c,d) 6 h after LPS exposure; e,f) 12 h after LPS injection; g,h) 18 h after LPS injection. a,c,e,g) x128, b,d,f,h) x640 (published at 53%). Arrows indicate TUNEL-positive cells. D, Decidua; DT, diploid trophoblast; ST, spongiotrophoblast



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  		FIG. 3. Quantitative analysis of TUNEL-positive cells in the placentas after LPS treatment for 6, 12, or 18 h (0 is saline treatment). The number of TUNEL-positive cells is expressed per 1000 trophoblastic cells. Values are expressed as mean ± SD. *P < 0.05, compared with placentas of mice injected with saline

Western Blot Analysis of Fas and Fas L

In the next series of experiments, we examined the expression of Fas and Fas L proteins in the placentas exposed to LPS by Western blotting with rabbit antisera raised against synthetic peptides of a part of mouse Fas and rat Fas L [38,39]. Figure 4 reveals that the injected LPS enhanced time-dependently the expression of Fas (45 kDa) by about 4-fold in the placentas 12 h after LPS injection (Fig. 4a), while Fas L (31 kDa) expression was gradually increased by about 4-fold up to 18 h after LPS injection.



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  		FIG. 4. Western blot analysis of the expression of Fas (a) and Fas L proteins (b) by LPS. Thirty micrograms of protein from placental cell lysates were electrophoresed, transferred to a membrane, and hybridized as described under Materials and Methods. Placenta exposed to LPS were prepared after 0, 6, 12, or 18 h (lanes 1–4, respectively). The reaction with specific polyclonal antibodies against Fas (a) and Fas L (b) (45 and 31 kDa, respectively) were detected in the placental lysates

Immunohistochemical Localization of Fas and Fas Lin the Placentas After LPS Injection

To identify positive cells for Fas and Fas L, we localized Fas and Fas L immunohistochemically in the paraffin sections at various time points after exposure to LPS [9]. As shown in Figure 5, staining for Fas was in the decidua and diploid trophoblasts in the basal zone of LPS-treated placentas, and the intensity and the number of positive cells were increased time-dependently after LPS injection, reaching a maximum level at 12 h after injection. In normal mouse placenta, essentially no positive cells were found. On the other hand, Fas L-positive cells were detected even in the normal mouse placenta, but their number gradually increased after LPS injection (Fig. 6). In the sections incubated with normal rabbit serum as a negative control, no staining was found (data not shown).



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  		FIG. 5. Immunohistochemical localization of Fas in the placenta. The sections of mouse placenta were subjected to reaction with anti-Fas antibody and counterstained with methyl green. a,b) Normal mouse placenta without LPS exposure; c,d) 6 h after LPS injection; e,f) 12 h after LPS injection; g,h) 18 h after LPS injection. a,c,e,g) x128, b,d,f,h) x640 (published at 53%). D, Decidua; DT, diploid trophoblast; ST, spongiotrophoblast



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  		FIG. 6. Immunohistochemical localization of Fas L in the placenta. The sections of mouse placenta were reacted with anti-Fas antibody and counterstained with methyl green. a,b) Normal mouse placenta without LPS exposure; c,d) 6 h after LPS injection; e,f) 12 h after LPS injection; g,h) 18 h after LPS injection. a,c,e,g) x128, b,d,f,h) x640 (published at 53%). D, Decidua; DT, diploid trophoblast; ST, spongiotrophoblast

In Situ Hybridization

To confirm the expression of Fas and Fas L mRNA, we carried out in situ hybridization using Fas and Fas L antisense oligo-DNA probes in the placental sections 12 h after LPS injection. The expression of Fas mRNA was observed in the decidua and diploid trophoblasts in the basal zone of placentas exposed to LPS (Fig. 7). On the other hand, the expression of Fas L mRNA was mainly observed in the spongiotrophoblasts of placentas exposed to LPS (Fig. 8). These findings revealed that Fas and Fas L mRNA well coincided with the localization of Fas and Fas L detected by immunohistochemistry. As a negative control, some sections were hybridized with Fas or Fas L sense probes, and with the Fas antisense probe in the presence of an excess amount of unlabeled oligo-DNA, resulting in no signal (Fig. 7, c and d, and Fig. 8c). Furthermore, in some sections digested with RNase-A (100 µg/ml, 37°C, 1 h) before the postfixation step, no staining was found (data not shown).



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  		FIG. 7. In situ hybridization of Fas mRNA. a) A section of normal mouse placenta without LPS treatment was hybridized with the Fas antisense probe. b–d) Sections of mouse placenta with LPS treatment for 12 h were hybridized with b) Fas antisense probe, c) Fas sense probe, d) Fas antisense probe in the presence of an excess amount of unlabeled Fas antisense oligo-DNA. x640 (published at 53%). DT, Diploid trophoblast.



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  		FIG. 8. In situ hybridization of Fas L mRNA. a) A section of mouse placenta without LPS treatment was hybridized with the Fas L antisense probe. b,c) Sections of mouse placenta with LPS treatment for 12 h were hybridized with b) Fas L antisense probe or c) Fas L sense probe. x640 (published at 53%). DT, Diploid trophoblast.

Expression of HNE-Modified Proteins and Effectof Oxidative Stress by LPS

To identify cell types exposed to oxidative stress from LPS, we studied the expression of HNE-modified proteins immunohistochemically in paraffin-embedded sections of placentas with or without LPS treatment. The expression of cytotoxic proteins was remarkably enhanced in the diploid trophoblasts and spongiotrophoblasts of placentas after 12 h of LPS treatment (Fig. 9). However, the immunostaining of HNE-modified proteins was not observed in the sections of placenta without LPS treatment.



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  		FIG. 9. Immunoreactivity of HNE-modified proteins in the placenta. The sections of mouse placenta were reacted with anti-HNE-modified proteins antibody, and counterstained with methyl green. Immunoreactivity was indicated by a brown color. a) A section of mouse placenta without LPS exposure. b) A section of mouse placenta with LPS exposure for 12 h. x128 (published at 53%). D, Decidua; DT, diploid trophoblast; ST, spongiotrophoblast


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study on the inflammatory placental dysfunction model induced by LPS injection, we have shown that apoptotic cell death occurred transiently in various kinds of placental cells after LPS injection, and that enhanced expressions of Fas and Fas L were spatially and temporally associated with the induction of apoptosis. Furthermore, we have also confirmed the possible involvement of oxidative stress in placental cell apoptosis. Fas was initially reported to be a key molecule in death-signal transduction in the immune system, and to induce apoptosis in Fas-positive cells through Fas L binding to the extracellular domain of Fas [7]. The crucial roles of the Fas system have been also emphasized recently in the induction of apoptosis under ischemia-reperfusion situations [15,44], in which oxidative stress has been postulated to be involved at least partially [44,45]. These accumulated results tempted us to raise the possibility that the successive events consisting of the generation of reactive oxygen species, the activation of the Fas system, and the induction of apoptosis could lead to the placental dysfunction in the LPS mouse model, though the different cascades leading to the induction of apoptosis are equally possible.

To assess the involvement of placental cell death in inflammatory placental dysfunction, we first examined the presence of apoptotic cells by means of biochemical and histochemical methods in the placentas of mice after LPS injection. Our results revealed that the DNA ladder, a hallmark of apoptosis, did appear, and its intensity increased time-dependently. In parallel with the increase in the ladder intensity, the number of TUNEL-positive cells in the placenta was increased in the decidua, diploid trophoblasts in basal zone, and spongiotrophoblasts, reaching a maximum level at 12 h after LPS injection. As a next step, we addressed whether the expression of Fas and Fas L might be associated with the induction of placental cell apoptosis, using Western blot, immunohistochemistry, and in situ hybridization. Western blot analysis revealed that the injection of LPS enhanced the expression of both Fas (45 kDa) and Fas L (31 kDa) time-dependently in the placentas of 13-day pregnant mice; the amount of Fas reached a maximum level (about 4-fold) at 12 h after LPS injection and that of Fas L increased gradually during the experimental period. Positive cells for Fas protein and mRNA were detected in the decidua and diploid trophoblasts in the basal zone of the placentas exposed to LPS, and their number increased also time-dependently after the treatment with LPS, reaching a maximum level at 12 h after the injection. Again, in parallel with the result of Western blotting, the positive cells for Fas L protein and mRNA were increased, and they were localized not only in the decidua and diploid trophoblasts in the basal zone but also in the spongiotrophoblasts. This finding indicates that the LPS-induced expression of Fas and Fas L in these tissues might lead to placental dysfunction or abruptio placentae, which are observed in inflammatory diseases such as infectious diseases or preeclampsia [22–27]. However, we failed to find Fas-positive cells in the spongiotrophoblasts, and therefore the molecular mechanism for inducing the apoptosis in the spongiotrophoblasts is not understood at present. In this context, it should be noted that LPS also induces expression of a variety of cytokines including tumor necrosis factor (TNF)-{alpha}, interleukin (IL)-1{alpha}, and IL-8 in placenta [28,29], which are involved in the induction of apoptosis. Moreover, histological examination indicated that Fas L positive cells in the spongiotrophoblasts of placentas exposed to LPS are predominantly lymphocytes, possibly activated T or natural killer cells, which express cytokines such as TNF-{alpha} and lymphotoxin-ß (TNF-ß) [39,46,47]. Therefore, the Fas system may regulate placental apoptosis singly or in a concerted manner with other cytokines, depending on the placental cell type.

The number of TUNEL-positive cells and the expressions of Fas and Fas L in placentas exposed to LPS (50 µg/ml, i.p.) increased time-dependently in this study model (a maximum level at 12 h for Fas and 18 h for Fas L after LPS injection). Although we did not examine the effect of LPS dose in our study, Kakinuma et al. [21] reported that the dose (50 µg/kg, i.p.) of LPS induces trophoblastic apoptosis effectively in mice. Moreover, it is enough to increase the serum level of various cytokines such as TNF-{alpha} and interleukin 1{alpha} (IL-1{alpha}), which are involved in the induction of apoptosis [21,48]. On the other hand, it should be noted that cytokines such as TNF-{alpha}, IL-1{alpha}, and IL-8 are also involved in producing ROS including superoxide anion (O2-), hydrogen peroxide (H2O2), and hydroxyl radicals (OH·) [30]. An increase in ROS or a depletion of cellular antioxidants can lead to apoptosis [48–51]. Interestingly, the accumulation of HNE-modified proteins (which are end products of unsaturated fatty acids subjected to oxidative stress and a marker of lipid peroxidation [52]) in placentas exposed to LPS correlated well with the expression of Fas and Fas L. Actually, the expression of the cytotoxic proteins was remarkably enhanced in the diploid trophoblasts and spongiotrophoblasts of placentas after 12 h of LPS treatment, indicating that LPS acts as an oxidative stress through the induction of lipid peroxidation.

In conclusion, the molecular mechanisms underlying the induction of placental apoptosis by LPS exposure seem to be quite complicated by many factors including cytokine networks and ROS. However, we have found the Fas system to be an active entity in the induction of placental apoptosis in the LPS model, leading to the placental dysfunction, and these findings may raise the possibility that artificial manipulation of Fas/Fas L interaction will possibly provide a new clinical approach to protect placenta against various toxic exposures by blocking the induction of placental cell apoptosis.


   FOOTNOTES
 
First decision: 3 June 1999.

1 This work was supported by a UOEH Research Grant for promotion of occupational health. Back

2 Correspondence: Takehiko Koji, Department of Histology and Cell Biology, Nagasaki University School of Medicine, 1-12-4, Sakamoto, Nagasaki, 852-8523, Japan. FAX: 81 958 497028; tkoji{at}net.nagasaki-u.ac.jp Back

Accepted: September 20, 1999.

Received: May 17, 1999.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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