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The Journal of Immunology, 2006, 177: 1257-1263.
Copyright © 2006 by The American Association of Immunologists

Chlamydia Heat Shock Protein 60 Induces Trophoblast Apoptosis through TLR41,2

Ozlem Equils3,*, Daning Lu*, Mary Gatter§, Steve S. Witkin||, Cristina Bertolotto*,{dagger}, Moshe Arditi*,{dagger}, James A. McGregor, Charles F. Simmons*,{dagger} and Calvin J. Hobel{dagger},{ddagger}

* Department of Pediatrics, {dagger} Steven Spielberg Pediatric Research Center, Burns and Allen Research Institute, Cedars-Sinai Medical Center, David Geffen School of Medicine at University of California, Los Angeles, and {ddagger} Department of Obstetrics and Gynecology at Cedars-Sinai Medical Center, Los Angeles, CA 90048; § Planned Parenthood Clinic, Los Angeles, CA 90033; and Department of Obstetrics and Gynecology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033; and || Department of Obstetrics and Gynecology, Weill Medical College of Cornell University, New York, NY 10021


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Intrauterine infection affects placental development and function, and subsequently may lead to complications such as preterm delivery, intrauterine growth retardation, and preeclampsia; however, the molecular mechanisms are not clearly known. TLRs mediate innate immune responses in placenta, and recently, TLR2-induced trophoblast apoptosis has been suggested to play a role in infection-induced preterm delivery. Chlamydia trachomatis is the etiological agent of the most prevalent sexually transmitted bacterial infection in the United States. In this study, we show that in vitro chlamydial heat shock protein 60 induces apoptosis in primary human trophoblasts, placental fibroblasts, and the JEG3 trophoblast cell line, and that TLR4 mediates this event. We observed a host cell type-dependent apoptotic response. In primary placental fibroblasts, chlamydial heat shock protein 60-induced apoptosis was caspase dependent, whereas in JEG3 trophoblast cell lines it was caspase independent. These data suggest that TLR4 stimulation induces apoptosis in placenta, and this could provide a novel mechanism of pathogenesis for poor fertility and pregnancy outcome in women with persistent chlamydia infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Chlamydia trachomatis is the most common reportable sexually transmitted bacterial species in the United States (1, 2). In pregnant women, cervical infection with C. trachomatis is independently associated with preterm premature rupture of the membranes, preterm labor, and low birth weight (reviewed in Ref. 3); however, the molecular mechanisms are not clearly known.

Chlamydia organisms are not acutely toxigenic, and in the majority of females, genital tract infection is asymptomatic and persistent. Recent evidence suggests that organism may persist for as long as 5 years in the genital tract (4). Interestingly, the organism has a distinct antigenic profile during persistent infection, where overall expression of all proteins decreases except for the 60-kDa heat shock protein (HSP60)4 (5), which is associated with the outer membrane complexes of Chlamydia and appears to be responsible for proinflammatory pathologic manifestations of human chlamydial disease in the reproductive tract (6, 7, 8, 9, 10).

TLRs have been identified as mediators of microbial Ag induced innate immune responses (11), regulators of adaptive immune responses (12), as well as inducers of apoptosis (13, 14, 15, 16, 17, 18, 19). TLR expression in the female reproductive tract has recently been examined. There is constitutive TLR1 to TLR6 mRNA and protein expression as well as MyD88 and CD14 mRNA expression in the fallopian tubes, uterine endometrium, cervix, and ectocervix (20, 21). TLR2 mRNA levels are highest in the fallopian tubes and cervical tissues, followed by endometrium and ectocervix. In contrast to TLR2, TLR4 expression declines progressively along the tract, with highest expression in the upper tissues (fallopian tubes and endometrium), followed by cervix and endocervix (20, 21). Fazeli et al. (21) have also shown the expression of a secretory form of TLR4 in the endocervical glands. TLR4 and TLR2 are also expressed in placenta cells (trophoblasts, fibroblasts, Hofbauer-placental macrophages), and levels of TLR4 expression in placenta increase in the presence of infection and vascular insufficiency (22, 23).

TLRs are increasingly documented to play a role in female reproductive tract physiology. Stimulation of TLR2, TLR3, TLR4, and TLR5 with their ligands has been shown to induce proinflammatory cytokine release in uterine epithelial cells (24, 25, 26). In placenta, binding of TLR2 with its ligand Gram-positive bacterial cell wall component peptidoglycan induces trophoblast apoptosis (27). Both maternal and fetal polymorphisms of the TLR4 gene have been associated with spontaneous preterm labor and preterm birth in certain populations (reviewed in Ref. 28).

We have previously shown that TLR4 mediates innate immune responses to C. trachomatis HSP60 (cHSP60) and that stimulation with cHSP60 induces innate immune responses in endothelial cells (29). Here, we show that cHSP60 treatment induces caspase-8, caspase-3, and caspase-9 activation in trophoblasts and leads to trophoblast apoptosis through TLR4. Because healthy trophoblast development is essential for healthy placenta and fetal development, these data provide a novel mechanism for Chlamydia-induced infertility—early pregnancy loss, preeclampsia, and preterm delivery.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell lines and reagents

JEG3, human syncytiotrophoblast cell line was obtained from American Type Tissue Culture Collection (ATCC). Cells were cultured in MEM (Invitrogen Life Technologies) supplemented with 10% FBS, 10 mM HEPES, 1 mM sodium pyruvate, and 100 nM penicillin/streptomycin (Invitrogen Life Technologies).

THP-1 cells were obtained from ATCC. They were grown in RPMI 1640 medium with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, and 1.0 mM sodium pyruvate and supplemented with 0.05 mM 2-ME (90%) and FBS (10%) (30). Highly purified, phenol-water extracted, and protein-free (<0.0008% protein) Escherichia coli K235 LPS was obtained from S. Vogel (University of Maryland, Bethesda, MD). The purity of this LPS preparation has been demonstrated previously (31, 32). This preparation of LPS is active on TLR4-transfected HEK 293 cells and not on TLR2 transfectants (S. N. Vogel, unpublished observation). Recombinant chlamydial HSP60 protein was isolated and purified as described earlier (33). C. trachomatis serovar A HSP60, fused with eight additional amino acids (arginine, serine, and six histidine residues) at the C terminus, was expressed in E. coli, and recombinant protein was purified by affinity chromatography with Ni-NTA resin, as previously described (33). The endotoxin concentration of this preparation was <0.04 EU/µg, as determined by Limulus amebocyte lysate assay (Associates of Cape Cod).

Neutralizing anti-TLR4 (catalog no. 14-9917; 20 µg/ml), anti-TNF-{alpha} (catalog no. 16-7348; 20 µg/ml), anti-TLR2 (catalog no. 14-9922; 20 µg/ml) mAb and nonspecific IgG1 (catalog no. 16-4724) control Ab were obtained from eBioscience. JEG3 cells were incubated with neutralizing or nonspecific control Ab for 1 h before stimulation with cHSP60. The pancaspase inhibitor (Z-VAD-FMK) was obtained from BD Pharmingen. Protein kinase C inhibitor calphostin was purchased from Sigma-Aldrich.

Isolation and culture of primary trophoblasts

Trophoblasts were isolated from early second-trimester placentas obtained according to National Institutes of Health, Cedars-Sinai Medical Center, and Planned Parenthood Institutional Review Board protocols as described previously (27). Briefly, tissue specimens were washed with cold HBSS (Invitrogen Life Technologies) to remove excess blood. Cells were scraped from the membranes, transferred to trypsin-EDTA (Invitrogen Life Technologies) digestion buffer and incubated at 37°C for 10 min with shaking. An equal volume of DMEM medium (Invitrogen Life Technologies) containing 10% FBS was added to inactivate the trypsin. This mixture was vortexed for 20 s and allowed to sediment, and the supernatant was collected. This was repeated twice and the collected supernatant was centrifuged at 1500 rpm for 10 min. Contaminating RBC were removed by resuspending the cellular pellet with HBSS, layering this over the same volume of Lymphocyte Separation Media (ICN Biomedicals), and centrifuging at 2000 rpm for 25 min. The cellular interface containing the trophoblast cells was collected and resuspended in DMEM supplemented with 10% normal human serum (Gemini Bio-Products) and cultured at 37°C/5% CO2 for three passages. Purity of the trophoblast cells was >98% as determined by immunostaining for cytokeratin-7 (Sigma-Aldrich) (27).

Western blot analysis

For TLR4 Western blots, JEG3 cells were lysed in immunoprecipitation lysis buffer containing 50 mM HEPES (pH 7.9), 250 mM NaCl, 20 mM beta-glycerophosphate, 2 mM DTT, 1 mM sodium orthovanadate, 1% Nonidet P-40, and 1:100 Protease Inhibitor Set III (Calbiochem). Protein concentration was determined using a colorimetric assay Bio-Rad DC protein assay. A total of 55 µg of protein was analyzed on a 10% Tris-HCl polyacrylamide gel (Bio-Rad). Membranes were blocked in 5% milk, 0.1% Tween 20 in TBS for 2–3 h at 4°C; incubated overnight at 4°C with anti-human TLR4 and anti-human TLR2 Ab (Santa Cruz Biotechnology) (1:250) followed by a 1-h incubation at room temperature with anti-rabbit HRP (1:2000); developed by Lumiglo (Cell Signaling); and exposed to radiographic film.

Caspase activity assay

We assessed caspase activation by using the Caspase-Glo assay (Promega) according to the manufacturer’s directions. Briefly, 10 µg of whole-cell lysates were incubated at room temperature in the dark for 1 h with either the caspase-3, -8, or -9 substrate. Following incubation, luminescence was measured using a TD-20/20 luminometer (Turner Designs). The amount of luminescence detected as relative light units was proportional to caspase activity.

FACS analysis of apoptosis

Apoptotic cells that are accompanied by phosphatidylserine exposure to the outer membrane were analyzed by incubation of cells with FITC-conjugated annexin V (Roche Molecular Biochemicals). Labeling procedures followed those suggested by the manufacturer’s manual. Briefly, cells were resuspended in annexin labeling solution containing 10 mM HEPES (pH 7.4), 140 mM NaCl, 5 mM CaCl2, and fluorescein-conjugated annexin V for 15 min. After being washed twice with PBS, cell pellets were resuspended in propidium iodide (PI) (2 µg/ml) containing PBS and analyzed by flow cytometry. At least 10,000 events were analyzed, and apoptosis was presented as percent positive cells stained with annexin V.

Statistical analysis

The experiments were set up in triplicate and were repeated on at least three separate occasions. Student’s t test was used to compare the means between medium and treatment groups. A value of p < 0.05 was reported as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Ex vivo cHSP60 stimulation of primary trophoblasts induces apoptosis

We examined the apoptotic effect of cHSP60 on primary trophoblasts isolated from early second-trimester elective termination placentas. Cells were incubated with medium, protein kinase C inhibitor calphostin (as positive control for apoptosis) and various concentrations of cHSP60 for 4 h. Cell death was assessed by staining with PI and FITC-labeled annexin V to detect necrotic and apoptotic cells, respectively, and by using flow cytometry.

We observed that treatment with cHSP60 (5 µg/ml) resulted in 5-fold increase (11.76–62.09%) in the number of apoptotic cells compared with medium-treated cells (Fig. 1). Because treatment with higher concentration of cHSP60 (20 µg/ml) increased double-positive cells (PI and annexin positive) that could be late apoptotic or necrotic (0.59–3.37%) (Fig. 1), we elected to use the lower concentration of cHSP60 (5 µg/ml) to study cHSP60-induced apoptosis in the remainder of the experiments. These data suggest that, in pregnant women with persistent chlamydia infection, the release of extracellular cHSP60 may lead to trophoblast cell apoptosis.


Figure 1
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  		FIGURE 1. Primary trophoblasts isolated from elective termination placentas were treated with various concentrations of cHSP60 for 4 h. Apoptosis was assessed by PI and FITC-labeled annexin V staining and flow cytometry. The data shown are representative of three independent experiments.

 
Chlamydia HSP60 stimulation of JEG3 trophoblast cells leads to apoptosis

We used the JEG3 trophoblast cell line to assess the molecular mechanisms involved in cHSP60-induced trophoblast apoptosis. To do this, we first demonstrated that the JEG3 human trophoblast cell line expressed 88-kDa protein corresponding to TLR4 and a 90-kDa protein corresponding to TLR2 (Fig. 2). Next, we examined the apoptotic effect of cHSP60 on JEG3 trophoblast cells by treating them with medium, calphostin, and cHSP60. Apoptosis was determined by staining with PI and FITC-labeled annexin V and performing flow cytometry. Treatment of the JEG3 trophoblast cell line with cHSP60 induced apoptosis (p < 0.05), but at a much reduced rate compared with primary trophoblasts (7.7–13.3%) (Fig. 3). These data suggest that cHSP60 induces apoptosis in both primary trophoblasts and JEG3 cells, and therefore JEG3 cell system can be used to study the molecular mechanisms of cHSP60-induced trophoblast apoptosis.


Figure 2
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  		FIGURE 2. We assessed TLR2 and TLR4 expression in JEG3 trophoblast cell line by using Western blot analysis. Protein isolated from THP-1 human monocytic cell line, known to express both TLR2 and TLR4, was used as positive control.

 

Figure 3
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  		FIGURE 3. JEG3 cells were treated with various concentrations of cHSP60 for 1 h, and the presence of apoptosis was assessed by PI and FITC-labeled annexin V staining and flow cytometry. The data shown are representative of three independent experiments.

 
Anti-TLR4 Ab blocks cHSP60-induced apoptosis in JEG3 cells

TLR4 mediates cHSP60-induced immune responses in macrophages and endothelial cells (29). We assessed the role of TLR4 in cHSP60-induced trophoblast apoptosis by treating JEG3 cells with function-blocking anti-TLR4, anti-TLR2, and nonspecific IgG1 control Ab before stimulation with cHSP60, and measuring apoptosis by FITC-labeled annexin V staining and flow cytometry. Pretreatment with anti-TLR4 Ab blocked the cHSP60-induced apoptosis, whereas control Ab and anti-TLR2 Ab had no effect (Fig. 4). These data suggest that TLR4 mediates cHSP60-directed trophoblast apoptosis.


Figure 4
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  		FIGURE 4. JEG3 cells were treated with anti-TLR4, anti-TLR2, anti-TNF, and nonspecific control IgG for 1 h before stimulation with cHSP60 (5 µM) for 1 h. The presence of apoptosis was assessed by FITC-labeled annexin V staining and flow cytometry. The experiment was repeated on at least three separate occasions. The data are presented as percent positive cells for annexin V. *, p < 0.05 apoptosis compared with medium-treated cells.

 
Stimulation with TNF-{alpha} induces apoptosis in trophoblasts (34). To assess whether cHSP60-induced apoptosis was secondary to TNF-{alpha} released, we treated JEG3 cells with anti-TNF-{alpha} Ab before stimulation with cHSP60, and assessed apoptosis by FITC-labeled annexin V staining and flow cytometry. We observed that pretreatment with anti-TNF-{alpha} Ab did not decrease cHSP60-induced apoptosis (Fig. 4). These data suggest that cHSP60-induced trophoblast apoptosis is not due to the autocrine effect of cHSP60-induced TNF-{alpha}.

TLR4 is the receptor for enteric Gram (–) bacterial LPS (35), and TLR4 signaling of HSP has recently been attributed to their LPS contamination (36, 37). To test the role of LPS contamination of cHSP60 in JEG3 apoptosis, we treated JEG3 cells with various concentrations of LPS that induce signaling in these cells (data not shown) and assessed apoptosis by using annexin V staining and flow cytometry. We observed that LPS treatment did not induce apoptosis in JEG3 cells (Fig. 4). These data suggest that cHSP60-induced JEG3 cell apoptosis is due to the direct effect of cHSP60 but not due to LPS contamination.

cHSP60 treatment activates capase-3, caspase-8, and caspase-9 in JEG3 cells

In the majority of cases, apoptosis is mediated through activation of caspases. Therefore, we assessed the effect of cHSP60 stimulation on caspase-3, caspase-8, and caspase-9 activity. Stimulation with cHSP60 but not LPS induced caspase-8, caspase-3, and caspase-9 activation in JEG3 cells (Fig. 5, A–C, respectively). These data suggest that caspases may mediate cHSP60-induced apoptosis in JEG3 trophoblasts.


Figure 5
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  		FIGURE 5. JEG3 cells were stimulated with cHSP60 for 1 h, and caspase-8 (A), capase-3 (B), and caspase-9 (C) activation was assessed by CaspaseGlo according to the manufacturer’s direction. The data presented are the mean ± SD of three independent experiments. *, p < 0.05 compared with medium-treated cells.

 
Next, we assessed the role of caspases in cHSP60-induced trophoblast apoptosis by treating JEG3 cells with various concentrations of pancaspase inhibitor (Z-VAD-FMK) before treatment with cHSP60 and assessing apoptosis by FITC-labeled annexin V staining and flow cytometry. We observed that, at the highest concentration of Z-VAD-FMK, inhibition of caspases only partially blocked the cHSP60-induced apoptosis in JEG3 cells (Fig. 6).


Figure 6
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  		FIGURE 6. JEG3 cells were treated with a high concentration of pancaspase inhibitor (Z-VAD-FMK) (250 µM) for 1 h before stimulation with cHSP60 for 1 h. The presence of apoptosis was assessed by FITC-labeled annexin V staining and flow cytometry. The data shown are representative of three independent experiments.

 
In parallel, we isolated primary fibroblasts from minced, PBS-washed second-trimester elective termination placentas cultured in DMEM medium with 10% FBS and antibiotics. Fibroblasts were then used in apoptosis experiments. Initial experiments suggested that Z-VAD-FMK was toxic to primary fibroblasts at the concentrations used in trophoblasts. Therefore, a lower concentration of Z-VAD-FMK (100 µM) was used in fibroblast experiments. Similar to trophoblasts, cHSP60 treatment led to apoptosis in fibroblasts and pretreatment with Z-VAD-FMK blocked the cHSP60-induced apoptosis (Fig. 7). These data suggest that caspase-dependent apoptotic pathways may mediate cHSP60-induced trophoblast and fibroblast apoptosis.


Figure 7
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  		FIGURE 7. Primary human placental fibroblasts were treated with pancaspase inhibitor Z-VAD-FMK (100 µM) for 1 h before stimulation with cHSP60 (5 µM) for 1 h. The presence of apoptosis was assessed by FITC-labeled annexin V staining and flow cytometry. The data shown are representative of two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Chlamydia is an obligate intracellular bacteria and has three major species: C. trachomatis, C. pneumonia, and C. psittaci. C. trachomatis is the etiological agent of the most prevalent sexually transmitted bacterial disease in the United States. There are roughly 3 million cases annually, most occurring in men and women < 25 years of age (38) with direct and indirect costs (mainly costs for complications) of ~$2.4 billion a year (39, 40).

Chlamydia are clearly known to be induce an immune response, and apoptosis in the neighboring uninfected cells (41). It is proposed that Chlamydia infection protects the host cell against apoptosis to provide survival advantage to this obligate intracellular organism (42, 43, 44, 45, 46, 47, 48). Recently, Zhong and Kihlstrom and coworkers (48, 49) have shown that, although chlamydia infection does not induce cell death in infected cells, there is significant apoptosis in the nearby uninfected adherent cells as measured by immunofluorescence. Eley et al. (50, 51) have shown that coincubation of human sperm with C. trachomatis leads to premature sperm death and that this is due primarily to chlamydial LPS. Dumrese et al. (52) have shown that C. pneumoniae infection of human aortic smooth muscle cells results in cell death with both apoptotic and necrotic characteristics. In addition, in RAW cells, infection with live C. pneumoniae or exposure to heat-killed or UV-inactivated C. pneumoniae led to aponecrosis, which was mediated via caspase-3-independent mechanisms (53). In a recent elegant review of Chlamydia pathogenesis, Byrne and Ojcius (54) state that "Chlamydia can elicit both the induction of host cell death, or apoptosis, under some circumstances and actively inhibit apoptosis under others. This subtle pathogenic mechanism highlights the manner in which these highly successful pathogens take control of infected cells to promote their own survival—even under the most adverse circumstances." In this study, we show that stimulation with an effector of Chlamydia, cHSP60, induces apoptosis in primary trophoblasts and JEG3 trophoblast cell line.

In our experiments, cHSP60-induced apoptosis was much lower in JEG3 cell line than in primary trophoblasts, suggesting that primary trophoblasts are more sensitive to apoptosis than the trophoblast cell line. This difference agrees with the observation that primary smooth muscle cells die through "aponecrosis" during C. pneumoniae infection (52), and that primary fibroblasts are more prone to die through apoptosis during C. trachomatis infection than epithelial cell line (55).

Zhong and coworkers (49) suggested that the overall rate of apoptosis (<15%) in chlamydia-infected cultures is low and would not have any biological significance. It has been suggested that the techniques used can account for some of the differences in interpretation regarding cell death during chlamydia infection (41). Besides, a 6- to 7-day-old blastocyst has on average 100–150 cells, from which the embryo and placenta develop (56). Our data suggest that cHSP60-induced trophoblast apoptosis at this early stage may have a significant impact on placental and fetal development, and consequently pregnancy outcome. Indeed, trophoblast apoptosis is increased in pregnancies complicated by preeclampsia and intrauterine growth restriction and are often associated with insufficient trophoblast invasion (57, 58, 59).

Chlamydia has several cell wall and outer membrane components that are immunogenic and may serve as TLR ligands. Chlamydial LPS (59) as well as cHSP (29, 60, 61) are ligands for TLR4. Recent papers describe TLR4-independent cytokine production from inflammatory cells exposed to live chlamydial elementary bodies (62, 63, 64), and a dominant role for TLR2 in the recognition process of C. pneumoniae (65), and C. trachomatis (66). In this study, we used recombinant cHSP60 to assess the effect of persistent chlamydial infection on placenta. HSP are a class of highly conserved proteins that have important functions in cellular metabolism and aid cells in dealing with adverse environmental stimuli. There are several families of HSP classified by the approximate m.w. of their constituents (67). Members of the HSP60 family are found both in prokaryotes and eukaryotes; in eukaryotes their presence appears mainly restricted to the mitochondria and chloroplast organelles, although under stress conditions extracellular HSP60 has also been identified. In bacteria, HSP60 is located in the cytoplasm (68); however, in chlamydia, large amounts of HSP60 can be obtained merely by washing the organism with isotonic solutions (69), which suggests that much of cHSP60 is associated with outer membrane, and can be exposed easily to induce immune activation of the neighboring host cells, and models of persistent chlamydial infection have demonstrated the release of cHSP60 into the extracellular milieu. Indeed, several studies have revealed a correlation between cHSP60 responses and the immunopathologic manifestations of human chlamydial disease (6). Witkin and coworkers (6, 7, 8, 9) have shown that in some women cervical anti-cHSP60 Abs may cross-react with endogenous hsp60, bind to HSP60 on the human embryo, and decrease the success rate of in vitro fertilization and embryo transfer. In this study, we show that cHSP60 stimulation induces apoptosis in trophoblasts. This, therefore, provides an alternative mechanism for chlamydia-induced early pregnancy loss.

Some of the effects of recombinant HSP have been attributed to LPS contamination (37, 38). The concentration of LPS measured in the undiluted recombinant cHSP60 protein used in our experiments was <0.06 ng/ml, which corresponds to <0.0012 ng/ml LPS in 10 µg of protein used in each well, a concentration well below what is needed for cellular activation (11). In addition, pretreatment with anti-TLR4 Ab blocked cHSP60-induced apoptosis in trophoblasts, whereas anti-TLR2 Ab treatment had no effect, and LPS treatment of JEG3 cells did not induce apoptosis or caspase activation, whereas cHSP60 did. These data suggest that the apoptotic effect of cHSP60 used in our experiments is not due to LPS contamination and is mediated through TLR4.

Mor and colleagues (27) have shown that LPS stimulation of TLR4 did not induce apoptosis in 3A and H8 trophoblast cell lines. In our hands, LPS did not induce apoptosis in JEG3 cells, whereas cHSP60 did. Similar to our findings, Li et al. (70) have shown that TLR4 stimulation with Taxol induced apoptosis in THP-1 human monocytic cell line but LPS did not. The differential apoptotic effect of different TLR4 ligands may be explained by the diverse downstream signaling events induced and differential use of adapter molecules by different TLR4 ligands.

Apoptosis is classically initiated through extracellular ligands that bind to cell surface receptors. One of the best characterized receptor is a member of the TNF family, Fas. Binding of Fas with its ligand, Fas ligand, recruits an intracytoplasmic protein adaptor (the Fas-associated death domain). This binding eventually leads to activation of caspase-8, which then activates caspase-3 and indirectly activates caspase-9 and leads to apoptosis characterized by nuclear fragmentation (71, 72, 73, 74). TLR4 and TLR2 adapter molecules, MyD88 and Toll/IL-1R domain-containing adaptor inducing IFN-beta (TRIF), have death domains, and interact with Fas-associated death domain to activate caspase-8 (14, 75). In this study, we show that TLR4 stimulation with cHSP60 induces apoptosis in primary trophoblasts and the JEG3 trophoblast cell line through caspase-8, caspase-3, and caspase-9. Inhibition of caspases blocked TLR2-peptidoglycan-induced trophoblast apoptosis (27). In our hands, inhibition of caspases with pancaspase inhibitor partially blocked the block TLR4-cHSP60-induced apoptosis in JEG3 trophoblast cells. We repeated the same experiments in primary human placental fibroblasts and showed that pretreatment with pancaspase inhibitor, Z-VAD-FMK, blocked the cHSP60-induced apoptosis. These data support the findings of Yaraei et al. (53) in RAW 264.7 cells and suggest that caspase-dependent and -independent pathways may play roles in cHSP60-induced apoptosis in trophoblasts.

Infection plays an important role in early pregnancy loss (76) and preterm delivery and its sequelae (1). It has been estimated that ~50% of all preterm deliveries and 80% of early preterm births (<32 wk) may be associated with intrauterine infection (2). Currently, the mechanisms of microbial Ag-induced preterm labor remain unknown. Based on our data, we propose that microbial Ag-induced trophoblast apoptosis may lead to "placental insufficiency" and play a role in early pregnancy loss in a group of susceptible women as well as development of preeclampsia, intrauterine growth restriction, and preterm delivery.


   Acknowledgments
 
We are grateful to the patients, nurses, and physicians at Bixby Planned Parenthood Clinic (Los Angeles, CA).


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health/National Institute of Child Health and Human Development (to C.J.H.). Back

2 The opinions expressed in this article do not necessarily reflect those of Planned Parenthood Federation of America, Inc. Back

3 Address correspondence and reprint requests to Dr. Ozlem Equils, Department of Pediatrics, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Room 4220, Los Angeles, CA 90048. E-mail address: ozlem.equils{at}cshs.org Back

4 Abbreviations used in this paper: HSP60, 60-kDa heat shock protein; cHSP60, Chlamydia trachomatis HSP60; PI, propidium iodide. Back

Received for publication August 1, 2005. Accepted for publication April 21, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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