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BOR - Papers in Press, published online ahead of print November 7, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.064444
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BIOLOGY OF REPRODUCTION 78, 497–505 (2008)
DOI: 10.1095/biolreprod.107.064444
© 2008 by the Society for the Study of Reproduction, Inc.

Female Reproductive Tract; 

ADRB3 Adrenergic Receptor Is a Key Regulator of Human Myometrial Apoptosis and Inflammation During Chorioamnionitis1

Fréderic Lirussi 3, Zo Rakotoniaina 3, Siham Madani 3, Françoise Goirand 3, Michelle Breuiller-Fouché 4, Marie-Josèphe Leroy 4, Paul Sagot 5, John J. Morrison 6, Monique Dumas 3, and Marc Bardou 2 3 7

Laboratoire de Physiologie et Pharmacologie Cardiovasculaires Expérimentales (LPPCE, EA279, IFR Santé-STIC),3 Faculté de Médecine, Université de Bourgogne, 21079 Dijon, France Unité INSERM U767,4 Faculté des Sciences Pharmaceutiques et Biologiques, Université René Descartes, 75006 Paris, France Department of Obstetrics and Gynecology,5 CHU du Bocage Dijon, 21079 Dijon, France Department of Obstetrics and Gynaecology,6 Clinical Science Institute, National University of Ireland Galway, University College Hospital Galway, Galway, Ireland Centre d'Investigation Clinique,7 Unité de Pharmacologie Clinique, 21000 Dijon, France

ABSTRACT

The pathophysiology underlying preterm labor triggered by inflammatory conditions such as chorioamnionitis remains largely unclear. It has already been suggested that beta-3 adrenergic (ADRB3) agonists might be of interest in the pharmacological management of preterm labor. Although there is evidence implicating ADRB receptors in the control of inflammation, there are minimal data relating specifically to ADRB3. To explore the cellular consequences of chorioamnionitis and detect apoptosis, we first performed immunostaining and Western blot experiments on human myometrial samples obtained from women with confirmed chorioamnionitis. We then developed an in vitro model of chorioamnionitis by incubating the myometrial samples obtained from uncomplicated pregnancies with Escherichia coli lipopolysaccharide (LPS). We observed that chorioamnionitis was associated with a significant increase in cleaved CASP3 protein expression, as well as chromatin condensation, which were reproduced experimentally by LPS stimulation (10 µg/ml, 48 h). Lipopolysaccharide stimulation of normal human myometrium also induced CASP3 transcripts, increased the proapoptotic marker BAX, and decreased the antiapoptotic marker BCL2. Lipopolysaccharide-induced apoptosis was antagonized by neutralization of secreted tumor necrosis factor by a specific antibody. Furthermore, LPS stimulation increased medium culture levels of proinflammatory cytokines interleukin 6 (IL6) and IL8. Lipopolysaccharide-induced apoptosis and cytokine production were prevented by the new and potent ADRB3 agonist SAR150640 in a concentration-dependent manner. SAR150640 by itself did not exhibit any effect on apoptosis or cytokine production in control tissues. This study shows that chorioamnionitis is associated with apoptosis of human myometrial cells. It emphasizes the potential therapeutic interest of ADRB3 agonists in the field of preterm labor and other inflammatory conditions.

apoptosis, bacterial infection, beta-adrenoceptors, cytokines, immunology, inflammation, lipopolysaccharide, pregnancy

INTRODUCTION

The incidence of premature birth has risen over the past 15 years [1], yet spontaneous preterm delivery remains relatively high in developed countries, despite preventative measures [2–4]. Around 6% to 12% of all pregnancies end prematurely. Spontaneous preterm labor, whether explained or unexplained, is one of the largest causes of preterm birth which, in turn, is the most frequent cause of infant death in the United States, accounting for at least one third of infant deaths [5]. Many cases of spontaneous preterm labor are unexplained, but a significant proportion are linked to genital tract infection or chorioamnionitis [6].

Several stimulatory and inhibitory pathways regulate the balance of uterine quiescence and contractile activity during pregnancy, but the specific changes that govern the switch between these opposing functional states are still poorly understood [7]. Inflammation has been implicated in the mechanisms responsible for preterm and term parturition, as well as for fetal injury [8, 9]. Of all of the suspected causes of preterm labor and delivery, infection and/or inflammation are the only pathological process for which both a firm causal link with preterm birth has been established and a molecular physiopathology defined [8]. Whereas it is well known that intrauterine infection affects placental development and function, and subsequently may lead to preterm delivery, the molecular mechanisms underlying this complication are not clearly known. Although controversial, the role of infection-induced apoptosis was recently suggested by Equils et al. [10], who stated that in vitro chlamydial heat shock protein 60 induces apoptosis in primary human trophoblasts and placental fibroblasts through Toll-like receptor 4 (TLR4) stimulation. In further support of the role of inflammation in the mechanism of spontaneous parturition at term, it was recently shown that labor at term induces gene expression changes consistent with localized inflammation, despite the absence of histologically detectable inflammation [11]. A few years ago, Charpigny et al. [12] showed that parturition was characterized by a downregulation of a large panel of developmental-, cell adhesion molecule-, and proliferation-related genes, along with the upregulation of inflammatory-, contraction-, and apoptosis-associated genes. In this study, one third of genes upregulated at term are linked to inflammatory-immune pathways. These findings were recently confirmed by Havelock et al. [13] and Bukowski et al. [14].

Apoptotic cell death can be initiated by two alternative convergent pathways: the extrinsic pathway, which is mediated by cell surface death receptors, and the intrinsic pathway, which is mediated by mitochondria [15]. In both pathways, cysteine aspartyl-specific proteases (caspases) that cleave cellular substrates are activated, and activation of the effector caspase 3 (CASP3) is important for the execution of apoptotic cell death. The BCL2 family members play a central role in the regulation of apoptosis. The multidomain proapoptotic proteins BAX and BAK together constitute a requisite gateway to apoptotic cell death, because cells doubly deficient for these proteins are resistant to several different intrinsic death stimuli [15]. Therefore, BAX, BCL2, and cleaved CASP3 are widely used to assess apoptosis [16, 17].

The pharmacologic management of preterm labor is based on the use of various drugs aimed at inhibiting myometrial contractions and known as tocolytic agents. Beta-2 adrenergic receptor (ADRB2) agonists have long been used as tocolytic agents for the treatment of preterm labor, but due to limited efficacy and toxicity they are no longer recommended as first-choice drugs. Oxytocin antagonists and calcium channel blockers are more recently developed drugs with a lower profile of adverse affects, and hence are more commonly used [18]. We have recently shown that ADRB3 is present and is the predominant ADRB in human myometrium [19–21], with an inhibitory action on spontaneous contractions. In contrast with ADRB2, ADRB3 has the interesting property of being resistant to long-term agonist induced desensitization [22]. A strong body of evidence supports an anti-inflammatory effect for ADRB2 stimulation [23–25], either in autoimmune [26], septic [27], or allergic [28] inflammatory states. It has been described that lipopolysaccharide (LPS) produces a large decrease in adipose tissue ADRB3 transcription and function [29]; nevertheless, there are no published data demonstrating an anti-inflammatory effect for ADRB3 stimulation. Since we have suggested its potential interest in the pharmacologic management of preterm labor, the aims of this study were: 1) to study the cellular consequences of chorioamnionitis in human myometrium; 2) to develop an in vitro model of chorioamnionitis induced by Escherichia coli LPS in human myometrium in order to analyze the myometrial consequences of chorioamnionitis and to assess the ability of the newly described selective ADRB3 agonist, SAR150640 (ethyl-4-(trans-4-[((2S)-2-hydroxy-3-(4-hydroxy-3[(methylsulfonyl)amino]phenoxy)propyl)amino]cyclohexyl)benzoate hydrochloride) [30] to oppose this LPS-induced chorioamnionitis.

MATERIALS AND METHODS

Drugs and Solutions

Lipopolysaccharide (E. coli 055:B5, ref: L2880) was purchased from Sigma-Aldrich and was dissolved in distilled water. SAR150640 was a gift from the Sanofi-Midy Research Center, Exploratory Research Department, Sanofi-Aventis S.p.A. It was dissolved in a mixture of absolute ethanol 30%, dimethyl sulfoxide (DMSO) 2%, and distilled water for the 1-mM solution and thereafter diluted in distilled water. The final maximal bath concentration was 0.3% for ethanol and 0.02% for DMSO.

Biologic Samples

Myometrial biopsies were obtained from women during pregnancy in four different clinical situations: 1) a woman with established chorioamnionitis undergoing postpartum hysterectomy; 2) a woman undergoing postpartum hysterectomy for postpartum hemorrhage in the absence of chorioamnionitis; 3) 3 women undergoing elective cesarean delivery with established chorioamnionitis; and 4) 10 women undergoing elective cesarean delivery for other reasons (cephalopelvic disproportion) in the absence of chorioamnionitis. For women undergoing cesarean delivery, the procedures were all performed prior to the onset of labor at a gestation period between 38 and 40 wk of pregnancy. Clinical chorioamnionitis was defined classically [31] as the presence of uterine tenderness and/or purulent or foul-smelling amniotic fluid with any two of the following: antepartum temperature of 37.8°C or more, maternal tachycardia (more than 120 beats/min), maternal leukocytosis more than 18 000 cells/mm3, or fetal tachycardia (more than 160 beats/min), and was confirmed in all cases included in the present study by either a positive culture of the placenta or an histological assessment of the placenta by a single pathologist using validated criteria [32]. Therefore, in the rest of this manuscript, chorioamnionitis refers to confirmed chorioamnionitis.

Myometrial strips were excised from an immediately subserosal site where the majority of the fibers are in a longitudinal orientation, at an antiplacental site, as previously described [21, 33]. This study was approved by the Comité Consultatif de Protection des Personnes pour la Recherche Biomédicale (CCPPRB, Dijon, France), and informed consent was obtained from all donors.

Tissues obtained from women with confirmed chorioamnionitis or postpartum hemorrhage were either used fresh to perform Western blot experiments or embedded in paraffin for histological assessment and Hoechst staining, as described below.

Myometrial biopsies obtained from uncomplicated pregnancies were used to develop the LPS experimental model mimicking the effects of chorioamnionitis.

Stimulation of Myometrial biopsies by E. coli LPS

Myometrial biopsies obtained from women with uncomplicated pregnancies were immediately transferred in sterile Dulbecco modified Eagle medium (DMEM) and washed twice with sterile phosphate-buffered saline (PBS). Biopsies were cut into small strips, each being placed in a 24-well plate containing 2 ml culture medium DMEM, without the use of antibiotics. Strips were then incubated at 37°C with 5% CO2 for 48 h in order to allow cytokine levels to return to basal values [34].

In order to reproduce with LPS (E. coli 055:B5) the findings on myometrial samples obtained from women with chorioamnionitis, myometrial strips were incubated with three different concentrations of LPS (50 ng/ml, 1 µg/ml, and 10 µg/ml) at three different times (8 h, 24 h, and 48 h).

In a second set of experiments, to assess the role of tumor necrosis factor (TNF) in LPS-induced apoptosis in myometrial tissue, the strips were incubated with LPS (10 µg/ml) for 48 h with or without anti-TNF antibodies (0.6 µg/ml; human TNF-alpha/TNFSF1A antibody; R&D Systems Europe, Lille, France: the concentration of the antibody used was chosen based on the manufacturer's recommendations).

In a third set of experiments aimed at assessing the ability of ADRB3 stimulation to oppose LPS-induced apoptosis, myometrial strips were incubated with LPS (10 µg/ml) for 48 h in the presence or absence of SAR150640, a selective ADRB3 agonist [30] (0.1 µM, 1 µM, and 10 µM), added immediately prior to begin LPS stimulation. Time-matched control experiments were performed with the solvent of SAR150640; that is, distilled water containing ethanol 0.3% and DMSO 0.02% as final bath concentration, both nonstimulated and LPS-stimulated myometrial samples.

At the end of the stimulation period, the supernatant samples and tissues were quickly frozen in liquid nitrogen and stored at –80°C.

Western Blotting Analysis

Snap-frozen myometrial tissues were homogenized with Ultra-Turrax in homogenization buffer (10 mM Tris-HCl [pH 7.4], 1 mM EDTA, 40 mg/ml–1 leupeptine, and 2 mM Pefabloc). After an initial centrifugation at 500 x g for 15 min at 4°C, total protein supernatant content was determined by the Bradford method, with BSA as standard. Samples (40 µg of protein by lane) were dissolved (vol/vol) in 2x Laemmli buffer [35] and boiled for 5 min before electrophoresis on a 10% SDS-PAGE. Proteins were transferred to a nitrocellulose membrane (Hybond-P; Amersham Biosciences). In order to block nonspecific antibody binding, membranes were incubated for 1 h in 10% nonfat dried milk powder in Tris-buffered saline/Tween 20 (TBST; 10 mM Tris, 150 mM NaCl, and 0.1% Tween 20, pH 7.8) at room temperature. Blocked membranes were washed three times with TBST. The blots then were incubated overnight at 4°C with a 1:200 dilution of primary cleaved CASP3 antibody (ASP175; Cell Signaling Technologies, Beverly, MA) or a 1:500 dilution of primary BAX polyclonal antibody (sc-493; Santa Cruz Biotechnology) or a 1:500 dilution of primary BCL2 polyclonal antibody (sc-7382; Santa Cruz Biotechnology) in 1% nonfat dried milk powder in TBST. After three washes with TBST, the blots were incubated for 45 min with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG; NA 934; Amersham Biosciences) or anti-mouse IgG (NA 931; Amersham Biosciences) whole antibody at a dilution of 1:10 000 at room temperature and thereafter washed five times with PTBST. Immunoreactive proteins were detected by chemiluminescence (ECL detection reagents; RPN2105; Amersham Biosciences) and exposure to an x-ray film (Hyperfilm; Amersham Biosciences). The intensities of the bands were analyzed densitometrically using the National Institutes of Health Image 1.62 program and normalized with the intensity of bands obtained with monoclonal antibody to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which was used as protein loading control. Results are expressed as the mean ± SEM in arbitrary density units (ADU).

Immunohistochemical Analysis

The myometrial strips were fixed for 1 h with paraformaldehyde 4%, then embedded in paraffin and cut into 5-µm-thick sections. After deparaffinization of myometrial sections and rehydratation, antigen retrieval was performed by incubating slides for 10 min in warm citric acid buffer, pH 6, with a pressure cooker. After endogenous peroxidase activity was blocked with hydrogen peroxide (H2O2) 3%, slides were incubated either with primary anti-cleaved CASP3 IgG (1:100), washed three times with PBS, and then incubated with biotinylated anti-rabbit (1:600) immunoglobulin, respectively, for 1 h. After a new washing, slides were incubated in peroxidase-labeled streptavidin (1:800) for 30 min and then with 3-amino-9-ethyl carbazole solution until a clearly visible color was developed. The reaction was stopped by extensive washing in double-distilled water. Subsequently, the preparations were counterstained with hematoxylin. The histological changes were evaluated in terms of variation of global intensity of staining. Negative controls were carried out by omitting the primary antibody.

Hoechst Staining

To distinguish between apoptotic and necrotic cells, chromatin condensation state was assessed by staining of nuclei with Hoechst-33342. Paraffin-embedded sections of myometrium samples were rehydrated as described above. Slides then were incubated for 2 min with Hoechst-33342 (2 µg/ml). The slides were rinsed with distilled water and mounted with Aquatex (VGD, Inc.). The stained nuclei were visualized with convert fluorescent microscope at a magnification of 100x using excitation light at 350 nm.

Real-Time Quantitative RT-PCR

Total RNA was prepared from five myometrial tissues obtained from five different women using Trizol solution (Invitrogen Life Technologies, Groningen, The Netherlands) according to the manufacturer's instructions. The integrity of RNA was verified by edithidium bromide staining of agarose gel analysis and by an optical density (OD) absorption ratio OD 260 nm/OD 280 nm ≥ 1.8. One microgram of total RNA was reverse transcribed with Super Script II RNAase H-reverse transcriptase (Invitrogen Life Technologies) using oligo(dT) according to the manufacturer's instruction. Real-time quantitative PCR analyses were performed using 25 ng of reverse-transcribed total RNA with 200 nM of both sense and antisense primers in a final volume of 25 µl using the SYBR Green PCR jumpstart reagent (Sigma, St. Louis, MO) in an iCycler iQ real-time detection system instrument (Bio-Rad, Marnes-la-Coquette, France). Polymerase chain reaction products were also analyzed on edithium bromide-stained agarose gel to ensure that a single amplicon of the expected size was indeed obtained.

Each reaction was performed in duplicate, and GAPDH was used in each experiment to control for variability in the quantities of cDNA. Relative quantification for any given gene, expressed as fold variation over control, was calculated after determination of the difference between cycle threshold (Ct) value of the given gene according to manufacturer's protocol using the formula 2–{Delta}({Delta}CtA – {Delta}CtB), in which {Delta}CtA is {Delta}Ct of the gene of interest; and {Delta}CtB is {Delta}Ct of GAPDH. {Delta}Ct = Ct of the experimental group – Ct of the control group. Polymerase chain reaction was performed using specific primers: GAPDH (forward, 5'-TGCACCACCAACTGCTTAGC-3' and reverse, 5'-GGCATGGACTGTGGTC-ATGAG-3') and CASP3 (forward, 5'-AGAACTGGACTGTGGCATTGAG-3' and reverse, 5'-GCTTGTCGGCATACTGTTTCAG-3').

Cytokine Level Measurement

Cytokine measurements were determined by Cytometric Bead Array (CBA; Bender MedSystems). Supernatants of five myometrial strips were incubated with labeled capture beads and detection reagent for 3 h in the dark at room temperature and were analyzed with a flow cytometer (FACSCalibur; BD Biosciences) using the respective CBA Analysis software (BD Biosciences) and Bender MedSystems software. Cytokine standards for quantification (pg/ml) as well as the blanks were handled in the same manner as the samples. Each experiment was performed in duplicate to ensure for reproducibility of results.

Statistical Analysis of Results

Differences among groups were determined by ANOVA followed by the Dunn multiple comparison test. Statistical analysis was carried out using GraphPad Instat version 3 (GraphPad Software, San Diego, CA). All differences were considered significant at P < 0.05.

RESULTS

Chorioamnionitis Is Associated with Apoptosis in Human Myometrium, Which Is Mimicked by In Vitro E. coli LPS Stimulation

Immunostaining of four myometrial tissues samples obtained from four different women with confirmed chorioamnionitis using cleaved CASP3 antibody showed an intense staining (Fig. 1A, one representative picture) that was observed neither in one control tissue obtained from a woman with postpartum hemorrhage [36] (Fig. 1B, one representative picture) nor in three tissues obtained at elective cesarean delivery (Fig. 1C, one representative picture). This staining was specifically located in myometrial cells, as it is shown in Figure 1D. Lipopolysaccharide stimulation in optimal conditions (i.e., 10 µg/ml for 48 h, determined after time course—8 h, 24 h, 48 h—and dose-response—50 ng/ml, 1µg/ml, 10 µg/ml—experiments of LPS stimulation; see Supplemental Fig. 1, available online at www.biolreprod.org) also was associated with an intense, cleaved CASP3 staining specifically located in myometrial cells (Fig. 1, E and F, one representative picture).


Figure 1
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  		FIG. 1. Immunohistostaining of human myometrium section using a cleaved CASP3 antibody. A, D) One representative picture of confirmed chorioamnionitis using x100 (A) or x500 (D) magnification. B, C) One representative picture of two negative controls postpartum hemorrhage (B) or elective cesarean delivery (C) using x100 magnification. E, F) One representative picture of LPS-treated myometrial explants (10 µg/ml for 48 h) using x100 (E) or x500 (F) magnification.

Hoechst-33342 staining of a myometrial section obtained from women with chorioamnionitis (n = 4), postpartum hemorrhage (n = 1), or elective cesarean delivery stimulated (n = 4) or not stimulated (n = 4) with LPS showed that chorioamnionitis and LPS stimulation (Fig. 2, A and B, respectively) were both associated with an increased proportion of cells with condensed chromatin, indicating apoptosis initiation, compared with postpartum hemorrhage or cesarean delivery (Fig. 2, C and D, respectively).


Figure 2
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  		FIG. 2. Assessment of chromatin condensation state by Hoechst staining (original magnification x500). A) Chorioamnionitis, one representative picture. B) Elective cesarean delivery, one representative picture. C) Postpartum hemorrhage, one representative picture. D) LPS (10 µg/ml, 48 h) stimulation, one representative picture.Arrows in A and B indicate chromatin condensation of nuclei undergoing apoptosis.

Western blotting of membranes prepared from pregnant myometrium obtained from the same three women with chorioamnionitis (two samples, one obtained after hysterectomy for chorioamnionitis and the other for postpartum hemorrhage, were not available for Western blot experiment, since we had only access to paraffin-embedded sections) or from five cesarean deliveries and stimulated with LPS or not stimulated (controls) revealed a 17-kDa band and a 19-kDa band corresponding to cleaved CASP3 (Fig. 3A). Densitometric immunoblot analysis indicated that cleaved CASP3 protein was significantly overexpressed both in case of chorioamnionitis and after LPS stimulation compared with elective cesarean delivery without LPS stimulation used as control (expressed in ADU: 921 ± 39, 941.6 ± 134, and 452 ± 50.6, for chorioamnionitis, LPS 10 µg/ml, and control group, respectively; P < 0.05; Fig. 3B). The overexpression of cleaved CASP3 induced by LPS stimulation was in the same extent as that observed in Western blot experiments performed in membranes prepared from pregnant myometrium women with chorioamnionitis (Fig. 3B) providing external validation of an in vitro LPS-induced model of chorioamnionitis.


Figure 3
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  		FIG. 3. Western blot analysis using cleaved CASP3 antibodies. One representative experiment (A) and semiquantification (B) of the expression of cleaved CASP3 protein expressed in ADU (mean ± SEM). Experiments were performed in myometrial samples obtained from three different women with chorioamnionitis, and with five myometria, which were obtained at cesarean delivery from five different women with uncomplicated pregnancies and either stimulated or not stimulated with LPS (LPS and control groups, respectively). *P < 0.05 vs. controls. LPS, LPS-treated (10 µg/ml, 48 h) group; CA, chorioamnionitis.

This overexpression of cleaved CASP3 was strongly antagonized by the blockade of TNF-alpha receptor with a selective TNF-alpha antibody, suggesting that LPS-induced apoptosis in human near-term myometrium involves a TNF-alpha signaling pathway (ANOVA P < 0.05, n = 3; Fig. 4).


Figure 4
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  		FIG. 4. Effect of TNF on cleaved CASP3 expression in LPS-treated myometrial explants. One representative experiment (A) and semiquantification (B) normalized to GAPDH and expressed in ADU (mean ± SEM). Experiments were performed on three myometria obtained at elective cesarean delivery from three different women. *P < 0.05 vs. LPS. Controls, time-matched experiments; LPS, 10 µg/ml LPS 48 h; TNF-ab, 10 µg/ml LPS + 0.6 µg/ml TNF antibodies, 48 h.

Our model of inflammation was validated by the measurement of supernatant cytokine production. In our experimental conditions, LPS stimulation was associated at 48 h with a significant increase of interleukin 6 (IL6; mean ± SEM in pg/ml: 28 860 ± 5257 and 61 860 ± 12 190 for control and LPS groups, respectively; n = 10 experiments from five different women; P < 0.05) and IL8 (in pg/ml: 8855 ± 1486 and 16 080 ± 2834 for control and LPS groups, respectively; n = 10 experiments from five different women; P < 0.05) but not IL1B, IL10, IL12B, or TNF levels.

This overexpression of cleaved CASP3 protein also was observed at a transcriptional level, since quantitative real-time RT-PCR showed that, compared with time-matched controls, incubation with 10 µg/ml LPS for 48 h was associated with a 2.36 ± 0.22-fold increase in transcript levels (n = 3 experiments performed with myometrial tissues obtained from three different women).

This weak effect on CASP3 mRNA expression might be explained by our experimental conditions (i.e., 48 h of incubation with LPS). Indeed, in an exploratory analysis we assessed time trend for CASP3 mRNA expression (3, 6, 12, 48, and 72 h) and found that the peak for mRNA expression occurred around 3 h of stimulation (in fold increase compared with controls: 8.6, 6.1, 2.3, 2.1, and 1.2, at 3 h, 6 h, 16 h, 48 h, and 72 h, respectively).

The Selective ADRB3 Agonist SAR150640 Reverses LPS-Induced Apoptosis and Cytokine Production in Human Near-Term Myometrium

In this set of experiments, Western blot experiments revealed that the selective ADRB3 agonist, SAR150640, was able to significantly antagonize LPS-induced changes in cleaved CASP3 expression in a concentration-dependent manner (ANOVA P < 0.001; Fig. 5). Furthermore, we observed that LPS-induced activation of the mitochondrial pathway of apoptosis, as expressed by BAX and BCL2 protein upregulation and downregulation, respectively, was antagonized in a concentration-dependant manner (ANOVA P < 0.01; Fig. 6). SAR150640 had no effect by itself on cleaved CASP3, BAX, and BCL2 expression in tissues not stimulated with LPS (data not shown). The solvent for the highest concentration of SAR150640 (i.e., distilled water containing ethanol 0.3% and DMSO 0.02%) had no effect on cleaved CASP3, BAX, and BCL2 expression in tissues either not stimulated or stimulated with LPS (data not shown).


Figure 5
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  		FIG. 5. Effect of SAR150640 on cleaved CASP3 expression in myometrial explants stimulated by LPS. One representative experiment (A) and semiquantification (B) expressed in ADU (mean ± SEM) and normalized to GAPDH. Experiments were performed on five myometria obtained at elective cesarean delivery from five different women. #P < 0.05 vs. controls; *P < 0.05 vs. LPS. Controls, time-matched experiments; LPS, 10 µg/ml LPS 48 h.


Figure 6
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  		FIG. 6. Effect of SAR150640 on BAX and BCL2 expression in myometrial explants stimulated by LPS. One representative experiment (A) and semiquantification (B) expressed in ADU (mean ± SEM) and normalized to GAPDH. Experiments were performed on seven myometria obtained at elective cesarean delivery from seven different women. #P < 0.05 vs. controls; *P < 0.05 vs. LPS; **P < 0.01 vs. LPS. Controls, time-matched experiments; LPS, 10 µg/ml LPS 48 h.

The effect of SAR150640 on CASP3 overexpression was at least partially explained at a transcriptional level, since quantitative real-time RT-PCR showed that ADRB3 treatment was associated with a decreased level of CASP3 transcripts (in fold increase, normalized to GAPDH, compared with controls 2.08 ± 0.63, 1.17 ± 0.81, 0.84 ± 0.53, and 0.93 ± 0.41, respectively, for LPS alone and with SAR150640 10–7, 10–6, and 10–5 M, respectively, ANOVA P < 0.05).

Finally, SAR150640 decreased IL6 and IL8 in a concentration-dependent manner, even though the effect was statically significant only for IL8 (ANOVA P = 0.01; Fig. 7). SAR150640 had no effect by itself on IL6 and IL8 release in tissues not stimulated with LPS (data not shown). TNF was under the lower level of detection for the flow cytometry technique at 48 h.


Figure 7
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  		FIG. 7. Effect of SAR150640 on IL6 and IL8 levels in culture supernatants of LPS-treated myometrial explants. Results (mean ± SEM in pg/ml) are from five experiments performed in duplicate in myometria obtained at elective cesarean delivery from five different women. #P < 0.05 vs. controls; *P < 0.05 vs. LPS; **P < 0.01 vs. LPS.

DISCUSSION

Inflammation, or chorioamnionitis, plays a well-established role in the etiology of preterm labor, with evidence that it simultaneously exerts an adverse effect on the developing fetal or neonatal brain, with resultant negative implications for development in later childhood. There is clear evidence of a link between genital tract infection and preterm labor, although infections are frequently difficult to detect.

A major finding in this paper is that chorioamnionitis is associated with an increased apoptosis of human myometrium. Significantly enhanced expressions of CASP3 and BAX proteins, associated with a decreased expression of BCL2 protein, were observed in myometrial samples obtained from women with chorioamnionitis, but not women with post-partum hemorrhage or uncomplicated pregnancies. The apoptotic nature of the observed effect is supported by Hoechst staining and by a recent report showing that CASP3 induces apoptosis [37], an effect that is inhibited by CASP3 mRNA antisense oligodeoxynucleotides [38]. Our findings expand those of Muhle et al. [39], who have shown that infection-induced labor in mice is characterized by the upregulation of inflammatory response factors and apoptosis-associated genes. Indeed, we have demonstrated that chorioamnionitis is associated with myometrial apoptosis. Whereas the relationship between clinical infection and apoptosis is a new finding in human near-term myometrium, it has already been reported in animal models that Gram-negative bacteria, such as E. coli, can activate both the intrinsic and extrinsic apoptotic cascades [40].

The present study strengthens a previous report from members of our group in which it was shown that intrauterine injection of E. coli LPS in 15-day pregnant mice induced a rise of amniotic fluid levels of TNF, IL1B, IL6, and IL10 and provoked massive preterm delivery and fetal demise that were antagonized by phosphodiesterase-4 inhibition [41].

Nevertheless, it has to be agreed that the relationships between infection and apoptosis may vary according to the germ involved and the model, as Chlamydia can both inhibit [42] or induce apoptosis [10]. The infection-induced apoptosis observed in our study is a key finding, since Shynlova et al. [43] have suggested that apoptosis by itself might be an important phenomenon in switching myometrium from a quiescent to a contractile status. Thus, targeting apoptosis, and not only looking at inhibition of contractions, might be interesting for drugs designed to prevent or treat preterm labor.

We have shown that our model of LPS-induced chorioamnionitis is associated with the same level of myometrial cleaved CASP3 immunostaining and protein expression as that observed in chorioamnionitis, providing validation of this in vitro model. We observed that LPS-induced apoptosis is 1) associated with an involvement of both terminal and mitochondrial pathways, the latter being recognized as an amplification mechanism, and 2) a TNF-dependant mechanism. These findings are in good agreement with a study on cultured myometrial cell apoptosis induced by conditioned medium obtained from LPS-treated choriodecidua explants [41]. In this study, TNF neutralization was able to inhibit cell apoptosis.

The present study was designed to assess apoptosis and not to specifically address the issue of TNF production. Indeed, we knew that at 48 h of LPS stimulation we were not going to be able to show any increase in TNF production, since we have already published that under the same experimental conditions as in the present study but using ELISA, a more sensitive assay than the Multiplex technique used here, LPS-induced TNF production was of minimal extent (maximal value 7 pg/mg wet weight) and peaked at 6 h and declined thereafter [44].

The onset of parturition is itself associated with increases in a number of proinflammatory mediators, many of which are regulated by the nuclear factor kappa-B (NFKB1) family of transcription factors. The role of TNF, a potent activator of NFKB1, in parturition has already been suggested by Chapman et al. [45], who have shown that levels of this cytokine are increased within the myometrium at term and have observed, using primary cultures of myometrial cells, that TNF or exposure to bacterial LPS repressed expression of G-alpha-s and, thus, the cAMP/protein kinase A pathway able to inhibit myometrial contractions. Taken together, these works suggest that TNF may strongly participate in the complex network of signaling processes associated with uterine contraction and involution.

The second key finding of this work is that the selective ADRB3 agonist SAR150640 is able to oppose LPS-induced apoptosis by decreasing both CASP3 mRNA and protein expression in a concentration-dependent manner, a strong argument in favor of a pharmacologically mediated effect. This is of particular interest, since we have previously suggested that ADRB3 agonists might represent a new class of drug of interest in the pharmacological management of preterm labor [19–22, 46]. This finding seems to contradict a vast body of literature suggesting that ADRB stimulation is associated with an increase in apoptosis that might explain, at least partly, the role of chronic catecholamine exposure in the development of cardiac failure, giving a rationale for the use of beta-blockers in heart failure [47]. But besides the fact that the observed effect is likely to be different according to the model and the condition, it has been described that the relationship between ADRB stimulation and apoptosis depends upon ADRB subtype. For example, Fajardo et al. [48] have described that ADRB2 appears to play a cardioprotective role, whereas ADRB1 plays a cardiotoxic role. Moreover, it has been shown that blocking ADRB pathway with propranolol, a selective antagonist, in septic mice increased the splenocyte apoptosis rate [49] and that, on the contrary, the use of isoproterenol, a nonselective ADRB1, ADRB2, and ADRB3 agonist, decreased apoptosis of T-cell subtype [50]. There are very few previous publications to support our ADRB3-mediated antiapoptotic effect, but Lindquist et al. [51] have shown that ADRB3 stimulation is able to reverse DNA fragmentation induced in brown adipose tissue by cessation of sympathetic stimulus. We can, although not formally, suggest that the previously reported cAMP increase in myometrium induced by ADRB3 stimulation [19, 22] is implicated in the observed inhibition of apoptosis. Indeed, Kim et al. have described that LPS-induced apoptosis of human umbilical vein endothelial cells is reversed by cilostazol, a phosphodiesterase type III inhibition, or by dibutyryl cAMP [52], and suggested that LPS-induced apotosis is mediated through an activation of cAMP-dependent protein kinase. Furthermore, in perfect agreement with our findings, in this study LPS treatment was associated with a decrease in BCL2 and an increase in BAX protein expression that was reversed by cilostazol or by dibutyryl cAMP.

However, it is widely accepted that cAMP/protein kinase A regulates apoptosis in a cell type-specific manner (for review see Iwai-Kanai and Hasegawa [53]). We did not perform experiments after the blockade of the ADRB3, since no good ADRB3 antagonist is currently available. Nevertheless, an effect not mediated through ADRB3 stimulation is unlikely to be involved, since we have recently shown that SAR150640 has a high affinity for ADRB3-binding sites and lower affinity for ADRB1- and ADRB2-binding sites [30]. In this paper, the absence of affinity of SAR150640 for ADRB1 and ADRB2 was confirmed by in vitro functional studies.

In the present study, we also observed that LPS induced the production of IL6 and IL8 and that SAR150640 opposed this increase in a concentration-dependent manner. This inhibitory effect of SAR150640 on cytokine production is of interest, since previous reports have shown that infection may trigger the production of inflammatory cytokines in premature delivery [54]. Interleukin 6 and IL8 are cytokines of interest during pregnancy, since their increase has been documented in amniotic fluid in cases of preterm labor, even with intact membranes [55]. Furthermore, IL6 and IL8 have been described as mediators of cervical ripening, a key event in delivery occurring both at term or preterm [56]. Furthermore, inhibiting cytokine production might be of interest also because cytokines have been related to major neonatal complications in premature infants, such as cerebral palsy [57] or chronic lung disease [58]. Data on the relationship between cytokine and ADRB are sparse. Nevertheless, previous reports have described that ADRB2 agonists might decrease cytokine production. For example, Shinkai et al. [59] have described in an LPS-treated premature delivery mouse model that formoterol, an ADRB2 agonist, was able to block LPS-induced premature delivery as well as prostaglandin F2-alpha, IL1-alpha, and IL6 increases in the amniotic fluid, and IL6 in the plasma. The inhibition of cytokine production has also been described with nonselective ADRB agonists, such as isoproterenol [60].

Therefore, to summarize our results, we have shown that: 1) chorioamnionitis is associated with apoptosis of human myometrium; 2) with experimental conditions reproducing chorioamnionitis, LPS-induced apoptosis is TNF mediated and involved both extrinsic and intrinsic apoptotic pathways; 3) SAR150640, a new ADRB3 agonist, is able to oppose apoptosis at a transcriptional level; and 4) that selective ADRB3 agonist is able to oppose, in a concentration-dependent manner, LPS-induced IL6 and IL8 overexpression, two key cytokines in chorioamnionitis and preterm labor. Together, these results allow for a better understanding of the myometrial consequences of human chorioamnionitis. In light of our previous reports [19–22, 30, 46], the present study emphasizes the potential interest of the ADRB3 agonist, SAR150640, in the pharmacological management of preterm labor, since it also is able to oppose the triggering mechanisms (i.e., inflammation and apoptosis) and their consequences (i.e., myometrial contractions) and suggest some interesting anti-inflammatory properties.

ACKNOWLEDGMENTS

The authors wish to express their gratitude to the Obstetric & Gynecology Departments of the Hôpital du Bocage, Dijon, France, for making tissue samples available to us.

FOOTNOTES

1Supported by a grant from the Conseil Régional de Bourgogne and the Centre Hospitalier Régional et Universitaire de Dijon. This research is integrated in the European Preterm Labour Group. Back

Correspondence: 2LPPCE, Faculté de Médecine 7, bd Jeanne d'Arc, BP 87900, 21079 Dijon, France. FAX: 33 380 393 293; e-mail: marc.bardou{at}u-bourgogne.fr

Received: 20 July 2007.

First decision: 21 August 2007.

Accepted: 19 October 2007.

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