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PDE4 Inhibition Prevents Preterm Delivery Induced by an Intrauterine Inflammation¹

Thomas Schmitz^*,,, Evelyne Souil^,, Roxane Hervé^*,, Carole Nicco^,¶, Frédéric Batteux^,¶, Guy Germain^||, Dominique Cabrol^*,,, Danièle Evain-Brion^*,, Marie-Josèphe Leroy^*, and Céline Méhats^2,*,

^* Institut National de la Santé et de la Recherche Médicale, Unité 767, Paris, France; Université René Descartes, Paris, France; Assistance Publique-Hôpitaux de Paris, Hôpital Cochin, Maternité Port-Royal, Paris, France; Institut Cochin, Plateforme de Morphologie/Histologie Animale, Unité 567, Unité Mixte de Recherche 8104, Paris, France; ^¶ Laboratoire d’Immunologie, UPRES 1833, Paris, France; and ^|| Institut National de la Recherche Agronomique, Unité Mixte de Recherche 1198, ENVA, Centre National de la Recherche Scientifique, Formation de Recherche d’Emergence 2857, Biologie du Développement et Reproduction, Jouy en Josas, France

	Abstract

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The aim of this study was to explore the anti-inflammatory properties of phosphodiesterase-4 (PDE4) inhibitors in vivo and their potential ability to prevent inflammation-induced preterm delivery. Indeed, intrauterine inflammation is the major etiology of very preterm delivery, the leading cause of neonatal mortality and morbidity. Intrauterine injection of Escherichia coli LPS in 15-day-pregnant mice induced an increase of PDE4 activity and PDE4B expression at the maternofetal interface, a rise of amniotic fluid levels of TNF-, IL-1, IL-6, and IL-10 and provoked massive preterm delivery and fetal demise. Selective PDE4 inhibition by rolipram prevented the rise in the proinflammatory cytokines. Following the nuclear translocation of the transcription factor NFB, as a marker of cellular activation after the inflammatory challenge, showed a time-dependent sequential activation of the gestational tissues, from the uterine mesometrial to the fetal compartment, particularly in the glycogen-trophoblastic cells of the placenta. This activation was disrupted by PDE4 inhibition, and inflammation-induced preterm delivery and fetal demise were prevented. PDE4 selective inhibitors may thus represent a novel effective treatment to delay inflammation-induced preterm delivery and to prevent adverse outcomes in infants.

	Introduction

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In industrialized countries, 5–11% of infants are born preterm, before 37 wk of postmenstrual age. Very preterm delivery (before 32 wk postmenstrual age) accounts for 70% of neonatal deaths and up to 75% of neonatal morbidity and contributes to long term neurocognitive deficits and pulmonary dysfunction. Intrauterine infection has been recognized as the primary cause of very preterm delivery and favors development of periventricular leukomalacia and bronchopulmonary dysplasia (1, 2, 3). Often clinically silent, intrauterine infection triggers at the maternofetal interface a feed-forward inflammatory mechanism involving the production of cytokines, potent uterotonics, and metalloproteases, which results in preterm cervical ripening, uterine contraction, and rupture of the membranes and ultimately ends by preterm delivery. Studies in humans demonstrated higher amniotic fluid concentrations of IL-6, TNF-, and many other cytokines and chemokines in patients who subsequently delivered preterm than in patients who delivered at term (4). Systemic or intrauterine injection of IL-1, TNF-, killed bacteria, or bacterial products induces preterm delivery in pregnant mice, rabbits, sheeps, or monkeys (5).

The cyclic nucleotides phosphodiesterases (PDE)³ hydrolyze the ubiquitous second messengers, cAMP and cGMP, involved in the regulation of a wide variety of physiological processes including smooth muscle contractility, inflammation, and neuronal development. Eleven families of PDE are described to date with different affinities for cyclic nucleotides, specific modulators, and different patterns of expression (6). Their expression and regulation define the duration and the intensity of the signal generated by the cyclic nucleotides, building thus the specificity of the biological response.

Selective inhibitors of some PDE families are currently used in clinical practice for the treatment of cardiovascular disorders and erectile dysfunction and other PDE inhibitors are under development for the treatment of CNS and inflammatory disorders. The PDE4 family is one of the major PDE families controlling inflammation. PDE4 represents the major cAMP hydrolyzing enzyme family in all immunocompetent cells. Human monocytes and neutrophils present a predominant PDE4 activity (7), and PDE4 inhibitors suppress monocyte and macrophage production of TNF-, adhesion and chemotaxis of neutrophils, as well as superoxide production and degranulation (8). The mammalian PDE4 family is encoded by four genes, A, B, C, and D, composed of multiple transcriptional units and multiple promoters, which generate at least 20 different PDE4s (9). PDE4B products are thought to be specifically involved in inflammation (7, 10, 11, 12).

Herein, we investigate the ability of a PDE4 selective inhibitor, rolipram, to prevent in vivo preterm delivery and fetal demise in a murine model of intrauterine inflammation.

	Materials and Methods

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Animals

Procedures that involved mice were approved by our Institutional Committee on Animal Use and Care and were conducted in strict accordance with guidelines for the use and care of laboratory research animals promulgated by the National Institutes of Health. CD-1 timed-pregnant mice were purchased from Charles River Laboratories. Animals were shipped on day 12 after mating and were acclimated in our facility for 3 days before use.

Mouse model of inflammation-induced preterm delivery

On day 15 of gestation (75% CD-1 gestation), a minilaparotomy was performed under general anesthesia in the lower abdomen. Ten micrograms of LPS of Escherichia coli (O127:B8; Sigma-Aldrich) were injected in 100 µl of PBS between the first two gestational sacs of the left uterine horn according to the technique of Elovitz et al. (13). Control animals received 100 µl of sterile PBS. Animals were recovered in individual cages and were observed closely for any sign of morbidity (piloerection, decreased movements, vaginal bleeding). Two hours after surgery, mice received an i.p. injection of 100 µl of PBS containing either rolipram (3 mg/kg) or vehicle (5% DMSO). Preterm delivery was defined by delivery of at least one pup within the 48 h after surgery. Forty-eight hours after surgery, undelivered mice were placed under general anesthesia, and the number of live and dead pups in each horn was recorded. Intrauterine fetal deaths were identified by white discoloration of the pup and placenta and lack of blood flow in the umbilical cord.

Histochemistry and immunohistochemistry

For tissue collections, mice were killed 2, 4, or 6 h after surgery. Cervical tissue and intact uteroplacental units were harvested and either fixed in 10% formalin and embedded in paraffin or snap frozen. Cervix structures were identified on 5-µm paraffin slides by Masson’s Trichrome stain. For uterine and placenta histology and immunohistochemistry, direct adjacent sections were processed either for periodic acid-Schiff reaction (PAS) for NFB or uterine NK (uNK) cell detection. Briefly, slides were deparaffinized through two rinses in xylene (Prolabo) and rehydrated through graded ethanol to PBS (pH 7.4) or to deionized water. To detect NFB expression, we used the polyclonal Ab directed against the p65 form of the protein (Biosciences) diluted to 1/1000 in 1% PBS-BSA, 0.05% Triton. To detect uNK cells, we used HRP-Dolichos biflorus lectin (Sigma-Aldrich) diluted to 200 µg/ml in 1% PBS-BSA (14). Incubation was processed for 24 h at 4°C; and after successives rinses, endogenous peroxidase activity was blocked with 3% hydrogen peroxide. Finally, immunoreactivity was detected using the avidin-biotin Vectastain ABC Elite kit (Vector Laboratories). Color development was performed using Sigma Fast diaminobenzidine, and tissue sections were counterstained with Mayer’s hematoxylin solution (Merck). Nonspecific labeling by NFB Ab was assessed on direct adjacent sections by incubation with normal rabbit serum instead of the primary Ab or by omitting the primary Ab. In both cases, immunostaining was abolished. NFB expression and uNK cell number were analyzed by two investigators under microscope equipped with a DC 300F camera (digital module R, IM 1000) (Leica) using different magnifications.

Quantification of immunohistochemistry

Positively stained nuclei were counted at x200 magnification. Nuclei were counted in five distinct random fields per section, limited to the placental junctional zone and the adherent decidua basalis. The average number of positive nuclei per square millimeter for each tissue sample was used for statistical analysis. All sections were counted by a single-blinded investigator for internal consistency. Random sections were counted by a blinded independent investigator for external verification of the results.

Cytokines measurement in amniotic fluid

The amniotic fluids from the two gestational sacs adjacent to the site of injection were collected and snap frozen. Measurements of IL-1, IL-6, TNF-, and IL-10 concentrations were performed using the Searchlight multiplex sample testing by Endogen, PerbioScience.

Progesterone measurement in dam plasma

Blood was collected by sinus orbital puncture under anesthesia, 6 h after LPS instillation. Plasma samples were analyzed for progesterone concentration in duplicate by direct RIA, as previously described by Schanbacher (15) with some modifications. Charcoal-dextran solution was used instead of polyethylene glycol for the separation of bound and free radioactivity. Tritiated progesterone (1,2,6,7-[³H]progesterone, sp. act. 88 Ci/mmol) was obtained from Amersham, and a specific antiprogesterone Ab was obtained from the Pasteur Institute (Paris, France). To minimize assay variability, all plasma samples were analyzed in a single RIA. The limit of assay sensitivity was 0.1 ng/ml, and the intra-assay coefficient of variation was <10%.

cAMP-phosphodiesterase assay

Uterine and decidual-placental units were homogenized in ice-cold hypotonic buffer (20 mM Tris-HCl (pH 8.0), 50 mM NaF, 1 mM EDTA, 0.2 mM EGTA, 10 mM Na₂PO₄, 5 mM 2-ME, and a protease inhibitor mixture: 0.5 µg/ml leupeptin, 4 µg/ml aprotinin, 50 mM benzamidine, 0.7 µg/ml pepstatin, 10 µg/ml soybean trypsin inhibitor, and 10 µg/ml PMSF added freshly before use) using an all-glass homogenizer. Aliquots of the homogenates were assayed for cAMP PDE activity according to the method of Thompson and Appleman (16). PDE activities were measured with 1 µM [³H]cAMP as a substrate. PDE4 activity was defined as the fraction of cAMP PDE activity inhibited by 10 µM rolipram. Protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad Laboratories) with BSA as a standard.

Western blot analysis

Samples (30 µg protein/lane) were boiled in Laemmli buffer, subjected to electrophoresis on a 10% SDS-PAGE, and blotted onto Hybond-P transfer membrane (Amersham Pharmacia Biotech). Membranes were blocked in TBS-Tween 20 0.1% containing 5% nonfat milk. Polyclonal anti-PDE4B, (K118, 1:1,000, gift of M. Conti, Stanford University) were used (17). Second-step HRP-conjugated anti-rabbit Abs (1:5,000) were purchased from Amersham (Amersham Pharmacia) and visualized by use of the ECL detection reagents (Amersham Biosciences).

Data analysis

Unless otherwise stated, the significance of the difference was assessed by two-way ANOVA followed by Student’s t test, two-tailed for unpaired samples using the Prism-Graph Pad software (GraphPad Software). The difference was considered significant when p was < 0.05.

	Results

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Blockade of inflammation-induced preterm delivery and reduction of fetal demise by rolipram

Intrauterine injection of LPS provoked preterm delivery in 11 of 13 treated animals with full emptiness of the two uterine horns at 48 h postsurgery. Despite maintaining pregnancies, the two remaining LPS-treated dams experienced massive in utero fetal deaths (75%; Table I). However, no maternal mortality or morbidity was observed. Uterine infusion of saline solution or rolipram alone resulted in minimal preterm delivery and in minimal fetal demise, observed only at the insert of the intrauterine injection. Treatment with rolipram 2 h after the LPS injection blocked preterm delivery and significantly reduced fetal demise (22%).

Cervical ripening is characterized by a dispersion of collagen fibers of the stroma and by increase in the mucin secretion (18). To document the onset of preterm labor induced by LPS and its prevention by rolipram, sections of cervical tissue from the four groups of animals, injected with saline or LPS with or without rolipram, were mounted and assessed for histological aspect and collagen organization with Masson’s Trichrome stain. In the LPS-injected group, 6 h postsurgery, the glandular epithelium showed increased volume of the epithelial glands containing secretory vacuoles. Trichrome staining revealed a loose array of disordered collagen fibers, which are histological features of compliant tissue (Fig. 1B). In contrast, animals injected with LPS and rolipram exhibited noncompliant tissue; denser, compact, heavily stained matrix of the collagen fibers associated with undeveloped glands; and absence of secretory vacuoles in the glandular epithelium as observed in the sham-operated control groups (Fig. 1, A–D).

View larger version (160K): [in this window] [in a new window]   FIGURE 1. Inhibition of inflammation-induced cervical ripening by rolipram. Cervical sections were assessed, 6 h after the inflammatory challenge, for histological aspect and collagen content (blue-green coloration) using Masson’s Trichrome stain. A, Saline-vehicle-treated animals; B, LPS-vehicle-treated animals; C, saline-rolipram-treated animals; D, LPS-rolipram-treated animals. Representative sections of the tissues obtained from five animals per group are shown. Scale bar, 400 µm.

To assess that the onset of preterm labor in LPS-injected dams was concomitant to an intrauterine inflammation and that this phenomenon was prevented in the presence of rolipram, we measured the concentration of cytokines in amniotic fluids, namely TNF-, IL-1, IL-6, and IL-10, which have been reported to be involved in inflammation-induced preterm delivery (19, 20). As depicted in Fig. 2, intrauterine injection of LPS induced a significant increase in the concentrations of these cytokines in the amniotic fluid, 6 h postsurgery, as compared with the saline-vehicle animals, demonstrating that an intrauterine inflammation has been mounted against LPS in these animals. The injection of rolipram alone did not significantly affect the concentrations of these factors; but it significantly diminished the increase of TNF-, IL-1, and IL-6 concentration in the amniotic fluids of LPS-injected animals while not significantly affecting IL-10 concentration.

View larger version (23K): [in this window] [in a new window]   FIGURE 2. Inhibition of inflammation-induced cytokine synthesis in amniotic fluid by rolipram. Concentrations of TNF-, Il-1, Il-6, and IL-10 were measured by multiplex ELISA in amniotic fluid of saline-vehicle, saline-rolipram, LPS-vehicle, and LPS-rolipram dams, 6 h after LPS challenge. Box-and-whisker representations indicate the median and the overall range of distribution of the measurements, the bars indicating the smallest and greatest values, obtained from 12 different animals. Significant difference from saline-vehicle group: *, p < 0.05; **, p < 0.01.

In the same mice, the concentration of circulating progesterone was evaluated 6 h postsurgery. Mice of each group demonstrated high plasma progesterone levels without significant difference between the four groups (Fig. 3).

View larger version (15K): [in this window] [in a new window]   FIGURE 3. Progesterone plasma level during intrauterine inflammation. Concentration of progesterone was measured by RIA in plasma of saline-vehicle, saline-rolipram, LPS-vehicle, and LPS-rolipram dams, 6 h after LPS challenge. Box-and-whisker representations indicate the median and the overall range of distribution of the measurements, the bars indicating the smallest and greatest values, obtained from 12 different animals.

To investigate the level of PDE4 activity in mouse gestational tissues at the time of the injection of rolipram, we measured PDE activity in whole homogenates of uterine tissues (myometrium and endometrium) or placental units, with the decidual cap still attached, collected 2 h after surgery. As reported in Fig. 4, PDE4 activity, gauged as rolipram-sensitive activity, represented almost one-half of the total cAMP-PDE activity in both tissues. Uterine tissues contained 10 times more cAMP-PDE and PDE4 activities than did decidual-placental units; however, no change was observed upon stimulation by LPS. Meanwhile, PDE4 activity increased significantly in the decidua-placental unit. Western blot analysis of PDE4B expression in the decidua-placental unit showed an increase in the signal of a short product of PDE4B gene at 72 kDa, corresponding to the PDE4B2 form. PDE4B2 was also expressed in uterine tissues, but no change upon LPS challenge was observed (Fig. 4).

View larger version (29K): [in this window] [in a new window]   FIGURE 4. Expression of PDE4B2 in decidual and placental units. A, Uterine tissues and decidual-placental unit from saline-vehicle, LPS-vehicle dams, 2 h after LPS challenge, were homogenized, and cAMP PDE activity was measured in the absence or the presence of 10 µM rolipram (Rol). Data are expressed as the mean ± SEM for three different experiments using a total of five animals per group. *, p < 0.05: significantly different from saline-vehicle group. B, Decidual-placental units from saline-vehicle or LPS-vehicle dams for the indicated time were homogenized. Thirty micrograms of the protein extracts were subjected to 8% SDS-PAGE and electroblotted. PDE4B protein was detected by incubation with the polyclonal Abs K118 and a peroxidase-conjugated secondary Ab. Representative blots of three different experiments performed with tissues from three different animals are shown.

Inhibition of LPS-induced NFB nuclear translocation in the gestational tissues by rolipram

LPS promotes nuclear translocation and activation of the transcriptional factor, NFB, most often found as a p50-p65 dimer unit. The p65 subunit is mainly responsible of the nuclear translocation of this complex, a nuclear localization signal being coded within its protein sequence (21). Thus, we looked for the cellular localization of p65-NFB subunit in gestational tissues using immunohistochemistry 2, 4, and 6 h after surgery (Fig. 5). In uterine tissues, p65-NFB was readily in the nucleus of endometrial cells in sham-operated animals at H0 and remained in the nucleus at 2, 4, and 6 h, whereas rare cytoplasmic signals were detected in the myometrium and were not affected by LPS treatment (data not shown). p65-NFB was found rarely within the nucleus of cells of the decidua-placental units in sham-operated animals at 2 (Fig. 5B), 4, and 6 h. Markedly, a significantly high number of stained nuclei was observed at 2 h in the maternal cells in the decidual cap of LPS-treated animals (Fig. 5C). This number of stained nuclei remained high at 4 h in the maternal compartment and was then also observed in the fetal cells of the placenta. At 6 h, only glycogen trophoblasts, recognized by their characteristic histological aspect and their staining with PAS (22 ; Fig. 5A), depicted a nuclear signal in LPS-treated animals (Fig. 5D). Treatment with rolipram abolished the nuclear signals in both maternal and fetal compartments (Fig. 5E). Quantitative analysis of nuclear NFB staining demonstrated significant inhibition of nuclear NFB translocation by rolipram 4 and 6 h after surgery (Fig. 6).

View larger version (109K): [in this window] [in a new window]   FIGURE 5. Nuclear localization of NFB in decidual and placental units during intrauterine inflammation. Diagram representing decidual-placental units on 15th gestational day. Square, regions photographed in the mesometrial side (M), junctional zone (JZ), and labyrinth (L). A, PAS staining of a saline-vehicle dam; B and C, NF-B immunostaining in saline-vehicle and LPS-vehicle dams, respectively, 2 h after the surgery. D and E, NF-B immunostaining in LPS-vehicle and LPS-rolipram dams, respectively, 6 h after the surgery. Arrowheads, glycogen trophoblasts in the junctional zone. Representative sections obtained from six different animals per group are shown. Scale bar, 100 µm.

View larger version (20K): [in this window] [in a new window]   FIGURE 6. Prevention of sustained NFB nuclear location by rolipram. Positively NFB nuclei were counted in the decidual-placental unit sections obtained from saline-vehicle, LPS-vehicle, saline-rolipram, and LPS-rolipram dams, for the indicated time after LPS challenge. Data are expressed as the mean ± SEM obtained from six animals per group. **, p < 0.01: significantly different from saline-vehicle group.

Because uNK cells are the main inflammatory cells within gestational tissues, we checked their presence in the decidual cap upon treatment using the highly selective D. biflorus lectin. An increase in the number of uNK cells in the mesometrial compartment (Fig. 7) 4 h after LPS treatment was observed. This increase was prevented in the presence of rolipram. However, the NFB nuclear signal and uNK cells were not superposed.

View larger version (127K): [in this window] [in a new window]   FIGURE 7. Prevention of uNK cell number increase in gestational tissues by rolipram. Decidual-placental units were assessed, 4 h after the inflammatory challenge, for the presence of uNK cells using the D. biflorus lectin. A, saline-vehicle-treated animals; B, LPS-vehicle-treated animals; C, saline-rolipram-treated animals, D, LPS-rolipram-treated animals. Representative sections of the tissues obtained from five animals per group are shown. Scale bar, 100 µm.

	Discussion

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Murine models of preterm delivery have been developed these last decades, based on systemic or local injection of heated-dead bacteria or bacterial products, including LPS at doses ranging from 10 to 250 µg, to provide new insights into the molecular events underlying causes of preterm labor (reviewed in Refs. 5 and 21). Indeed, in humans, the ethical status of the fetus and large discrepancies in clinical variables among patients at the time of asserted preterm labor prohibit any possibility of conducting experimentally controlled basic research. The murine preterm labor model provides a powerful means for maintaining tight experimental controls in an in vivo context. Using such a model, we showed that an injection of a low dose of LPS in the uterus between two gestational sacs caused 85% of preterm delivery with full emptiness of the uterus and 75% of fetal demise in the undelivered dams within 48 h. PDE4 inhibition 2 h after the intrauterine inflammatory challenge blocks preterm labor as documented by the absence of morphological changes in the cervix and subsequent preterm delivery. Furthermore, PDE4 inhibition diminished to more than one-third the rate of fetal demise in undelivered dams (22% vs 75%), though this figure underestimates the rate of intrauterine death before expulsion in the LPS-injected group.

We also showed increase in the amniotic fluid concentration of key cytokines 6 h after the inflammatory challenge, namely, TNF-, IL-1, IL-6, and IL-10. Elevated levels of TNF-, IL-1, and IL-6 are found in amniotic fluid during pregnancies complicated by infection and preterm delivery in humans (23). The IL-6 level has been proposed as a marker for the prediction of preterm delivery (24). TNF-, IL-1, and IL-6 can induce the production of both prostaglandins and metalloproteases leading to preterm delivery (19, 20). Double knockouts for TNF- and IL-1 receptors are refractory to bacterially induced preterm labor (22). Conversely, the anti-inflammatory cytokine IL-10 has been shown to prolong gestation and to diminish fetal demise in bacterially treated dams when administered alone or in combination with antibiotics (25). In our model, PDE4 inhibition blocked rise in TNF-, IL-1, and IL-6, whereas it had no significant effect on IL-10 level. Manipulation of these cytokines by PDE4 inhibition may provide a mechanism by which rolipram prevents both inflammation-induced delivery and fetal demise.

Several compounds have been shown to delay inflammation-induced preterm delivery, lactoferrin, -methasone, pharmacological doses of progesterone, or a progestin derivative, medroxyprogesterone acetate, antioxidant N-acetylcysteine, platelet-activating factor antagonist, metalloprotease inhibitor phosphoramidon, prostaglandin G/H synthase (PGHS) inhibitors such as indomethacin, PGHS-1 and PGHS-2 inhibitors, and PGF₂ receptor antagonist (13, 23, 24, 26, 27, 28, 29, 30). Each of these compounds interacts with one or more of the factors believed to have a role in the inflammatory cascade leading to preterm birth. However, in all these studies, most of the undelivered fetus died in utero, the rate of fetal demises ranging to 60–100%, although the drugs were given before the inflammatory challenge, in the contrary to the present study where PDE4 inhibition starts 2h after LPS challenge.

One major discrepancy between humans and mice gestation consists on the fact that maintenance of gestation in mice depends on the production of progesterone by the corpus luteum all along the gestation (31). However, despite the fact that physiological progesterone supplementation prolongs the interval to delivery in ovariectomized animals, it has no effect in bacterially exposed animals, implying that progesterone withdrawal is not the priming event of inflammation-induced preterm delivery. Consistent with others’ studies, we documented no significant decrease in plasmatic progesterone concentration after intrauterine inflammatory challenge at a time when labor has started with cervical modification (13, 32).

PDE4 inhibition impacts both uterine and decidua-placental tissues, insofar as we detected PDE4 activity in these tissues. We documented that PDE4B2, a PDE4 isoform involved in inflammation, is induced in the decidual-placental unit by LPS, concomitantly to a significant increase in PDE4 activity. These data suggest a role for PDE4B2 in this compartment upon LPS inflammatory cascade, while no change of PDE4 activity or PDE4B2 expression was observed in the uterine tissues.

To identify, within the materno-fetal interface, the cells involved in the LPS inflammatory cascade, we searched by immunohistochemistry the localization of NFB. In our model, we observed a sequential nuclear translocation of NFB from the maternal to the fetal compartment. We evidenced nuclear localization of NFB 2h after the inflammatory challenge in the uterine mesometrial compartment (33). We also confirmed an increase of the number of uNK cells after the LPS challenge, consistent with earlier report (34). As PDE4B2 expression is reported high in immunocompetent cells (35), increase of the number of uNK cells may then account for the increased presence of PDE4B2 within the decidual-placental unit. The presence of active uNK cells has been shown to be controlled by IL-10 (34). The IL-10 knockout strain displays a high number of uNK cells in the uterine mesometrial compartment. Moreover, this KO strain is highly sensitive to very low doses of LPS, which induce massive fetal demise, not seen in the wild-type littermates. It has been speculates that uNK cells may serve as a failsafe mechanism to terminate pregnancy, when excessive inflammatory or other insults are experienced (34).

Four hours after the LPS insult, NFB was also localized in the nucleus of a specific trophoblast in the fetal compartment, the glycogen trophoblast. In mice, glycogen trophoblasts appear in the junctional zone of the placenta at 12 days and invade the uterine mesometrial compartment until term (36, 37). Their functions remain largely unknown; however, the absence of differentiation of these cells in IGF-2 KO strain delays the onset of term labor (38). Further studies on these specialized cells are required to decipher their role in nourishing the feed-forward mechanisms of intrauterine inflammation to preterm delivery.

We showed that the nuclear translocation of NFB is prevented by PDE4 inhibition in the cells of the maternofetal interface, with no more nuclear signal 6 h after LPS challenge. In a LPS-induced uveitis model in rat, p65-NFB was also excluded of the nucleus of the iris-ciliary body of LPS-treated animals by rolipram (39). Therefore, prevention of nuclear NFB translocation may represent a hitherto unnoticed molecular mechanism by which rolipram or PDE4 inhibition exerts its anti-inflammatory effect.

The therapeutic strategy in case of preterm labor, namely tocolysis, is aimed to delay birth by inhibiting uterine contractions (40). However, conventional methods of tocolysis are ineffective on the inflammation component of preterm labor and expose the fetus longer to stimuli noxious for the immature brain. Novel tocolytic agents that down-regulate intrauterine inflammation may then offer a solution for the prolongation of pregnancy, safe for the fetus. We showed previously in humans that PDE4 inhibition blocks spontaneous contractions of myometrial strips. It also abolished the activation in fetal membranes of metalloproteases and prostaglandin syntheses that may lead to preterm premature rupture of the membranes (41, 42, 43). Here, we report that PDE4 inhibition blocks intrauterine inflammation, which may injure the fetus if uncontrolled. PDE4 inhibitors may thus represent a novel class of tocolytic, also able to control inflammation and its consequences for the infant.

	Acknowledgments

 
We are indebted to Dr. Michelle Breuillet-Fouché for helpful discussions, Dr. André Malassiné for his expertise on placental development, and Professor Marco Conti for the PDE4B-specific Abs.

	Disclosures

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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 March of Dimes Birth Defects Foundation Grant 6-FY03-6.

² Address correspondence and reprint requests to Dr. Céline Méhats, Faculté de Pharmacie, Institut National de la Santé et de la Recherche Médicale, Unité 767, 4 Avenue de l’Observatoire, 75270 Paris cedex 06, France. E-mail address: mehats{at}cochin.inserm.fr

³ Abbreviations used in this paper: PDE, phosphodiesterase; uNK, uterine NK; PAS, periodic acid-Schiff; PGHS, prostaglandin G/H synthase.

Received for publication September 11, 2006. Accepted for publication November 7, 2006.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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