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Regular Article |
a Department of Biology,
b Section of General Physiology, University of Ferrara, 44100-I Ferrara, Italy Department of Morphology and Embryology,
c Section of Human Anatomy, University of Ferrara, 44100-I Ferrara, Italy Department of Biomedical Sciences and Advanced Therapy, Section of Obstetrics and Gynecology, University of Ferrara, 44100-I Ferrara, Italy
ABSTRACT
The presence of binding sites for formyl-methionyl-leucyl-phenylalanine (fMLP), its effect on prostaglandin E (PGE) release, and the signal transduction pathway activated by the peptide were investigated in human amnion-derived WISH cells. Our results demonstrate that specific binding sites for fMLP are present on WISH cells and that the peptide induces a significant increase of prostaglandin (PG)E2 release. The kinetic properties of binding are similar to those previously found in amnion tissue prior to the onset of labor, i.e., only one population of binding sites with low affinity for the peptide is present. Binding of 3H-fMLP in WISH cells is inhibited by N-t-butoxycarbonyl-methionyl-leucyl-phenylalanine, an fMLP receptor antagonist, with an IC50 value very close to that shown by nonlaboring amnion. The fMLP-induced PGE2 output is inhibited by indomethacin, quinacrine, and U-73122, inhibitors of cyclooxygenase, phospholipase A2, and phospholipase C, respectively. As regards the transduction pathway activated by fMLP, we demonstrate that phospholipase C activation, followed by an increase of intracellular calcium concentration ([Ca2+]i), is involved in response to the peptide. Our results add further evidence to the role of proinflammatory agents in the determination of labor. Furthermore, because WISH cells appear to behave like nonlaboring amnion tissue, they represent the ideal candidate for in vitro investigation of the events triggering the mechanism of delivery.
calcium, cytokines, parturition, pregnancy, signal transduction
INTRODUCTION
It is well known that gestational tissues increase prostaglandin production in both term and preterm labor, as suggested by their elevated concentrations in the amniotic fluid and maternal plasma [1, 2]. These prostanoids, in particular prostaglandin (PG)E2 and PGF2
, induce uterine contractions by means of a paracrine mechanism [3]. Prostaglandin E2 is the main prostanoid produced by gestational tissues, and its biosynthesis is affected by several agents, among which oxytocin, growth factors, and cytokines such as several interleukins (ILs) and tumor necrosis factor- (TNF), as well as other factors [4–6]. It has been demonstrated that the above-mentioned cytokines, through an increase of amniocyte phospholipase (PL)A2 and cyclooxygenase 2 (COX 2) expression [7, 8], act as potent physiological regulators of PG biosynthesis in normal labor at term, but they are also involved in pathological conditions such as preterm labor, whether dependent on or independent of intrauterine infections.
In addition to cytokines, formyl-methionyl-leucyl-phenylalanine (fMLP) must be considered a factor involved either directly or indirectly in PG production by fetal membranes. Indeed, in a previous report we provided the first evidence for the presence of specific binding sites for 3H-fMLP in crude plasma membranes obtained from term human amnion, as well as for a direct action of the peptide on amniotic PGE2 release [9]. Furthermore, it has been reported that the peptide-induced activation of granulocytes and mononuclear cells leads to the release of cytokines that stimulate PGE2 production by amniocytes [10].
In addition to amniocytes, WISH cells that are human amnion-derived cells have also been shown to release PGs and cytokines in response to different stimuli [11–14]. This cell line hence represents an ideal candidate for an in vitro study of the events triggering the onset of labor.
In this study we investigated the presence of fMLP binding sites, their possible effect on PGE2 release, and the transduction pathway activated by the peptide in WISH cells.
MATERIALS AND METHODS
Cell Culture and Treatments
Amnion-derived WISH cells were obtained from the American Type Culture Collection (ATCC CCL-25) and maintained in the laboratory. Cells were grown at 37°C in an atmosphere of 5% CO2 and 95% air, in a mixture of Ham's F12 and Dulbecco modified Eagle medium (F12/DMEM) (1:1 vol/vol) supplemented with 10% fetal bovine serum (FBS), 30 µg/ml gentamicin, and 0.25 µg/ml amphotericin B [11].
To evaluate the effects of fMLP alone or in combination with other agents on PGE2 production, WISH cells were seeded in 24-well plates at 2 x 105 cells/well in F12/DMEM + 10% FBS and grown to confluence (2–3 days); the medium was then replaced with fresh serum-free medium for 24 h. This medium was removed and again replaced with fresh serum-free medium, containing increasing concentrations of fMLP (10 nM to 10 µM) or 50 µM N-t-butoxycarbonyl-methionyl-leucyl-phenylalanine (Boc-MLP), an fMLP receptor antagonist. Indomethacin, quinacrine, and U-73122, inhibitors of COX, PLA2, and phospholipase C, respectively, were added 30 min before the addition of fMLP. After incubation of samples for the indicated time, the media were collected and stored at -80°C until PGE2 RIA was performed. The fMLP binding receptor assays to whole WISH cells were carried out. Cells were harvested (107 cells/flask) by trypsinization, suspended in PBS for neutralization, and pelleted by centrifugation at 500 x g for 5 min.
Binding Assay
Radioreceptor assay for fMLP binding was performed as described previously [9]. Briefly, 106 cells were incubated in Dulbecco medium, pH 7.4, in the presence of 5 nM [3H]fMLP alone to determine the specific binding of fMLP or in combination with 10 nM to 50 µM unlabeled fMLP for Scatchard analysis. Nonspecific binding was determined by adding 10-4 M unlabeled fMLP in both the above tests. To determine the ligand specificity of the receptor, competitive binding assays were performed with Boc-MLP (10-8 to 10-4 M).
Prostaglandin E2 RIA
The amount of E series PGs was assayed in the collected media by an RIA procedure, as previously described [9]. A specific antiserum for PGE1 and PGE2 (cross reactions 100% and 165%, respectively) was used. Labeled [3H]PGE2 (3 nCi) was added to each tube. Assay sensitivity was 15 pg/tube, and the intra- or interassay coefficients of variation were <10%. Data were expressed as nanograms of PGE2 produced/106 cells.
Measurements of Ca2+ with Fluorescent Indicators and Confocal Imaging in WISH Cells
WISH cells, grown to 40–60% confluence (2 days after plating) at 37°C in an atmosphere of 5% CO2 and 95% air on glass coverslips 12 mm in diameter, were washed three times with 1 ml of medium containing: 125 mM NaCl, 5 mM KCl, 1.5 mM CaCl2, 1 mM MgCl2, and 10 mM glucose, buffered to pH 7.4 with 25 mM Hepes.
Cells were loaded at 37°C for 30 min with the Ca2+-sensitive fluorescent dye Fluo-3 acetoxymethyl ester (Fluo-3/AM) that was diluted in the medium to a final concentration of 5 µM from stock solutions (10 mM) frozen in dimethylsulfoxide.
After being loaded, the glass coverslip was placed in a 500-µl bath chamber, and loaded cells were left to hydrolyze for 15 min to ensure the complete hydrolysis of acetoxymethyl ester groups; the chamber was then placed on an inverted microscope. Samples were imaged by an LSM410 inverted confocal microscope (Zeiss, Oberckochen, Germany) coupled with a 25-mW multiline argon ion. Laser light was attenuated to 3% of transmission with a neutral density filter to limit bleaching of Fluo-3 fluorescence, and to avoid cell damage. Settings for all experiments were rigorously maintained as described previously [15]. Intracellular calcium concentration ([Ca2+]i) was measured as previously described [11]. Quantitative fluorescence mean values in different cells, expressed as arbitrary units, were calculated from a sequence of images obtained throughout the experiment and were plotted as different diagrams.
Data Expression and Analysis
Binding data were analyzed using Radlig version 4 by Dr. G.A. McPherson (Biosoft, Cambridge, UK). This program utilized a nonlinear least-squares curve-fitting algorithm and assumed the simultaneous contribution of one or more independent binding sites. Data are expressed as mean ± SEM. Statistical significance was assessed by one-way ANOVA, followed by Dunnett's t-test; P < 0.05 was considered significant.
Chemicals
[5,6(n)-3H]PGE2 (181 Ci/mmol) and [3H]fMLP (71.5 Ci/mmol) were purchased from Amersham Pharmacia Biothec Srl (Milan, Italy) and NEN Life Sciences Products, Inc. (Milan, Italy), respectively. Whatman GF/B glass fiber filters were from Whatman Int. (Maidstone, Kent, England). The fMLP, Boc-MLP, quinacrine, PGE2, anti-PGE-BSA serum, and indomethacin were from the Sigma Chemical Co. (St. Louis, MO). The U-73122 was from Biomol Research Lab, Inc. (Plymouth Meeting, PA). All tissue culture media and sera were purchased from Gibco BRL (Paisley, Scotland). The Fluo-3/AM was obtained from Molecular Probes (Eugene, OR). All other chemicals were the highest reagent grades commercially available.
RESULTS
Binding of 3H-fMLP
The specific binding of 3H-fMLP to amnion-derived WISH cells was rapid, reaching a maximum at 15 min; this value was stable until at least 120 min at 37°C. Binding was saturable, and nonspecific binding, evaluated in the presence of 10-4 M unlabeled fMLP, never exceeded 10% of total binding. A Scatchard analysis was performed, where nonlinear iterative curve-fitting elaboration of binding data revealed the existence of a single class of binding sites. The mean values of the dissociation constant (Kd) and the maximal density of binding sites (Bmax) were 6.3 ± 0.6 µM and 23 100 ± 2080 sites/cell, respectively.
Under the same experimental conditions, the fMLP receptor antagonist Boc-MLP was then utilized in competition binding experiments, where increasing concentrations of the peptide (10-8 to 10-4 M) progressively inhibited [3H]-fMLP binding to WISH cells. The calculated IC50 was 56.4 ± 5.1 µM.
Prostaglandin E2 Release from WISH Cells
In a first series of experiments, we tested the ability of fMLP to affect PGE2 release from WISH cells. In Figure 1, the time-courses of basal and fMLP-induced PGE2 output are illustrated. Both basal and fMLP-stimulated release progressively increased with time, reaching a plateau after 30 min of incubation. The addition of 10-6 M fMLP increased PGE2 output at each time tested; this effect became statistically significant at 15 min of incubation and reached maximal stimulation at 30 min. In the subsequent experiments, the reactions were therefore carried out for 30 min.
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Concentrations of fMLP, ranging from 10-8 to 10-5 M, dose-dependently increased PGE2 production; the effect became statistically significant (P < 0.05) at 10-7 M and reached the maximum (+67%, with respect to basal value) at 10-6 M. No further increase in PGE2 output was observed in the presence of higher peptide concentrations (Fig. 2).
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Figure 3 reports the effect of 5 x 10-5 M Boc-MLP (a concentration close to IC50, the value obtained from the binding studies) on both basal and fMLP-stimulated PGE2 output. As shown, the receptor antagonist was ineffective in inhibiting basal PGE2 release but very efficaciously prevented activation by fMLP.
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To provide evidence that fMLP-induced PGE2 synthesis is a COX-driven event, we preincubated WISH cells with various concentrations (10-9 to 5 x 10-6 M) of indomethacin for 30 min. Cells were then incubated with or without 10-6 M fMLP. As shown in Figure 4, indomethacin dose-dependently reduced both basal and fMLP-evoked PGE2 output, the effect becoming statistically significant (P < 0.05) at 10-7 M and reaching the maximum (about -85% with respect to values obtained in the absence of indomethacin) at 10-6 M; inhibition remained almost unchanged at higher doses of the COX inhibitor.
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We then tested the effect of increasing concentrations (10-8 to 10-5 M) of quinacrine, a PLA2 inhibitor [16], added 30 min before fMLP. The drug was ineffective in inhibiting basal PGE2 release but dose-dependently reduced fMLP-evoked PGE2 output; the effect was statistically significant (P < 0.05) at 5 x 10-7 M, reaching the maximum (-40% of the value obtained in the absence of the inhibitor) at 10-5 M (Fig. 5).
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In order to evaluate the role of phospholipase C (PLC) activation on fMLP-induced PGE2 increase, WISH cells were preincubated for 30 min with various concentrations (10-8 to 10-5 M) of U-73122, a PLC inhibitor [17], and then incubated with or without 10-6 M fMLP. As shown in Figure 6, basal PGE2 release was unaffected by all the tested concentrations of the drug, but fMLP-induced PGE2 production was dose-dependently inhibited; the inhibitory effect of U-73122 became statistically significant at 5 x 10-7 M (P < 0.05) and maximum at 10-5 M (-40%).
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An fMLP-Induced [Ca2+]i Transient in Single WISH Cells
We assessed the intracellular level of Ca2+ indicator dyes in confocal laser scanning microscopy-imaged WISH cells loaded with Fluo-3/AM. Scanned images were obtained from a single optical slice passing through the plane of the cell nucleus with a temporal resolution of 0.5 sec and were analyzed as a function of time.
Treatment with 10-6 M fMLP, the most effective dose for inducing PGE2 release, evoked a rise in intracellular calcium, as evidenced by the increase in fluorescence level (Fig. 7, B and C). We observed that fMLP stimulation resulted in an increased level of intracellular [Ca2+] that appeared at a defined time. Data obtained from computer processing of fluorescence images showed that fMLP-induced [Ca2+]i enhancement was 50% above the basal value and was statistically significant for the majority of cells analyzed (Fig. 7, B and C), while a weak response in the fluorescence intensity levels was observed in nearly 39% of the cells (Fig. 7D). The rise of [Ca2+]i levels started immediately after fMLP exposure, and took 15 sec to reach the maximum level, indicating that Ca2+ is involved in a response that requires different steps in order to be activated.
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Furthermore, the role of PLC-mediated [Ca2+] level oscillations was investigated by employing the specific U-73122 PLC inhibitor. As can be observed in Figure 7E, no generation of [Ca2+]i oscillations was produced, thus showing that a PLC-mediated response is required for [Ca2+]i rise after fMLP treatment.
DISCUSSION
The peptide fMLP is prototypic for a series of chemoattractants that are isolated from bacterial culture suspensions [18] and are possibly released by disrupted mammalian cells [19]. Granulocytes and mononuclear cells are considered the conventional target for fMLP actions, but it has recently been shown that the peptide exerts effects that do not depend on its chemoattractant action. For instance, a direct effect of fMLP in the synthesis of acute phase proteins in HepG2 cells has been reported [20]. Furthermore, in coronary arteries the peptide modulates the arterial tone, producing a transient contraction, without involving peripheral leukocytes: this response is mediated by the generation of thromboxane A2 and prostaglandin I2 in endothelial and smooth muscle cells [21].
As regards human pregnancy, we have previously demonstrated that the peptide causes an enhancement of amniotic PGE2 release, and that the amount of the prostanoid before labor is significantly lower than that observed after spontaneous term delivery. Moreover, two populations of specific binding sites, with high and low affinity for fMLP, are found in laboring amnion, while only the low affinity type is present in nonlaboring tissue [9].
The results of the present study demonstrate that WISH cells show a specific binding for 3H-fMLP, with kinetic properties very close to those reported for amniotic membrane obtained prior to the onset of labor. In fact, Scatchard analysis reveals the presence of only one binding site population with low affinity toward the peptide. Moreover, the IC50 value, calculated for the inhibition of formyl peptide binding by the antagonist Boc-MLP, is similar to that observed in amnion obtained prior to the onset of labor. However, fMLP-induced PGE2 synthesis from WISH cells is greater than that observed in nonlaboring amnion, probably due to differences in the experimental conditions employed.
As for fMLP signal transduction pathways leading to biosynthesis of the prostanoid, it has been reported that, in neutrophils, the peptide stimulates PLA2, either directly or through the activation of PLC [22]. This mechanism is operative also in our model. In fact: 1) indomethacin, a COX inhibitor, dose-dependently decreases both spontaneous and fMLP-stimulated PGE2 release; 2) quinacrine, a PLA2 inhibitor, dose-dependently inhibits fMLP-induced PGE2 production, but does not affect basal release; 3) U-73122, an inhibitor of PLC, behaves like quinacrine. These results seem to exclude a direct influence of fMLP on PLA2 and suggest that the peptide directly activates PLC that, in turn, seems to stimulate PLA2. To elucidate further the involvement of PLC in this action, we tested the effect of fMLP on [Ca2+]i mobilization. The concentration that induces the maximal PGE2 release, 10-6 M fMLP, is able to increase [Ca2+]i with a rapid and transient pattern. Because U-73122 counteracts Ca2+ mobilization, it can be concluded that the cation enhancement induced by PLC activation plays a crucial role in fMLP-induced amniotic PGE2 synthesis. However, an involvement of PLC also through the activation of protein kinase C cannot be excluded.
The existence of specific binding sites on human amniocytes implies that endogenous fMLP is presumably produced in both physiological and pathological conditions. As regards pregnancy, these may be represented by recurrent rupture of amniochorial or decidual cells following mechanical events, such as distension of the uterus as a consequence of gestational growth, multiple pregnancy, or polyhydramnios, as well as dynamic processes, such as Braxton-Hicks contractions and premature labor. Indeed, it has recently been reported that fetal membranes and WISH cells respond to distension with up-regulation of IL-8 [23, 24]; moreover, it has long been known that mitochondrial N-formylmethionine-containing peptides are released from degenerating mitochondria at sites of tissue damage [19]. Thus, a possible fMLP release following stretching or cell rupture could represent a mechanism whereby amnion tissue contributes to both term and premature delivery. Further pathologic conditions for fMLP release are represented by infection, which represents a well-known cause of the majority of premature births. In fact, it has been reported that fMLP-activated granulocytes and mononuclear cells release cytokines that, in turn, stimulate PGE2 production from amnion cells [10].
In our opinion, the response of amnion tissue to fMLP may therefore represent one step in the chain of events leading to the determination of labor. Extensive investigation is needed to establish the peptide's role in physiological and pathological delivery, to recognize the conditions able to control its release and to determine the full expression of its amniotic receptor. At present the factors that could favor the expression of the high-affinity form of fMLP receptors are unknown. However, possible candidates are glucocorticoids that are known to enhance the expression of cytokine receptors [25], and whose levels dramatically increase at term [26]. Moreover, because it has been demonstrated that fMLP receptors are up-regulated by prolonged adenosine 3',5'-cyclic monophosphate exposure of neutrophils [27], the numerous agonists whose receptors on amniocyte membrane are positively coupled to adenylyl cyclase could be involved in this response.
On the basis of the above considerations, it cannot be excluded that fMLP and fMLP antagonists may represent new tools in the future management of premature labor, a major cause of maternal and fetal morbidity and mortality.
ACKNOWLEDGMENTS
We are grateful to Mrs. Linda Bruce, a qualified mother tongue English teacher, for revision of the text.
FOOTNOTES
First decision: 6 September 2000.
1 Supported by grants from Ministero dell'Università e della Ricerca Scientifica e Tecnologica and a grant for biomedical research from Azienda Ospedaliera "Arcispedale Sant'Anna," University of Ferrara. 
2 Correspondence: Carla Biondi, Department of Biology, Section of General Physiology, University of Ferrara, via L. Borsari, 46, 44100-I Ferrara, Italy. FAX: 0532 207143; clm{at}dns.unife.it
Accepted: October 23, 2000.
Received: August 1, 2000.
REFERENCES
metabolite levels in plasma, amniotic fluid, and urine during pregnancy and labor. Am J Obstet Gynecol 1979; 133:886–890[Medline]This article has been cited by other articles:
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  S. Fiorini, M. E. Ferretti, C. Biondi, B. Pavan, L. Lunghi, G. Paganetto, and L. Abelli 17{beta}-Estradiol Stimulates Arachidonate Release from Human Amnion-Like WISH Cells through a Rapid Mechanism Involving a Membrane Receptor Endocrinology, August 1, 2003; 144(8): 3359 - 3367. [Abstract] [Full Text] [PDF]
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