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a Department of Biology, Section of General Physiology, University of Ferrara, 44100-I Ferrara, Italy
b Department of Biomedical Sciences and Advanced Therapy, Section of Obstetrics and Gynecology, University of Ferrara, 44100-Ferrara, Italy
c Department of Biology, University of Bologna, 40100 Bologna, Italy
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, that in turn induce prostaglandin synthesis by decidua and fetal membranes ([1] and citations therein). The prostanoids are directly responsible for uterine contractions. However, growing evidence suggests that the mediators of inflammation mentioned may also exert a physiological role in the determination of labor. As a matter of fact, even in the absence of infection, amniotic fluid—at term and preterm—contains considerable amounts of proinflammatory cytokines, and fetal membranes as well as decidua produce significant amounts of the same substances [2, 3]. These observations suggest that the mechanism for the onset of labor at term may, in some way, be similar to mechanisms involved in the inflammatory response. In addition to cytokines, N-formyl peptides released from bacterial [4] or cellular proteins [5] exert a crucial role in the inflammatory process. The prototype of these peptides is N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), recognized as the major chemotactic factor produced by Escherichia coli [6]. Furthermore, it has recently been demonstrated that fMLP-activated granulocytes and mononuclear cells release cytokines that stimulate PGE2 production from amnion cells [7].
Although the fMLP receptor was originally found in polymorphonuclear and mononuclear phagocytes, its expression has been demonstrated in non-myeloid cells, such as human spermatozoa [8], hepatocytes [9], coronary arteries [10], astrocytes, and microglia [11], as well as denditric cells [12]. Moreover, a partial cDNA sequence for the fMLP receptor has been isolated from human brain [13]. On the basis of these observations, it can be hypothesized that fMLP influences cellular mechanisms that are independent of the inflammatory response.
All these findings prompted us to verify whether fMLP, in addition to its indirect action on the amnion, mediated by cytokines released from granulocytes and mononuclear cells, could exert a direct effect on this tissue, thus playing a physiological role in the determination of delivery. To this purpose, we investigated the presence of fMLP binding sites and their action on E-type prostaglandin release in human amniotic membranes before and after spontaneous term labor.
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MATERIALS AND METHODS |
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Amniotic membranes were obtained from term physiological pregnancies after spontaneous delivery and after delivery of normal infants by cesarean section at term prior to the onset of labor. The selection of our cases was based on clinical assessment including repeated biochemical and hematologic profiles, as well as urinary and vaginal microbiologic cultures to rule out the presence of infection. In all the cases of cesarean delivery, the indication for the procedure was represented by a cesarean delivery in the previous pregnancy. The absence of any cervical change was ascertained by pelvic examination, and the lack of uterine contractions was tested by cardiotocography with the aim of excluding from the study any cases in which labor had already started.
For binding experiments, amniotic crude plasma membranes were used. For this purpose, after gentle washing several times with cold NaCl (0.15 M), amnion was homogenized in 6 vol (v:w) of ice-cold buffer with an Omni-Mixer (Sorvall, Norwalk, CT) at 200 rpm for at least three 30-sec bursts in an ice bath. Homogenization was completed by a Teflon (DuPont, Wilmington, DE) glass homogenizer. The homogenizing buffer contained 250 mM sucrose, 1 mM EGTA, and 10 mM Tris-HCl, pH 7.2. After filtering through four layers of gauze, the homogenate was centrifuged at 500 x g for 10 min. The supernatant was then recentrifuged at 40 000 x g for 20 min, and the pellet was suspended by gentle homogenization with the homogenizing buffer to obtain amniotic crude plasma membranes.
For perifusion experiments, amnion was minced with scissors; approximately 0.25 g (wet weight) of fragments per sample was utilized.
Binding Assay
Amniotic crude plasma membranes (300 µg of proteins) were incubated in 50 mM Tris-HCl, pH 7.4, in the presence of 5 nM 3H-fMLP and various concentrations of unlabeled fMLP (from 10-12 M to 5 x 10-5 M) or N-t-butoxycarbonyl-methionyl-leucyl-phenylalanine (Boc-MLP; from 10-8 M to 10-4 M). Incubations were carried out at 37°C for 15 min in a final volume of 200 µl. Total and nonspecific binding was determined in the absence or presence of 10-4 M unlabeled fMLP, respectively. At the end of incubation, samples were immediately filtered by rapid vacuum filtration on a Brandel cell harvester MB-48 (Biomedical Research and Development Labs., Gaithersburg, MD) through Whatman GF/C glass fiber filters, presoaked in the incubation buffer. Filters were washed three times, and bound radioactivity was measured by scintillation spectrometry (LS 6500; Beckman Instruments, Palo Alto, CA) after the addition of scintillation cocktail.
Perifusion of Amnion Fragments
Amnion fragments were preincubated for 30 min at 37°C in 5 ml of pseudoamniotic fluid, containing: 118.5 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2, 1.15 mM KH2PO4, 1.15 mM MgSO4, and 25.0 mM NaHCO3, supplemented with 2.0 mM glucose, 6.0 mM urea, and 0.2% BSA, pH 7.0, previously gassed with a 95% O2:5% CO2 mixture. For each experiment, the amniotic fragments, derived from a single patient, were mixed with polyacrylamide resin Bio-Gel P4 (Bio-Rad Labs., Hercules, CA) and transferred into 4 perifusion columns, each containing approximately 0.25 g of amniotic tissue (wet weight); the columns were supplied with pseudoamniotic fluid at a constant flow rate (70 µl/min). The pH (7.0) and temperature (37°C) were kept constant throughout the experiment. The rate of PGE release from the amniotic fragments during perifusion was stable over a 4-h period; the tissue was allowed to stabilize for 1 h before collection of samples. Test substances were dissolved in gassed pseudoamniotic fluid and infused into the columns by means of a peristaltic pump. Fractions of perifusate were set apart every 15 min, and the tubes were immediately frozen until assay.
Prostaglandin Assay
The amount of E type prostaglandin contained in the collected fractions was measured by a specific RIA procedure, as previously described [14]. A specific antiserum for both PGE1 and PGE2 (cross-reactions 110% and 100%, respectively) was used at a final dilution of 1:2000. Labeled 3H-PGE2 (6 nCi) was added to every tube. The level of PGE in each serially diluted sample was determined by comparison with a displacement standard curve (5–250 pg). Incubation was performed for 90 min at 4°C. Bound and free radioligand were separated by dextran-coated charcoal and centrifugation of tubes for 10 min at 2000 x g. Intra- and interassay coefficients of variations were < 10%. Results are presented as percentage of basal release.
Protein Determination
Protein concentrations were determined according to the method of Lowry et al. [15] using BSA as standard.
Data Expression and Analysis
Binding data were analyzed using Radlig Ver. 4 by Dr. G.A. McPherson (Biosoft, Cambridge, UK). This program utilized a nonlinear least-squares curve-fitting algorithm and assumes the simultaneous contribution of one or more independent binding sites. Data are expressed as mean ± SEM.
Results for each perifusion experiment were calculated as the mean of PGE release (-SEM) established from at least three independent experiments. PGE output is expressed as a percentage of the basal values calculated as the mean of three samples (45 min), taken just before infusion of the test substance. Statistical significance was assessed by one-way ANOVA followed by pair-wise multiple comparison procedures (Bonferroni's test); p < 0.05 was considered significant.
Chemicals
3H-PGE2 (specific activity 181 Ci/mmol) and 3H-fMLP (specific activity 71.5 Ci/mmol) were purchased from Amersham Pharmacia Biothec Srl. (Milan, Italy) and DuPont NEN (Milan, Italy), respectively. Liquid scintillation solution Filter Count was obtained from Canberra Packard (Milan, Italy). Whatman GF/C glass fiber filters were from Whatman Int. (Maidstone, Kent, England, UK); fMLP, BocMLP, PGE2, anti-PGE-BSA were from Sigma Chemical Co. (St. Louis, MO). All other chemicals were of the highest reagent grades commercially available.
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RESULTS |
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The specific binding of 3H-fMLP to crude plasma membranes of amnion derived from spontaneous delivery and cesarean section was rapid, reaching a maximum at 15 min; this value was stable until at least 30 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 (not shown).
A representative competition experiment, carried out on both laboring and nonlaboring amnion, is reported in Figure 1. As shown, increasing concentrations (10-12–5 x 10-5 M) of fMLP progressively displaced 3H-fMLP from its binding sites, more efficaciously in laboring than in nonlaboring amnion (Fig. 1a). Scatchard analysis and nonlinear iterative curve-fitting elaboration of binding data revealed the existence of two populations of binding sites in spontaneous delivery samples (Fig. 1b), while showing that a single class of binding sites was present in cesarean section samples (Fig. 1c). The mean values of the dissociation constant (Kd) and of maximal density of binding sites (Bmax), calculated from the data of five different experiments, are reported in Table 1.
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The fMLP receptor antagonist Boc-MLP [16] was then utilized in competition binding experiments, carried out under the same experimental conditions. As shown in Figure 2, increasing concentrations of the peptide (10-8–10-4 M) progressively inhibited 3H-fMLP binding to crude plasma membranes of amnion derived from both spontaneous labor and cesarean section. The calculated IC50 values were 12.1 µM and 60.3 µM, respectively.
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PGE Output from Amniotic Tissue
Perifusion experiments on amnion fragments were carried out to evaluate the effect of fMLP on PGE release. Peptide concentrations close to the Kd values, calculated from 3H-fMLP binding experiments, were chosen.
As shown in Figure 3, 10-8 M fMLP increased PGE release in amnion from spontaneous delivery (+59%, peak value vs. basal level); this effect became more evident (+76%, peak value vs. basal level) at 10-6 M fMLP. The stimulatory effects were statistically significant when compared either to the basal value or to one another.
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As for nonlaboring amnion, fMLP at 10-8 M was completely ineffective, as expected, while at 10-6 M (the concentration closest to the Kd value calculated for 3H-fMLP binding in this tissue), it induced a small but statistically significant stimulation (+28%, peak value vs. basal level) of PGE production (Fig. 4).
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The effect of fMLP was rapid and reversible in both laboring and nonlaboring amnion; in fact, full recovery of basal PGE output was observed after the termination of each pulse.
The selective fMLP receptor antagonist Boc-MLP, which was per se ineffective in provoking PGE release, completely abolished PGE output induced by 10-6 M fMLP in amnion derived from both spontaneous delivery (Fig. 5) and cesarean section (Fig. 6). The antagonist was tested at 10-5 M and at 5 x 10-5 M in laboring and nonlaboring amnion, respectively, as these are concentrations close to the IC50 values calculated in the two preparations (see Fig. 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several recent reports demonstrate that fMLP, a proinflammatory mediator, exerts effects independent of its action as a chemoattractant [9, 10]. Moreover, it has been reported that, in human neutrophils, the formyl peptide stimulates phospholipase A2 either directly or through the activation of phospholipase C [17]; in addition, the peptide evokes prostanoid output not only from its conventional target, i.e., the human neutrophil [18], but also from non-myeloid cells, such as endothelial and smooth muscle cells of coronary arteries [10]. A role for fMLP in triggering labor can therefore be hypothesized. On the basis of these considerations, we decided to search for fMLP binding sites on human amnion and verify their influence on PGE release, which represents a specific response of this tissue during the event of delivery.
Our results demonstrate the presence of specific binding sites for 3H-fMLP in crude plasma membranes obtained from the amnion of laboring and nonlaboring patients, as well as the inhibition of formyl peptide binding by the neutrophil receptor antagonist Boc-MLP. Scatchard plot analysis demonstrated that two classes of binding sites, with high and low affinity toward the formyl peptide, are present in laboring amnion, whereas only the low-affinity population appears in amnion from nonlaboring women. Similarly, different affinity states have been demonstrated for the fMLP receptor in human neutrophils, where they are responsible for the multiple functional responses of these cells [19]. The high-affinity binding sites, found in laboring amnion, show kinetic properties similar to those originally described for the fMLP receptor in cells of myeloid lineage [20] and in human coronary arteries [10]; but they show an affinity about one order of magnitude less than that reported in liver cells [9]. As for the low-affinity binding sites, they behave similarly to FPR2, a granulocyte protein highly homologous to the human formyl peptide receptor [21].
In this study we also investigated the effect of fMLP on PGE synthesis and found that the peptide is able to significantly increase prostanoid release in laboring and, to a lesser extent, nonlaboring amnion. This action could be mediated by the activation of phospholipases A2 and C, as already demonstrated in human neutrophils [17]. PGE release is completely abolished by Boc-MLP in both cases. The discrepancy between the dramatic effect of Boc-MLP on fMLP-induced PGE release and its slight efficacy in displacing 3H-fMLP from its binding sites could be explained by hypothesizing that the occupancy of only a few fMLP binding sites is already sufficient to evoke the biological response. Alternatively, since it has been reported that butoxycarbonyl analogues of fMLP are not stable [22], the continuous supply of the fMLP antagonist to the perifusion system makes it possible to avoid partial degradation of the peptide, as would otherwise occur in binding experiments.
The presence of fMLP-specific binding sites responsible for PGE release from amnion, together with the appearance of a receptor high-affinity state during the progression of labor, suggests a role for the peptide in human delivery. Moreover, since our results were obtained in cases of normal pregnancy, in the absence of any clinical and laboratory sign of infection, we hypothesize that fMLP can be released from healthy fetal or maternal tissues. This is suggestive of a possible physiological role of the peptide during normal delivery. The differences in the kinetic properties of fMLP receptor and the pattern of PGE release in spontaneous and cesarean delivery are a consequence of unknown events that occur in a relatively short time, most probably immediately before or during labor. In this regard, it has been reported that the initiation of parturition is associated with the up-regulation of oxytocin receptors in rabbit amnion [23] and with the appearance of entire IL-6 receptors in the human placenta that lead to spontaneously occurring labor at term and also to preterm labor in the absence of intrauterine infection [24].
However, as fMLP stimulates granulocytes and mononuclear cells to produce cytokines that in turn induce PGE2 production from amnion cells [7], it should also be admitted that this indirect mechanism may become operative during labor. On the other hand, fMLP production by bacteria could represent the link between infection and premature delivery through the same pathway.
If our data speak in favor of a possible role for fMLP in normal delivery, the possibility cannot be excluded that the same peptide could also be involved in other physiological and pathological conditions, such as the regulation of uteroplacental blood flow, abortion, premature delivery, and preeclampsia. Indeed, as has been shown in the case of coronary arteries, fMLP modulates arterial tone by influencing the synthesis of several arachidonic acid metabolites, among them thromboxane A2 and prostaglandin I2 [10]. Therefore, if the same mechanisms were operative also in gestational tissues, the peptide could exert a role in regulating the resistance of uterine arteries and hemostasis in pregnancy, in addition to its effect on smooth muscle contraction.
In conclusion, our data provide the first evidence for a direct action of fMLP on amniotic PGE release, thus implying its role in the determination of labor and its addition to the list of factors and cytokines involved in the release of prostanoids from fetal and maternal tissues.
Further knowledge of the regulation of these mechanisms is essential in order to improve the management of normal and pathological labor. In this connection, the search for the physiological source of fMLP as well as the factors responsible for the appearance of high-affinity binding sites in gestational tissues is of paramount importance.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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: December 21, 1998.
Received: September 11, 1998.
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