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Regular article |
a Magee-Womens Research Institute and Department of Obstetrics, Gynecology, and Reproductive Sciences, Division of Maternal Fetal Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213
ABSTRACT
Intra-amniotic infection leads to preterm labor and is associated with the local release of inflammatory cytokines by fetal membranes, resulting in the production of uterotonic prostaglandins. Oxytocin, however, also plays a key role in the initiation of labor. Short-term exposure of myometrium to interleukin (IL)-1 enhances oxytocin signaling and contractility. With intrauterine infection, however, myometrium is exposed to inflammatory cytokines for prolonged periods. The present study was conducted to demonstrate that myometrial oxytocin signaling is significantly impaired following prolonged exposure to IL-1. Myometrial cells were treated with IL-1 for 24 h. Oxytocin-stimulated inositol trisphosphate (IP3) production was measured in tritiated myoinositol-loaded myometrial cells. Arachidonic acid (AA) release was measured in tritiated AA-loaded myometrial cells. Increases in intracellular calcium were measure with fluo-3. Prostaglandin (PG) F2
and 6-keto-PGF1 were measured by ELISA assay. Prolonged exposure of myometrial cells to IL-1 resulted in a significant reduction in oxytocin-mediated signaling as measured by IP3 production and AA release, as well as a decrease in intracellular calcium. Prolonged exposure of myometrial cells to IL-1, however, resulted in enhanced PG release. Oxytocin may not contribute significantly to the labor-inducing action of IL-1 in the setting of preterm labor with prolonged infection.
cytokines, hormone action, oxytocin, parturition, signal transduction, uterus
INTRODUCTION
Inflammatory cytokines, principally interleukin (IL)-1, IL-6, and tumor necrosis factor alpha, are known to play key roles in the initiation of preterm labor in the setting of choriodecidual infection. The pathophysiology of infection-mediated preterm labor has been extensively studied. Intra-amniotic infection is associated with the production of inflammatory cytokines by decidua, chorion, and amnion [1, 2]. These cytokines are known to up-regulate the inducible form of cyclo-oxygenase (COX)-2 in amnion, decidua, and myometrium, resulting in increases in uterine prostaglandin (PG) production and the onset of labor [3, 4].
In addition to the role of PGs, oxytocin also contributes to the maintenance of both term and preterm labor [5–7]. Oxytocin receptors increase in the myometrium and decidua prior to the onset of both term and preterm labor in all animals studied, including humans. Several studies have examined both the short-term and long-term effects of inflammatory cytokines on oxytocin receptor concentration and function in myometrium. Molnar et al. [8] demonstrated that IL-1 treatment of myometrial tissues for a short duration increases arachidonic acid (AA) release and potentiates oxytocin-mediated contractility. In contrast, we have shown that prolonged exposure of myometrial cells to IL-1 results in down-regulation of the oxytocin receptor [9]. Following 24 h of IL-1 treatment, myometrial cells have 90% fewer oxytocin receptor binding sites. No studies have previously measured oxytocin receptor signaling in myometrium after prolonged exposure to cytokines. Such studies are relevant because the myometrium may be exposed to inflammatory cytokines for a prolonged period with intrauterine infection. The present study was therefore conducted to investigate the effect of IL-1 on myometrial oxytocin signaling after 24 h of treatment. This study investigated several important second-messenger pathways involved in oxytocin signal transduction, including inositol trisphosphate (IP3) production, resulting in intracellular free calcium increases as well as AA release, and increases in PG release. Prolonged IL-1 treatment, which results in oxytocin receptor down-regulation, also results in a 75% decrease in oxytocin-stimulated IP3 production and intracellular calcium, and a 50% decrease in AA release. PG release into the media is enhanced by IL-1 treatment.
MATERIALS AND METHODS
Materials
Collagenase P was purchased from Boerhinger-Mannheim (Indianapolis, IN). Hanks balanced salt solution, Earles salt solution, minimal essential media, and all additives were obtained from Gibco-BRL (Grand Island, NY). Tritiated myoinositol, tritiated AA, and Renaissance Chemiluminescence Kits were purchased from New England Nuclear (Boston, MA). NS-398 was obtained from Biomo Research Laboratories, Inc. (Plymouth Mission, PA). Fluo-3 and pluronic F-127 were purchased from Molecular Probes (Eugene, OR). Oxytocin was obtained from Peninsula Laboratories (San Carlos, CA). Dowex and prestained Kaleidoscope protein standards were obtained from BioRad Laboratories (Hercules, CA). Prostaglandin F2 and 6-keto-PGF1 ELISA kits were obtained from Cayman Chemical Co. (Ann Arbor, MI). Antibodies to G q/11 and PLCß3 were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). Goat anti-rabbit immunoglobulin (Ig)G horseradish peroxidase-conjugated antibody was obtained from Jackson Immuno Research (West Grove, PA).
Human Uterine Smooth Muscle Cell Culture
Samples of human uterine myometrium were obtained at the time of cesarean section from women with uncomplicated pregnancies undergoing elective cesarean section at term (37–40 wk). All patients consented to this study in accordance with the Magee-Womens Hospital Human Use and Experimentation Committee. Segments of the upper margin of the lower uterine segment (0.5 x 0.5 x 3 cm) were placed in modified Hanks balanced salt solution (500 ml of calcium- and magnesium-free Hanks balanced salt solution with 5 ml heparin [1000 U/ml]) and kept at 4°C prior to processing. Using sterile technique, the tissue samples were minced, washed with Earles balanced salt solution, and placed in a 50-ml conical tube with 20 ml of collagenase type P solution (1 mg/ml). The suspension was incubated at 37°C for 2 to 3 h with occasional pipetting. The suspension was then passed through fine-meshed gauze and individual cells were collected by centrifugation at 500 x g for 5 min. The cells were washed twice with media (minimal essential media with nonessential amino acids, sodium bicarbonate [26 mM], pyruvate [1 mM], gentamicin [50 µg/ml], penicillin G [100 U/ml], streptomycin [100 µg/ml], L-glutamine [2 mM], and charcoal-stripped fetal calf serum [FCS; 10% v/v]). The cells were then plated in 75 cm2 flasks and maintained at 37°C in humidified 5% CO2. The media was changed every 2 to 3 days and the cells subcultured at confluence after 5 to 7 days. All experiments were performed at passage 2 to 3.
Inositol Trisphosphate Measurements
Myometrial cells (5 x 106) were grown to confluence in T75 plates. Cells were treated with either IL-1 (5 ng/ml) or control for 24 h and simultaneously labeled with [3H]myoinositol (3.5 µCi/ml) for 24 h in media with 5% FCS. We previously determined that there was no difference between IL-1- and control-treated cells with respect to the total counts per min incorporated into cells during this 24-h period. Cells were then removed from the plates with 0.05% trypsin/0.53 mM EDTA, and washed with Hanks balanced salt solution with 20 mM HEPES buffer (pH 7.4) and 5 mM cold-unlabeled myoinositol. Cells were then incubated in HEPES-Hanks buffer containing 10 mM LiCl at 37°C for 10 min. Oxytocin (0–5000 nM) was then added with 5 mM MgCl2 at 37°C for 10 min. The reaction was stopped with the addition of methanol, chloroform, and hydrochloric acid (50:100:1 by volume). After centrifugation, the aqueous phase was separated over Dowex resin columns. The final wash, representing total IP3, was collected with 1 M ammonium formate/0.1 M formic acid and was counted by scintillation. All experiments were performed in triplicate with cells isolated from six different subjects.
Intracellular Free Calcium Measurements
Cells were grown to confluence in media with 10% FCS in T160 plates. Cells were treated with either IL-1 (5 ng/ml) or control for 24 h. Cells were removed from the plates with 0.05% trypsin/0.53 mM EDTA and resuspended at a concentration of 2 x 106 cells/ml in Krebs buffer with 4 µM fluo-3 and 0.02% pluronic F-127, and incubated at 37°C for 20 min. The cells were then centrifuged and washed with Krebs buffer and resuspended in Krebs buffer. The cell suspension was then divided into five aliquots, oxytocin (0, 1, 10, and 100 nM) or A23187 (1 µM) was added for 10 sec, and the peak fluorescence was measured by spectrofluorometry with excitation at 485 nm and emission at 530 nm. Baseline fluorescence was measured for 1 min prior to the addition of oxytocin or A23187. Experiments were performed in triplicate with cells isolated from six subjects. Results are expressed as the percent of maximum calcium release obtained after A23187 treatment.
Arachidonic Acid Release Measurement
Myometrial cells (2 x 105) were grown to confluence in 24-well plates in media with 5% FCS. Cells were treated with either IL-1 (5 ng/ml) or control for 24 h and simultaneously labeled with 3H-AA (0.4 µCi/well) for 24 h in media with 2% FCS. We previously determined that there was no difference between IL-1- and control-treated cells with respect to the total counts per min incorporated into cells during this 24-h period. Cells were washed with HEPES/Hanks, including 0.2% BSA. Oxytocin (0–5000 nM) was added at 37°C for 10 min. The media was collected and the plated cells lysed with NaOH (0.5 N). The cell lysate was centrifuged to remove cellular debris, the supernatant was combined with the media, and counted by scintillation. Maximum AA release was observed at 5 µM oxytocin. Experiments were performed in triplicate with cells isolated from six different subjects.
Western Blot
Cells were exposed to media alone or IL-1 (5 ng/ml). After treatment for 24 h the cells were scraped from the plates in buffer (10 mM Tris pH 7.4, 0.15 M NaCl, 1% Triton, 2 mM phenylmethylsulfonyl fluoride, 0.02 mg/ml leupeptin) and pelleted at 500 x g for 5 min. The cell pellet was lysed with sonication in reducing loading buffer. Protein was determined using the BioRad Protein Assay (Hercules, CA). Samples as well as a protein standard (6.9 to 202 kDa) were then denatured at 95°C in reducing loading buffer and electrophoresed on an 8% or 10% polyacrylamide gel with 4% stacking gel. After electrophoresis the proteins were transferred to nitrocellulose. The membranes were first stained with Poncheau-S (1% in 5% acetic acid) to visualize the protein bands and evaluate the blot for equal loading of protein per lane. The membrane was then blocked in 5% milk solution and incubated with either the rabbit polyclonal G q/11 or the rabbit polyclonal PLCß3 antibody and a secondary goat anti-rabbit IgG horseradish peroxidase-conjugated antibody. Bands were detected with chemiluminescence using Renaissance Chemiluminescence and exposure to x-ray film.
Prostaglandin Enzyme-Linked Immunosorbent Assay
Myometrial cells (5 x 105) were grown to confluence in 12-well plates in media with 5% FCS. Cells were treated with either IL-1 (5 ng/ml) or control with and without the selective COX-2 inhibitor, NS-398 (16 µM) for 24 h. The media was replaced with fresh media and oxytocin (1–500 nM) was added to the cultures for 4 h. The media was collected and frozen at -70°C. Prostaglandin F2 and 6-keto PGF1 were measured using ELISA from conditioned media without extraction. The sensitivities for PGF2 and 6-keto PGF1 were 14.2 pg/ml, and 15.4 pg/ml, respectively.
Statistical Analysis
All dose-response experiments were performed in triplicate on myometrial cells obtained from six subjects. Comparisons were made in the all of the dose-response experiments using a two-way ANOVA using STAT VIEW (Abacus Concepts, Berkeley, CA). Western blot experiments were compared using the Student's t-test of densitometry data obtained from the chemiluminescence images. Significance was achieved at P < 0.05.
RESULTS
Inositol Trisphosphate Production
Total IP3 was measured in myometrial cells treated with IL-1 or media alone for 24 h (Fig. 1). Basal IP3 production was not different either in cells treated with IL-1 (4837 ± 785 cpm) or controls (4486 ± 829 cpm). In both IL-1- and control-treated cells, oxytocin treatment resulted in a dose-dependent increase in IP3 production. At oxytocin concentrations from 20 to 5000 nM, there was significantly less IP3 production in the IL-1-treated cells. The maximal increase in IP3 after oxytocin (5000 nM) treatment was 40% in control- and 13% in IL-1-treated cells. To test whether this smaller increase in IP3 production after IL-1 pretreatment was specific to oxytocin treatment, cells were also stimulated with G-protein activator, NaF. There was no difference in the maximal IP3 production after stimulation with NaF in control cells (% increase 43 ± 12) and IL-1-treated cells (% increase 38 ± 10). There was also no change in G q/11 protein as measured by Western blot between IL-1- and control-treated cells (Fig. 2A). A single band was detected at the expected molecular weight of 43 kDa.
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Intracellular Free Calcium Release
Intracellular free calcium concentrations were measured in cells treated with IL-1 and media alone for 24 h. At each of three concentrations of oxytocin (1, 10, and 100 nM), there was a significant decrease in percent of maximal calcium release in IL-1-treated cells compared with controls (Fig. 3). Maximal calcium release was determined by treating cells with the calcium ionophore, A23187. There was no difference in fluorescence maximum between cells treated with IL-1 (0.212 ± 0.019) and controls (0.227 ± 0.020).
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Arachidonic Acid Release
The release of AA was measured in cells treated with IL-1 and media alone for 24 h. There was a significant decrease in AA release between controls and IL-1-treated cells after oxytocin treatment at 10 to 500 nM (Fig. 4). Previous studies have demonstrated an increase in cytoplasmic phospholipase A2 in myometrial cells following IL-1 treatment and a corresponding increase in AA release. We did not detect any difference in basal AA release between controls (50 426 ± 6928 cpm) and cells treated with IL-1 (47 514 ± 4485 cpm). Because phospholipase C is also a major source of AA release from myometrial cells after oxytocin stimulation, we measured the major myometrial isoform, PLCß3, by Western blot (Fig. 2B). A single band was detected at the expected molecular weight of 155 kDa. There was no difference in PLCß3 between controls and cells treated with IL-1.
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Prostaglandin Release
Cells treated with IL-1 released more PGF2 and 6-keto PGF1 into the culture media than control-treated cells (Fig. 5). As expected, NS-398, a selective COX-2 inhibitor, completely blocked the increased PG release in IL-1-treated cells. No differences in PG release was found between control- and IL-1 plus NS-398-treated cells. Oxytocin did not have any effect on PGF2 release. Oxytocin treatment significantly increased 6-keto PGF1 release only in control-treated cells.
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DISCUSSION
The present study demonstrates a significant impairment in oxytocin receptor signaling following prolonged exposure to IL-1. Inositol triphosphate production and intracellular free calcium were reduced by 75% after IL-1 treatment. In comparison, the concentration of oxytocin receptors per cell was reduced by 90% after 24 h of IL-1 treatment [9]. This impairment in signaling appears to be solely due to a reduction in oxytocin receptors and not through changes in second-messenger signaling proteins. We did not demonstrate any change in the total amount of G q/11 or PLCß3 protein (the major isoforms in human myometrium) in the myometrial cell extracts after IL-1 treatment. Others have detected no change in G q/11 or PLCß3 protein with homologous desensitization of the oxytocin receptor [10–12]. We did not, however, measure PLC activity. In addition, we did not observe any change in IP3 production following stimulation with NaF. Hirata et al. [13] previously demonstrated a correlation between oxytocin receptor concentration and IP3 production in rabbit myometrium. Similarly, Phaneuf et al. [10, 11] demonstrated a significant correlation between oxytocin receptor concentration and IP3 production and intracellular calcium increase in human myometrium. The results of our study are similar to those reported after homologous oxytocin-mediated desensitization in human myometrium.
Arachidonic acid release from membrane phospholipids by oxytocin is believed to occur through two pathways; hydrolysis of diacylglycerol (DAG) by DAG lipase, and also through the activation of PLA2 by increases in intracellular free calcium [14]. We demonstrated a marked reduction in oxytocin-stimulated IP3 production and intracellular free calcium. Both a decrease in DAG as well as the reduction in intracellular free calcium would therefore explain the marked decrease in AA release by IL-1-treated myometrial cells. The impairment in oxytocin-stimulated AA release is, however, not as pronounced as that seen with IP3 production. Interleukin-1 treatment of myometrial cells is known to up-regulate PLA2 and to release AA, and most likely accounts for the finding in this study [8, 15, 16].
We also demonstrated enhanced production of PGs by prolonged IL-1 treatment. A specific COX-2 inhibitor, NS-398, completely blocked the increases in PGF2 and 6-keto PGF1 in this study. Previous studies have also shown that IL-1 up-regulates COX-2 (the inducible form of cyclo-oxygenase) in myometrium, amnion, and decidua [3, 4, 17]. We were unable to demonstrate any dose-related increases in PGF2 with oxytocin treatment. Shrey et al. [18] had previously shown that oxytocin increases AA release and incorporation of AA into PGE2 and PGF2; however, Fuchs et al. [19] and Hertelendy et al. [14] showed no significant increase in PGF2 or PGE2 with oxytocin stimulation. We did demonstrate dose-related increases in 6-keto PGF1 production with oxytocin stimulation. Others have similarly shown that prostacyclin is the principal PG produced by myometrium [20, 21].
Prolonged exposure of myometrial cells to IL-1 results in a significant decrease in receptor concentration as well as a significant decrease in oxytocin-stimulated increases in intracellular free calcium [9]. Other studies have shown that oxytocin-stimulated increases in intracellular calcium and contractility correlate with the concentration of oxytocin receptor binding sites [22, 23]. The degree of oxytocin-stimulated uterine contractions also correlates with the magnitude of increase in intracellular free calcium. Although not measured in this study, oxytocin-mediated myometrial contractility would be significantly decreased by prolonged exposure to IL-1. Clinically, the uteri of women with severe chorioamnionitis are less sensitive to infused oxytocin. Severe chorioamnionitis results in a higher rate of dysfunctional labor and increased incidence of postpartum atony and hemorrhage, which are unresponsive to oxytocin [24].
The results of this study suggest that with prolonged intrauterine infection, myometrial contractions may be generated independent of oxytocin stimulation. In the setting of prolonged infection, contractions are likely to result from continued PG production by COX-2. The mechanism by which IL-1 results in down-regulation of the oxytocin receptor and the impairment in oxytocin signaling is not known. Our results are, however, similar to those reported following homologous down-regulation of the oxytocin receptor. Because IL-1 is known to induce the release of oxytocin by neuronal tissues, IL-1 may also induce the autologous release of oxytocin by myometrium and subsequent down-regulation of the oxytocin receptor [25]. Such a mechanism would explain the results of this study.
FOOTNOTES
First decision: 14 February 2000.
1 This work received financial support from the National Institutes of Health, grant HD34373-03. 
2 Correspondence: Phillip N. Rauk, Magee-Womens Research Institute, Laboratory 240, 204 Craft Avenue, Pittsburgh, PA 15213. FAX: 412 641 5425; rsipnr{at}mail.magee.edu
Accepted: April 18, 2000.
Received: December 28, 1999.
REFERENCES
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