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The Journal of Immunology, 2005, 175: 6509-6516.
Copyright © 2005 by The American Association of Immunologists

IL-1{beta}-Mediated Proinflammatory Responses Are Inhibited by Estradiol via Down-Regulation of IL-1 Receptor Type I in Uterine Epithelial Cells1

Todd M Schaefer2, Jacqueline A. Wright, Patricia A. Pioli and Charles R. Wira

Department of Physiology, Dartmouth Medical School, Lebanon, NH 03756


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The objective of this study was to examine the effects of sex hormones on IL-1{beta}-mediated responses by uterine epithelial cells. The mRNA expression and secretion of human {beta}-defensin-2 and CXCL8 by uterine epithelial cells was examined following stimulation with IL-1{beta} in the presence of estradiol or progesterone. Estradiol inhibited the IL-1{beta}-mediated mRNA expression and secretion of human {beta}-defensin-2 and CXCL8 by uterine epithelial cells while progesterone had no effect. Inhibition of the IL-1{beta}-mediated response by estradiol was dose dependent, with maximal inhibition observed using 10–7 to 10–10 M, and was shown to be mediated through the estrogen receptor because addition of a pure estrogen receptor antagonist abrogated this effect. The mechanism by which estradiol inhibits IL-1{beta}-mediated responses by uterine epithelial cells appears to be the down-modulation of the IL-1R type I, thereby reducing the uterine epithelial cell’s ability to respond to IL-1{beta}. These results suggest that the inhibitory effect of estradiol on IL-1{beta}-mediated inflammatory responses by uterine epithelial cells indicates a link between the endocrine and immune systems and may be crucial for dampening proinflammatory responses during the time of ovulation or pregnancy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The human uterine endometrium has evolved to orchestrate many essential functions for the host, ranging from fertilization, implantation, and pregnancy to defense from sexually transmitted diseases and other invading pathogens. In particular, the endometrium must protect itself from sexually transmitted pathogens while allowing for the presence of allogeneic spermatozoa and the immunologically distinct fetus. Uterine epithelial cells are the first line of defense against invading pathogens and have evolved innate immune antimicrobial functions, as well as the ability to modulate the recruitment and activity of immune cells of both the innate and adaptive immune systems (1).

Uterine epithelial cells secrete antimicrobial factors, such as {beta}-defensins and secretory leukocyte inhibitory factor, that eliminate potential bacterial and viral pathogens (2, 3, 4, 5). Uterine epithelial cells also secrete a wide variety of cytokines, such as IL-6 and TNF-{alpha}, and chemokines, such as CXCL8, CCL2, and CCL5, that stimulate and recruit immune cells to the sites of infection (6, 7, 8, 9). Furthermore, these cells express a wide variety of antiviral molecules, including IFN-{beta}, 2',5'-oligoadenylate synthetase, and myxovirus resistance gene A (9). Because epithelial cells play a significant role in developing innate and adaptive immunological responses, a further understanding of the events leading to the stimulation or activation of epithelial cells and how these are regulated is needed.

IL-1 is a cytokine that is integral to the generation of a rapid and potent inflammatory response by uterine epithelial cells. IL-1 is a prototypic proinflammatory cytokine that regulates a wide spectrum of immunological processes and also plays a role in endometrial and reproductive functions, particularly those related to endometrial receptivity and implantation (10, 11, 12). IL-1 is present in two forms, IL-1{alpha} and IL-1{beta}, both of which bind to two distinct plasma membrane receptors: IL-1R type I (IL-1RtI),3 crucial for IL-1-mediated activation, and IL-1R type II (IL-1RtII), a decoy receptor with no signaling capabilities (13, 14, 15). Cells capable of producing IL-1{alpha}{beta}, such as macrophages, T lymphocytes, and fibroblasts, are found throughout the endometrial stroma or as lymphoid aggregates in the stratum basalis (16, 17, 18). IL-1{beta} induces a systemic and local response to pathogenic invasion or injury of tissues by inducing transcription or enhancing mRNA stability of a wide variety of proinflammatory genes. Stimulation of epithelial cells with IL-1{beta} leads to the enhanced expression of many immunologically relevant genes, such as human {beta}-defensin (HBD)2, CCL5, CCL20, CXCL1, CXCL2, CXCL3, CXCL8, TNF-{alpha}, IL-6, and GM-CSF (16, 19, 20, 21, 22, 23). Furthermore, IL-1 enhances the expression of genes associated with inflammation. In particular, inducible NO synthase, type 2 cyclooxygenase, and type 2 phospholipase A2 appear to be sensitive to IL-1 and are potent proinflammatory mediators (16). By increasing gene expression and synthesis for these enzymes, the effects of IL-1 are prolonged several hours after triggering the cell.

However, in addition to its protective effects, the immune response can also be harmful to the host. IL-1{beta} is a major factor in the initiation of infection-related preterm labor and delivery and has been associated with preeclampsia (24, 25, 26). IL-1{beta} levels are elevated in the amniotic fluid of pregnancies complicated by intra-amniotic infection, and IL-1{beta} may be capable of causing preterm labor by up-regulating PG production leading to myometrial contractions (27, 28, 29). Elevated IL-1{beta} expression is also observed in the placentas of women with preeclampsia and may be associated with placental hypoxia (26). IL-1 may also play an important role in the pathophysiology of endometriosis, a gynecological disease that is associated with a chronic immunoinflammatory process (30, 31, 32). In women with endometriosis, peripheral blood monocytes (33), as well as peritoneal macrophages (34), were found to be more activated than in normal women and to secrete elevated levels of IL-1. Increased concentrations of IL-1 were detected in the peritoneal fluid of women suffering from endometriosis (33, 34, 35). More recently, examination of women with endometriosis revealed a deficiency in the ability of endometrial cells to down-regulate their response to IL-1 (36). In the female reproductive tract of healthy women, the endometrium has needed to evolve immune mechanisms to protect against potential pathogens, in part through the effects of IL-1, without compromising fetal survival or normal physiological processes. For this reason, the effects of IL-1 are stringently controlled in vivo and are likely regulated by the sex hormones estradiol and progesterone.

There is little information currently available about the regulation of IL-1{beta}-mediated responses by sex hormones and, in particular, if sex hormones modulate the expression of the IL-1RtI. The goal of this study was to examine the effects of sex hormones on IL-1{beta}-mediated responses. For these studies, we used the human uterine epithelial cell line ECC-1 that expresses a functional progesterone receptor and estrogen receptor (ER){alpha}. ER{beta} is also expressed by ECC-1 cells but to insignificant levels (37, 38, 39, 40). Using ECC-1 cells, we examined the mRNA expression and secretion of HBD2 and CXCL8 following stimulation of these cells with IL-1{beta} in the presence of sex hormones. Furthermore, we examined IL-1RtI mRNA expression in the presence of sex hormones.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell culture and reagents

To establish a cell culture system of polarized human uterine epithelial cells with both apical and basolateral compartments, the human uterine epithelial cell line ECC-1 (originally established by Dr. P. Satyaswaroop, Milton S. Hershey Medical Center, Hershey, PA; and provided by Dr. G. Olt, Penn State College of Medicine, Milton S. Hershey Medical Center, Hershey, PA) was cultured in Falcon cell culture inserts in 24-well culture dishes designed for these cell inserts (Fisher Scientific). For these experiments, apical and basolateral compartments had 300 and 850 µl of complete medium, respectively. The medium was changed every 2 days. Complete medium was supplemented with 20 mM HEPES, 50 U/ml penicillin, 50 mg/ml streptomycin, 2 mM L-glutamine (all from Invitrogen Life Technologies), and 10% heat-inactivated stripped FBS (HyClone) and did not contain phenol red. Following 24-h treatment with or without IL-1{beta} (5 ng/ml) (PeproTech), ECC-1 apical and basolateral supernatants were centrifuged for 5 min at 10,000 x g.

Hormone and antagonist preparation and treatment

Estradiol (Calbiochem), progesterone (Calbiochem), and ICI 182,780 (ICI) (Tocris) were each dissolved in 100% ethanol, evaporated to dryness, and resuspended in complete medium. To control for the alcohol present in steroid or antagonist preparation, an equivalent amount of ethanol was evaporated in vials used to prepare control medium. Once epithelial cells achieved high transepithelial resistance (TER) (>2000 ohms/well), medium was removed from the apical and basolateral compartments and replaced with fresh medium alone or medium containing steroid or antagonist/inhibitor at the appropriate concentrations for 72 h. In experiments involving ICI, the antagonist was added in combination at the same time as estradiol. In those experiments, estradiol/ICI remained in culture throughout the experiment.

Measurement of transepithelial resistance

As an indicator of tight junction formation of epithelial cell monolayers, TER was assessed periodically using an EVOM electrode and Voltohmmeter (World Precision Instruments), as described previously (41).

Measurement of HBD2 and CXCL8 secretion

Concentrations of HBD2 in the apical and basolateral supernatants were determined by ELISA (PeproTech). Concentrations of CXCL8 in the apical and basolateral supernatants from ECC-1 cells were determined with an ELISA test kit (R&D Systems) according to the manufacturer’s protocol. The HBD2 and CXCL8 ELISAs had minimum detection levels of 2.3 pg/well. Standards for each ELISA were resuspended in cell culture medium. Calculations of HBD2 and CXCL8 were determined from a standard curve after OD measurements at 450 nm on an ELISA reader (Dynex).

TaqMan real-time RT-PCR

Real-time RT-PCR was done with a two-step protocol as described previously (42). Total RNA was isolated from cells using TRIzol reagent according to the manufacturer’s recommendations (Invitrogen Life Technologies) and purified with RNeasy columns (Qiagen). Coincident with RNA purification was DNase digestion using the RNase-Free DNase set (Qiagen). For each specimen, 600 ng of total RNA was reverse transcribed using the iScript cDNA synthesis kit, according to the manufacturer’s recommendations (Bio-Rad), in a 20-µl volume. Relative expression levels of HBD2, CXCL8, and IL-1RtI were measured using the 5' fluorogenic nuclease assay in real-time quantitative PCR using TaqMan chemistry on the ABI 7300 Prism real-time PCR instrument (Applied Biosystems). The HBD2, CXCL8, IL-1RtI, and CD71 primer/MGB probe sets were obtained from Applied Biosystems assays-on-demand (ID no. Hs00823638, Hs00174103, Hs00168392, and Hs99999911, respectively). Primers and probe used for detection of HBD3 are as described previously (3). PCR was conducted using the following cycle parameters: 95°C 12 min for 1 cycle, 95°C 20 s, 60°C 1 min for 40 cycles. Analysis was conducted using the sequence detection software supplied with the ABI 7300. The software calculates the threshold cycle (Ct) for each reaction and this was used to quantitate the amount of starting template in the reaction. The Ct values for each set of duplicate reactions were averaged for all subsequent calculations. A difference in Ct values ({Delta}Ct) was calculated for each gene by taking the mean Ct of gene of interest and subtracting the mean Ct for CD71 for each cDNA sample. Assuming that each reaction functions at 100% PCR efficiency, a difference of one Ct represents a 2-fold difference in mRNA expression. Relative expression levels were expressed as a fold increase in mRNA expression and calculated using the formula 2–{Delta}Ct.

Lysate preparation and immunoblotting

ECC-1 cells were washed twice with 1x PBS and lysed by addition of M-PER reagent (Pierce). Proteins were extracted from cells by resuspending pellets in 20 µl of M-PER and centrifuging at 13,000 x g for 15 min. Following centrifugation, supernatants were aliquoted and stored at –80°C as whole cell lysates. Protein concentrations were determined by bicinchoninic acid protein assay (Pierce). Proteins (60 µg of each sample) were resolved by 10% SDS-PAGE and electrotransferred to nitrocellulose membrane in Towbin’s buffer. Immunoblots were washed with Tris-buffered saline, 0.1% Tween 20, and blocked in 5% BSA overnight at 4°C. Membranes were then probed with an affinity-purified biotinylated Ab directed against human IL-1R (BAF269) (R&D Systems) and a streptavidin HRP-conjugated secondary Ab (BD Biosciences). Reactive Ags were visualized with SuperSignal chemiluminescence substrate (Pierce). Individual bands were scanned using the FluorChem 8900 (Alpha Innotech), and the intensity was quantified by using AlphaEaseFC software (Alpha Innotech). The amount of IL-1RtI was normalized to GAPDH levels and plotted as a percentage of the control (no estradiol treatment).

Statistics

The data are presented as the mean ± SE. A one-way ANOVA with Bonferonni’s posttest was performed using GraphPad InStat version 3.0a (GraphPad Software). A value of p < 0.05 was taken as indicative of statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Estradiol inhibits IL-1{beta}-mediated secretion of HBD2 by uterine epithelial cells

Because IL-1{beta} induces the expression HBD2 in primary uterine epithelial cells as well as uterine epithelial cell lines (23), the secretion of HBD2 was used to examine the effects of sex hormones on IL-1{beta}-mediated stimulation of uterine epithelial cells. ECC-1 cells were grown on cell inserts until TER measurements indicated that tight junctions had occurred (>2000 ohms/well). Cell inserts without ECC-1 cells had mean TER values of 130–150 ohms/well. Polarized ECC-1 cells were pretreated with and without physiologically relevant doses of estradiol (5 x 10–8 M), progesterone (1 x 10–7 M), or a combination thereof for 72 h before stimulation with IL-1{beta} at 5 ng/ml. Following 24 h of stimulation, both the apical and basolateral conditioned medium were collected and analyzed for HBD2 secretion. In addition, total RNA was also collected from the treated cells so that HBD2 mRNA expression could be examined by real-time RT-PCR.

As shown in Fig. 1A, constitutive HBD2 secretion by ECC-1 cells was undetectable in both apical and basolateral conditioned medium. Stimulation of ECC-1 cells with IL-1{beta} resulted in the secretion of HBD2 protein into both the apical and basolateral compartments, with HBD2 being preferentially released from the basolateral surface (8.40 pg (apical) vs 35.0 pg (basolateral); Fig. 1). Preincubation of ECC-1 cells with estradiol before stimulation with IL-1{beta} resulted in a significant reduction of HBD2 secretion into both apical (5.6-fold) and basolateral (5.5-fold) compartments compared with cells treated with IL-1{beta} alone (Fig. 1A). Similarly, pretreatment of epithelial cells with a combination of estradiol and progesterone inhibited IL-1{beta}-mediated apical and basolateral secretion of HBD2. Although there was a marked reduction of HBD2 secretion in the presence of estradiol, both alone and with progesterone, a preferential release of defensin protein into the basolateral chamber persisted. Preincubation with progesterone had a slight inhibitory effect on IL-1{beta}-mediated HBD2 basolateral secretion by ECC-1 cells but had no effect on HBD2 secretion from the apical surface. Inhibition of IL-1{beta}-mediated HBD2 secretion by estradiol was the direct result of reduced HBD2 mRNA expression (Fig. 1B). Although stimulation of ECC-1 cells with IL-1{beta} greatly enhanced HBD2 mRNA expression (33-fold), preincubation of the uterine epithelial cells with estradiol or a combination of estradiol and progesterone before stimulation resulted in a marked reduction of HBD2 mRNA expression (4.3- and 3.8-fold increase; Fig. 1B). Pretreatment of ECC-1 cells with progesterone alone had no effect on HBD2 mRNA expression.



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  		FIGURE 1. IL-1{beta}-mediated HBD2 secretion and mRNA expression by uterine epithelial cells is inhibited by estradiol. ECC-1 cells were preincubated with the indicated sex hormones for 72 h before stimulation with IL-1{beta} (5 ng/ml). A, Apical and basolateral conditioned medium was collected following 24 h of stimulation and analyzed for HBD2 protein secretion by ELISA. The results are shown as the mean ± SEM. *, Significantly different (p < 0.05) from control. ***, Significantly different (p < 0.001) from control. UD, undetectable; <2.3 pg/well. Hatched bars, Apical conditioned medium; {blacksquare}, basolateral conditioned medium. B, Real-time RT-PCR was used to determine the relative levels of expression of HBD2 normalized against an endogenous control, CD71. The data were further normalized by using values from the control for calibration (representative of four experiments).

 
Estradiol inhibits IL-1{beta}-mediated secretion of CXCL8 by uterine epithelial cells

To determine whether estradiol was specifically inhibiting HBD2 secretion or generally inhibiting IL-1{beta}-mediated responses, we examined the effect of sex hormones on IL-1{beta}-mediated CXCL8 secretion by uterine epithelial cells. The proinflammatory chemokine CXCL8 is a potent chemoattractant and activator of neutrophils and T lymphocytes and is produced by a variety of cells types, including monocytes, fibroblasts, lymphocytes, and epithelial and endothelial cells (43, 44, 45). Following IL-1{beta} stimulation, ECC-1 cells secreted copious amounts of CXCL8, with preferential secretion occurring from the basolateral surface of the cells (Fig. 2A). As shown in Fig. 2A, estradiol, alone or in combination with progesterone, significantly reduced both apical and basolateral IL-1{beta}-mediated secretion of CXCL8 by ECC-1 cells. Preincubation of ECC-1 cells with progesterone had no effect on IL-1{beta}-mediated secretion of CXCL8. Of note, estradiol abrogated the observed preferential release of CXCL8 into the basolateral compartment following IL-1{beta} stimulation. Examination of CXCL8 mRNA expression revealed a pattern of inhibition similar to that of CXCL8 secretion (Fig. 2B). Stimulation of ECC-1 cells with IL-1{beta} enhanced CXCL8 mRNA expression 5.3-fold, while preincubation of the epithelial cells with estradiol, alone or in combination with progesterone, abrogated this increase. Preincubation of epithelial cells with progesterone had no effect on CXCL8 mRNA expression. Interestingly, treatment of ECC-1 cells with estradiol alone or in combination with progesterone, in the absence of IL-1{beta} stimulation, resulted in a 4.4-fold reduction in CXCL8 mRNA expression (data not shown). These results demonstrate that estradiol has an inhibitory effect on IL-1{beta}-mediated stimulation of uterine epithelial cells, resulting in reduced mRNA expression/secretion of the innate antimicrobial molecule HBD2 and the proinflammatory chemokine CXCL8.



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  		FIGURE 2. IL-1{beta}-mediated CXCL8 secretion and mRNA expression by uterine epithelial cells is inhibited by estradiol. ECC-1 cells were preincubated with the indicated sex hormones for 72 h before stimulation with IL-1{beta} (5 ng/ml). A, Apical and basolateral conditioned medium was collected following 24 h of stimulation and analyzed for CXCL8 protein secretion by ELISA. The results are shown as the mean ± SEM. ***, Significantly different (p < 0.001) from control. Hatched bars, Apical conditioned medium; {blacksquare}, basolateral conditioned medium. B, Real-time RT-PCR was used to determine the relative levels of expression of CXCL8 normalized against an endogenous control, CD71. The data were further normalized by using values from the control for calibration (representative of four experiments).

 
Dose-dependent inhibition of IL-1{beta}-mediated HBD2 and CXCL8 mRNA expression by estradiol

We observed potent inhibition of IL-1{beta}-mediated HBD2 and CXCL8 protein secretion and mRNA expression when uterine epithelial cells were pretreated with estradiol at 5 x 10–8 M. To determine the minimal concentration of estradiol that would exert an inhibitory affect on IL-1{beta}-mediated HBD2 and CXCL8 expression, we pretreated ECC-1 cells for 72 h with estradiol at concentrations ranging from 1 x 10–7 to 1 x 10–12 M before stimulation with IL-1{beta}. Potent inhibition of both HBD2 and CXCL8 mRNA expression was observed when cells were pretreated with estradiol at concentrations ranging from 1 x 10–7 to 1 x 10–10 M (Fig. 3). Slight inhibition of IL-1{beta}-mediated HBD2 and CXCL8 mRNA expression was observed when using estradiol at 1 x 10–11 M, and no effect was observed when incubating cells with estradiol at 1 x 10–12 M (Fig. 3).



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  		FIGURE 3. Dose-dependent inhibition of IL-1{beta}-mediated HBD2 and CXCL8 mRNA expression by estradiol. ECC-1 cells were preincubated with various concentrations of estradiol for 72 h, stimulated with IL-1{beta} for 24 h, followed by isolation of total RNA. Real-time RT-PCR was used to determine the relative levels of expression of HBD2 and CXCL8 normalized against an endogenous control, CD71. The data were further normalized by using values from the control for calibration (representative of four experiments).

 
Inhibition of HBD2 and CXCL8 secretion by estradiol is mediated through the ER

To gain a better understanding of the mechanism by which estradiol inhibits the secretion of HBD2 and CXCL8, we used the pure ER antagonist ICI to determine whether the inhibitory effects of estradiol are mediated through the ER. ICI causes the rapid degradation of the ER leading to down-regulation of ER cellular protein levels (46). ECC-1 cells were pretreated with estradiol (5 x 10–8 M), ICI (1 x 10–6 M), or a combination thereof, for 72 h before IL-1{beta} stimulation for 24 h, at which time apical and basolateral conditioned medium was collected and analyzed for HBD2 and CXCL8 protein secretion. As shown in Fig. 4, pretreatment of ECC-1 cells with estradiol resulted in potent inhibition of IL-1{beta} mediated HBD2 and CXCL8 protein secretion. Pretreatment of these cells with ICI had no effect on IL-1B-mediated apical secretion of HBD2 or CXCL8 but exhibited a slight but significant enhancement of their secretion from the basolateral surface. ECC-1 cells preincubated with estradiol and ICI abrogated the estradiol inhibitory effect, indicating that inhibition of IL-1{beta}-mediated HBD2 and CXCL8 protein secretion by estradiol is mediated through the ER (Fig. 4).



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  		FIGURE 4. Inhibition of IL-1{beta}-mediated HBD2 and CXCL8 secretion by estradiol occurs through the ER. ECC-1 cells were preincubated with estradiol, the pure ER antagonist ICI, or a combination of steroid and antagonist for 72 h before stimulation with IL-1{beta} (5 ng/ml). Apical and basolateral conditioned medium was collected following 24 h stimulation and analyzed for HBD2 and CXCL8 protein secretion by ELISA. The results are shown as the mean ± SEM. *, Significantly different (p < 0.05) from control. *|P8, Significantly different (p < 0.01) from control. ***, Significantly different (p < 0.001) from control. Hatched bars, apical conditioned medium; {blacksquare}, basolateral conditioned medium.

 
Estradiol inhibits IL-1{beta}-mediated HBD2 and CXCL8 production by down-regulation of the IL-1RtI

IL-1{beta} is the ligand for IL-1RtI, and it is this receptor that is crucial for IL-1{beta}-mediated activation. ECC-1 cells, as well as primary endometrial epithelial cells, express at their cell surface the IL-1RtI (47, 48). Because estradiol inhibits IL-1{beta}-mediated expression of two immunologically relevant but unrelated genes, this suggested that the mode of inhibition mediated by estradiol was not by targeting the expression of these specific genes following IL-1{beta} stimulation but rather inhibiting the IL-1{beta} response directly, possibly through modulation of the IL-1RtI. ECC-1 cells were treated with estradiol (5 x 10–8 M), progesterone (1 x 10–7 M), and a combination thereof for 72 h, at which time total RNA was isolated for examination of IL-1RtI mRNA expression by real-time RT-PCR. Incubation of uterine epithelial cells with estradiol resulted in a ~20-fold decrease in IL-1RtI mRNA expression (Fig. 5). This effect was also seen when cells were incubated with a combination of estradiol and progesterone. Treatment of cells with progesterone alone had no effect on IL-1RtI mRNA expression. Because we demonstrated that the inhibitory effect of estradiol on IL-1{beta}-mediated responses was mediated through the ER, ECC-1 cells were also treated with the ER antagonist ICI and a combination of ICI and estradiol. In agreement with data shown in Fig. 4, ICI completely blocked estradiol-dependent down-regulation of IL-1RtI mRNA expression while ICI alone had no effect (Fig. 5). Because up-regulation of the decoy receptor IL-1RtII can reduce reactivity of cells to IL-1{beta}, we also examined the effect of sex hormones on IL-1RtII mRNA expression. Interestingly, treatment of ECC-1 cells with estradiol also resulted in the significant down-regulation of IL-1RtII (6.4-fold), and these estradiol-mediated effects were completely blocked by the addition of ICI (Fig. 5). Progesterone had no effect on IL-1RtII mRNA expression.



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  		FIGURE 5. Estradiol down-regulates IL-1RtI and IL-1RtII. ECC-1 cells were preincubated with the indicated steroids/antagonists for 72 h, followed by isolation of total RNA. Real-time RT-PCR was used to determine the relative levels of expression of IL-1RtI and IL-1RtII normalized against an endogenous control, CD71. The data were further normalized by using values from the control for calibration (representative of four experiments).

 
Because we previously showed that the inhibition of IL-1{beta} responses by estradiol was dose dependent, we treated ECC-1 cells with a similar dose range of estradiol for 72 h and examined IL-1RtI mRNA expression by real-time RT-PCR. As shown in Fig. 6 and concordant with data shown in Fig. 3, IL-1RtI mRNA expression was greatly reduced in the presence of estradiol at 1 x 10–7 M down to 1 x 10–10 M. In agreement with Fig. 3, which showed a partial inhibition of HBD2 and CXCL8 secretion by estradiol at 1 x 10–11 M, the mRNA expression of IL-1RtI was reduced to a lesser extent (5.5-fold) as well (Fig. 6). Treatment of ECC-1 cells with estradiol at 1 x 10–12 M had no effect on IL-1RtI mRNA expression. These data further support the physiological relevance of this effect, demonstrating that at doses as low as 1 x 10–10 M, a level easily attained in uterine tissues, estradiol exerts a dampening effect on IL-1{beta}-mediated responses by down-regulation of the IL-1RtI.



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  		FIGURE 6. Down-regulation of IL-1RtI by estradiol is dose dependent. ECC-1 cells were preincubated with various concentrations of estradiol for 72 h, followed by isolation of total RNA. Real-time RT-PCR was used to determine the relative levels of expression of IL-1RtI normalized against an endogenous control, CD71. The data were further normalized by using values from the control for calibration (representative of four experiments).

 
In an attempt to demonstrate a reduction in total IL-1RtI protein, Western blot analyses were performed on whole cell lysates from ECC-1 cells treated for 72 h with estradiol at concentrations ranging from 1 x 10–8 to 1 x 10–12 M. As shown in Fig. 7, IL-1RtI protein expression is greatly reduced in the presence of estradiol, with nearly a 97% reduction observed when ECC-1 cells were preincubated with estradiol at a concentration of 1 x 10–10 M. These data demonstrate that estradiol, acting through the ER, down-regulates the mRNA expression of IL-1RtI, resulting in greatly reduced levels of IL-1RtI protein. The result of this is that uterine epithelial cells would then have a diminished capacity to generate a response following IL-1{beta} stimulation and thereby the expression/secretion of specific downstream genes, such as HBD2 and CXCL8, are reduced.



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  		FIGURE 7. Estradiol inhibits IL-1RtI protein expression. Whole cell lysates were generated from ECC-1 cells incubated with various concentrations of estradiol for 72 h. Proteins were resolved by 10% SDS-PAGE and detected with an anti-IL-1RtI Ab. Individual bands were scanned, and the intensity was quantified by computer analysis. The amount of IL-1RtI was normalized to GAPDH levels and plotted as a percentage of the control (no estradiol treatment) (representative of two experiments).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
This study demonstrates that IL-1{beta}-mediated responses by uterine epithelial cells, in particular the secretion of HBD2 and CXCL8, are inhibited by estradiol. Inhibition by estradiol was dose dependent, with maximal inhibition observed using 10–7 to 10–10 M, and mediated through the ER because addition of a pure ER antagonist abrogated this effect. The mechanism by which estradiol inhibits IL-1{beta}-mediated responses by uterine epithelial cells appears to be the down-modulation of the IL-1RtI, thereby reducing the cell’s ability to respond to IL-1{beta}. The inhibitory effect of estradiol on IL-1{beta}-mediated inflammatory responses generated by uterine epithelial cells, such as the secretion of HBD2 and CXCL8, indicates a link between the endocrine and immune systems and may be crucial for dampening proinflammatory responses during the time of ovulation or pregnancy.

In the present study, we demonstrate that estradiol inhibits IL-1{beta}-mediated mRNA expression and secretion of HBD2 and CXCL8 by uterine epithelial cells in a dose-dependent manner. The regulation of IL-1{beta}-mediated responses by estradiol is of particular importance during menstruation, ovulation, and pregnancy. During the onset of menstruation, when estradiol levels exhibit a precipitous decline and IL-1{beta} levels are at their peak (49), IL-1{beta} induces the expression of CXCL8, CCL2, and CCL5 by uterine epithelial cells. These chemokines are not only important for leukocyte migration, required for efficient degradation of the extracellular matrix, but also can enhance apoptosis of endometrial glandular and stromal cells (50). During ovulation and pregnancy, when estradiol levels are at their peak, IL-1{beta}-mediated proinflammatory responses would be dampened, likely facilitating the survival of the allogenic sperm and fetus, respectively.

Recognizing that mRNA expression and secretion of HBD2 and CXCL8 are regulated by estradiol, one could postulate that other genes whose expression is induced by uterine epithelial cells following IL-1{beta}-stimulation, such as HBD2, CCL5, CCL20, CXCL1, CXCL2, CXCL3, CXCL8, TNF-{alpha}, IL-6, and GM-CSF, may also be regulated by estradiol (16, 19, 20, 21, 22, 23). These proinflammatory factors facilitate the recruitment of immune cells, exhibit antimicrobial activities (51, 52), induce B cell responses and enhance T cell help (53, 54), stimulate proliferation, differentiation, maturation, and antimicrobial functions of neutrophils, as well as augment the functions of monocytes, macrophages, and dendritic cells (55, 56). The impact of IL-1{beta} on the generation of proinflammatory responses is quite evident and without proper regulation of this cytokine the host is likely to have serious physiological defects with regard to reproduction and chronic inflammatory disease.

To further understand the mechanism by which estradiol inhibits IL-1{beta}-mediated HBD2 and CXCL8 expression, we examined the effects of sex hormones on the IL-1RtI. Although it is known that sex hormones orchestrate cyclic endometrial changes and regulation of cytokine expression in the endometrium, little is known about modulation of the IL-1RtI on endometrial epithelial cells by sex hormones. Some evidence indicates that the IL-1RtI follows a triphasic pattern of expression, expressed to a low level in the proliferative phase, moderately expressed during ovulation, and highly expressed at the end of the menstrual cycle (57, 58), possibly correlating with increasing levels of progesterone, but other evidence suggests that the expression of IL-1RtI mRNA remains unchanged throughout the menstrual cycle (59). To the best of our knowledge, our findings are the first demonstration that in vitro the expression of the IL-1RtI mRNA by uterine epithelial cells is down-regulated by estradiol in a dose-dependent manner. Furthermore, we demonstrated that estradiol had a similar effect on IL-1RtII mRNA, suggesting that both types of the IL-1R may share a common mode of regulation. Significant inhibition was observed within a wide dose range of estradiol (10–7 to 10–10 M), and partial inhibition was observed at 10–11 M. In agreement with a significant decrease in the transcription of IL-1RtI, we observed a decrease in IL-1RtI protein by Western blot analysis. At estradiol concentrations of 10–8 to 10–12 M, IL-1RtI protein levels were greatly reduced with nearly a 97% reduction observed at 10–10 M. Our findings of ~20-fold decrease in IL-1RtI mRNA expression and 97% reduction in IL-1RtI protein levels in the presence of estradiol and a subsequent decrease in IL-1{beta}-mediated HBD2 and CXCL8 secretion suggest that cell surface expression of IL-1RtI is down-modulated, diminishing the cell’s ability to respond to IL-1{alpha}{beta}.

Dysregulation of IL-1{beta}-mediated responses can have severe consequences on the outcomes of pregnancy and also lead to chronic immunoinflammatory disease. IL-1{beta} plays a major role in the initiation of infection-related preterm labor and delivery (24, 25). Although normal term labor directly correlates with the expression levels of IL-1{beta}, IL-6, and CXCL8 (60, 61, 62, 63, 64), premature induction of these proinflammatory factors often results in preterm delivery (65). In addition, enhanced levels of neutrophil defensins correlate with impending preterm delivery, likely a product of increased neutrophils migration following aberrant CXCL8 production (66). Our observation that estradiol inhibits IL-1{beta}-mediated CXCL8 mRNA expression and secretion and that estradiol inhibits IL-1{alpha}{beta}-mediated induction of IL-6 production by endometrial stromal cells (67) suggests a mechanism by which stimulation of endometrial cells is minimized to ensure the viability of the fetus. IL-1 may also play an important role in the pathophysiology of endometriosis (30, 31, 32). IL-1{beta}-mediated CCL2 production by endometrial epithelial cells is elevated in women with endometriosis, which can result in macrophage activation and infiltration of eutopic endometrial tissue and enhanced angiogenesis (68, 69). Interestingly, not only are women with endometriosis deficient in their ability to down-regulate responses to IL-1 (36), estrogen appears to augment IL-1{beta}-mediated MCP-1 production (68). Although the underlying mechanisms of endometriosis remain unclear, IL-1{beta}-mediated responses appear to be involved, and regulation of these responses by estradiol may be important in healthy women at preventing disease.

Although the mechanism by which estradiol mediates the down-regulation of the IL-1RtI was not further explored in this study, we can speculate on how estradiol exerts its effects. Estradiol exerts its effects via an intracellular and membrane-bound ER (70). Estrogen-bound ER up-regulates or down-regulates the transcription of various genes by binding to the estrogen-responsive element of the genes or by interacting with other transcription factors (71, 72). However, estradiol-dependent effects can also be mediated through estrogen-responsive element containing mismatches (73), the NF-{kappa}B site, or from AP1 (72) and SP1-like elements (74), sequences of which are all found in the IL-1RtI promoter (75). It has been proposed that the NF-{kappa}B site may mediate IL-1RtI mRNA down-regulation by estradiol because ER can interact with NF-{kappa}B proteins to repress NF-{kappa}B-activated genes (76). Further clarification of the sites found within the IL-1RtI promoter that are necessary for estrogen-mediated effects is currently being addressed.

In conclusion, our studies suggest that participation of uterine epithelial cells in generating or sustaining an IL-1{beta}-mediated proinflammatory response is regulated by estradiol. Stimulation of uterine epithelial cells with IL-1{beta} induces the mRNA expression and secretion of HBD2 and CXCL8 and likely many more proinflammatory factors that are crucial for the defense of the female reproductive tract during pathogenic invasion but may be deleterious during times of ovulation and pregnancy. The presence of estradiol inhibits the secretion of these molecules by uterine epithelial cells following IL-1{beta}-stimulation, and the mechanism by which this occurs is via estrogen-dependent down-regulation of the IL-1RtI. During ovulation and pregnancy, when estradiol levels are at their peak, uterine epithelial cells would have a decreased capacity to respond to IL-1{beta} due to reduced levels of IL-1RtI on the cell surface. Although it is likely that other steroids or soluble factors secreted by other cells types, such as TGF-{beta} or IL-10, may play a role in regulation of proinflammatory responses in the uterus, we have demonstrated here that the inhibitory effect of estradiol on uterine epithelial cells may be directly related to reproductive fitness.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
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 National Institutes of Health Grant AI51877 (to C.R.W.) and Norris Cotton Cancer Center Grant CA23108. Back

2 Address correspondence and reprint requests to Dr. Todd M Schaefer, Department of Physiology, 710W Borwell, 1 Medical Center Drive, Lebanon, NH 03756. E-mail address: todd.m.schaefer{at}dartmouth.edu Back

3 Abbreviations used in this paper: IL-1RtI, IL-1R type I; HBD, human {beta}-defensin; ER, estrogen receptor; TER, transepithelial resistance; ICI, ICI 182,780; Ct, threshold cycle. Back

Received for publication April 21, 2005. Accepted for publication September 1, 2005.


   References
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 Abstract
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 Disclosures
 References
 

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J. Immunol., November 1, 2007; 179(9): 6284 - 6290.
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