The endometrial epithelial cell lines, AN3-CA, HEC-1-A, KLE, and RL95-2, and the breast adenocarcinoma cell line, MCF7, were obtained from the American Type Culture Collection (ATCC) (Manassas, VA). KLE and RL95-2 cells were maintained in phenol red-free DMEM-F12, HEC-1-A cells in McCoy's 5a, AN3-CA cells in MEM, and MCF7 cells in EMEM. AN3-CA, HEC-1-A, KLE, and RL95-2 cells were supplemented with 2 mM L-glutamate and 50 μg/ml gentamicin (American Pharmaceutical Partners, Inc., Schaumburg, IL). MCF7 cells were additionally supplemented with 1 mM sodium pyruvate, 1 mM non-essential amino acids (NEAA), and 0.15 mg/ml sodium bicarbonate. All media contained 5% fetal bovine serum (FBS) (US Bio-Technologies, Inc., Parkerfield, PA). Forty-eight hours prior to experiments, media was replaced with CD-F12, a phenol red-free DMEM/F12 containing charcoal/dextran treated FBS (Hyclone, Logan, UT) and the previously mentioned additives specific for each cell line. Adherent cell lines were harvested using 0.05% trypsin/0.53 mM EDTA in HBSS. Unless otherwise indicated, all reagents were obtained from Invitrogen (Carlsbad, CA).

17β-estradiol (E₂) and progesterone (P) (Calbiochem, San Diego, CA) were resuspended in ethanol to make a 1 mM stock solution of E₂ and P. Prior to experiments, E₂ and P stock solutions were diluted to the indicated concentrations using CD-F12. E₂ and P concentrations were determined by performing a dose response. The chosen concentrations demonstrated the highest level of cytokine and chemokine suppression after stimulation, based on preliminary experiments, and were also physiologically relevant. Cells were examined for presence of hormone receptors by endpoint RT-PCR using primers listed in Table 1 prior to performing each experiment. Following 48 hours culture in CD-F12, cells were plated at 0.2 × 10⁶ cells/well/ml in 12 well plates. The following day (~12 hours), CD-F12 media was aspirated and replaced with CD-F12 media containing either 0.001% ethanol vehicle (V), 10^-8 M E₂, 10^-7 M P, or a combination of 10^-8 M E₂ and 10^-7 M P. Triplicate wells were used for each hormone treatment condition and stimulation condition. For hormone receptor antagonist experiments, cells were pretreated for 2 hours with CD-F12 media containing V, 10^-6 M ICI 182, 780 or 10^-6 M RU486 (Tocris, St. Louis, MO). Following antagonist treatment, media was aspirated and replaced with CD-F12 containing V, 10^-8 M E₂, or 10^-7 M P, with antagonist vehicle, 10^-6 M ICI 182, 780, or 10^-6 M RU486. Hormone and antagonist combinations are indicated for in each figure demonstrating hormone and/or antagonist results. Triplicate wells were used for each condition. After 48 hours treatment, the triplicate wells were stimulated with ligand. Supernatants and/or cells for each well were harvested after 18 hours of stimulation. Cells were stimulated with the synthetic TLR3 ligand, polyinosinic-polycytidylic acid (Poly I:C, 10 μg/ml) (Amersham Pharmaceutical Biotech, Piscataway, NJ), the double-stranded DNA negative control, polydeoxyinosinic-deoxycytidylic acid (Poly dI:dC, 10 μg/ml) (Amersham Pharmaceutical Biotech, Piscataway, NJ), or the positive control, phorbol-12-myristate-13-acetate (Calbiochem, San Diego, CA) plus Ionomycin (Calbiochem, San Diego, CA) (PMA/I, 100 ng/ml/500 ng/ml). Supernatants were centrifuged at 1400 rpm for 8 minutes at 4°C, transferred to fresh tubes, and stored at -20°C until use. Cells were washed once with 1× PBS prior to use.

IL-6, IL-8, and Interferon-inducible protein 10 (IP-10) matched antibody pairs were purchased from either BioLegend (San Diego, CA) or BD Biosciences (San Diego, CA), and ELISA was performed according to manufacturer's instructions with 100 μl of cell free supernatant. IL-6 detection limit was 5 pg/ml, IL-8 was 8 pg/ml, and IP-10 was 10 pg/ml (the lowest standard used in each standard curve). Absorbance at 450 nm was read with the SPECTRAMax 190 microplate spectrophotometer, and results were analyzed by SOFTMax Pro software (Molecular Devices, Sunnyvale, CA). Sample concentrations were determined by interpolation from the standard curve. Triplicate samples for each treatment condition were analyzed in each experiment. Readings for the triplicate treatment conditions were averaged and standard deviation was calculated. Statistical analysis between treatment conditions was performed as described below. All experiments were performed a minimum of three times.

Endometrial epithelial cell lines were grown to confluence in 12 well plates and total RNA was isolated using the RNAqueous kit (Ambion, Austin, TX) per manufacturer's instructions. RNA was treated with 11 μl of Turbo DNase buffer, 2 μl of Turbo DNase, and 22 μl of Turbo DNase inactivation reagent (Ambion, Austin, TX). RNA was quantified using the RiboGreen^® RNA Quantitation kit (Molecular Probes, Eugene, OR), and fluorescence was measured using a 485 nm excitation filter and a 535 nm emission filter on a Fusion™ Universal Microplate Reader (Perkin-Elmer, Wellesley, MA). RNA concentrations were interpolated from a standard curve. Samples were measured in triplicate.

A total of 100 ng of RNA was used to synthesize cDNA using the random primers provided in the ImProm-II™ Reverse Transcriptase System (Promega, Madison, WI). Briefly, 100 ng of RNA was denatured at 65°C for 15 minutes and immediately placed on ice for 5 minutes. RNA was combined with master mix containing 4 μl ImProm-II 5× reaction buffer, 2 μl 25 mM MgCl, 1 μl 10 mM dNTP mix, 1 μl random primers, 0.5 μl RNasin, and water. Master mix was prepared with or without AMV reverse transcriptase (RT) in a total reaction volume of 20 μl. The reaction was run using an Eppendorf^® Master Gradient Thermocycler (Brinkman, Westbury, NY). Polymerase chain reaction (PCR) using gene specific primers was performed using 1 μl cDNA in 25 μl total reaction volume. PCR amplification was performed using Eppendorf^® MasterTaq Polymerase (Brinkman, Westbury, NY). Primers for TLRs 1–10, ERα, PRB, and PRT were obtained from the literature and are listed in Table 1252829. Primers for ERβ, also listed in Table 1, were designed using Primer3 software http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi (Whitehead Institute for Biomedical Research, Cambridge, MA). Loading dye (5 μl) was added to the reaction and 10 μl of sample was run on a 2% agarose 3:1 (Amresco, Solon, OH) gel to separate PCR products. Gels were stained with SYBR^® Green (BioWhittaker, Rockland, ME) for 45 minutes and visualized by ultraviolet transillumination at 302 nm. Digital images were obtained using a GelLogic 100 (Kodak, Rochester, NY).

Real time RT-PCR was performed using cDNA synthesized as described for end point RT-PCR. cDNA was combined with primer/probe sets and Taqman^® Universal PCR Master Mix (Applied Biosystems, Inc., Foster City, CA). Primer/probe sets for TLR3, ERα, and hypoxanthine phophoribosyltransferase (HPRT) were purchased and designed using TaqMan Gene Expression Assays™ (Applied Biosystems, Inc., Foster City, CA). Real time assays were run on an ABI 7000 (Applied Biosystems, Inc., Foster City, CA). Samples were normalized internally using the cycle threshold (CT) of the housekeeping gene, HPRT, as follows: ΔCT = (CT TLR3 or CT ERα) - (CT HPRT). Quantitative values for ERα mRNA are demonstrated using ΔCT, where the samples with the highest quantitative level of ERα mRNA have the lowest value and the samples with the lowest quantitative level of ERα mRNA have the highest value. For analysis of quantitative TLR3 mRNA values, the mean CT of RNA from untreated cells (Norm 1) was set to a relative quantity (RQ) value of 1 using the ΔΔCT calculated as follows: ΔΔCT = mean ΔCT(Norm 1) - mean ΔCT(Norm 1) and RQ = 2^(-1*ΔΔCT). All other samples were compared to the mean RQ value for the untreated cells (Norm 1) using the following equation: ΔΔCT = ΔCT(sample) - mean ΔCT(Norm 1). RQ values were calculated as follows: RQ = 2^(-1*ΔΔCT).

For preparation of protein lysates from endometrial epithelial cell lines for western blot analysis, cells were lysed by adding ice-cold RIPA lysis buffer (50 mM Tris, 150 mM NaCl, 50 mM NaF, 1 mM Na₄P₂O₇·10 H₂O, 0.1% DOC, 1.0% NP-40, 50 μl Na₃VO₄, and 100 μl Halt Protease Inhibitor Cocktail (Pierce Biotechnology, Inc, Rockford, IL)). After 30 min on ice with shaking, the lysates were centrifuged at 15000 × g for 10 min at 4°C. Supernatants were stored at -80°C until use. For western blot analysis, protein concentrations were determined using the BCA Protein Assay (Pierce Biotechnology, Inc, Rockford, IL). Equal amounts of unboiled protein were loaded onto a 4–20% Tris-HCl Precast Gel (BioRad) with 10 μl of sample or Blue Ranger marker (Pierce Biotechnology, Inc, Rockford, IL), and transferred to two identical 0.45 μM PVDF membranes (Millipore, Billerica, MA). The membranes were washed in Tris-buffered saline containing 0.05% Tween 20 (TBS-T) and non-specific binding sites were blocked by immersing the membrane in block reagent (1:7.5 mL Sea Block·TBS blocking buffer) (Pierce Biotechnology, Inc, Rockford, IL) for 1 hour at room temperature on a shaker. The membranes were then washed with TBS-T. One of two identical membranes was incubated overnight at 4°C with the isotype antibody in blocking buffer. The other membrane was incubated overnight at 4°C with ERα clone H-184 in blocking buffer. ERα clone H-184 was generously provided by Dr. Mark Hannink (University of Missouri-Columbia, Columbia, MO). Membranes were then washed and incubated with secondary antibody for 1 hour at room temperature. After incubation, the membranes were washed in TBS-T. Bound antibodies were detected by a chemiluminescent detection system (West Femto; Pierce Biotechnology, Inc, Rockford, IL) as recommended by the manufacturer's instructions. For exposure (10 seconds, 30 seconds, 1 min, 5 min, and up to 1 h) we used CL-XPosure™ Film (Pierce Biotechnology, Inc, Rockford, IL).

Cells were plated in 12 well plates and grown to confluence in CD-F12 with or without hormones. After 72 hours, cells were harvested, counted, and 0.5 × 10⁶ cells were labeled with either PE-labeled mIgG1α hTLR3 antibody (clone TLR3.7) (eBioscience, San Diego, CA) or PE-labeled mIgG1 isotype control (BD Biosciences, San Diego, CA). For intracellular staining, cells were fixed using Cytofix/Cytoperm buffer (BD Biosciences, San Diego, CA), and all washes and incubations were performed in the saponin-containing Perm/Wash buffer (BD Biosciences, San Diego, CA). Cells were analyzed using a Fluorescence-Activated Cell Sorter (FACScan) instrument and analysis was performed using CellQuest software (BD Biosciences, San Diego, CA). Ten thousand cells were counted, and viability was determined by generating forward scatter (FSC) versus side scatter (SSC) density plots and setting the gate to exclude dead cells. Histogram plots included only gated live cells. Markers indicating TLR3 positive cells were set so that less than 10% of isotype control cells were included in the positive marker.

Mean values for treatment conditions in triplicate were compared using one-way analysis of variance (ANOVA) and the Tukey post-hoc test to determine statistical significance. Differences were considered to be statistically significant at P < 0.05.

To examine the presence of TLR3, ERα, ERβ, PRA, and PRB in endometrial cell lines, mRNA was subjected to RT-PCR. The human breast adenocarcinoma cell line, MCF7, was used as a positive control for expression of mRNA coding for estrogen receptor (ER) and progesterone receptor (PR) 303132. We have previously demonstrated expression of TLR3 mRNA in RL95-2 cells26. We confirmed expression of TLR3 mRNA in the RL95-2 cells, and demonstrated expression of TLR3 mRNA in the cell lines HEC-1-A, KLE, and MCF7 (Table II). Expression of ER and/or PR has been previously demonstrated in the HEC-1-A, KLE, MCF7, and RL95-2 cells 3031323334. Receptor expression in these cell lines was confirmed and receptor expression was documented in AN3-CA cells. As summarized in Table II, many of our cell lines express detectable levels of mRNA for TLR3 and hormone receptors. We observed that the band intensity for all cell lines expressing hormone receptors was high, with intensity for the MCF7 cells being the highest. To determine if the differences observed by endpoint RT-PCR were due to quantitative differences in ERα mRNA expression, we subjected cDNA synthesized from 100 ng RNA from each of the cell lines to Real Time RT-PCR. As demonstrated in Figure 1A, our cell lines express varying quantities of ERα mRNA. The AN3-CA and MCF7 cells expressed the highest levels of ERα mRNA, with values significantly greater than values for the RL95-2, KLE, and HEC-1A cells. The RL95-2 and KLE cells expressed the second highest levels of ERα mRNA, which were significantly lower than values for the AN3-CA and MCF7 cells, but greater than values for the HEC-1A cells, which did not express ERα mRNA. Thus, the results in Figure 1A confirm the endpoint RT-PCR results shown in Table 2 and demonstrate differential expression of ERα mRNA in endometrial and breast epithelial cell lines.

To determine whether hormone treatment altered expression of ERα mRNA, we subjected the RL95-2 cell line to hormone treatment and analyzed ERα mRNA by Real Time RT-PCR. Hormone concentrations were determined by dose response as described in Methods. As demonstrated in Figure 1B, hormone treatment does not alter levels of ERα mRNA in the RL95-2 cells.

In addition to endpoint and Real Time RT-PCR, we also analyzed our cell lines for presence of ERα protein. The MCF7 breast epithelial cell line was used as a positive control (Figure 1C, lanes 3 and 4). All cell lines, with the exception of the HEC-1A cells and high passage RL95-2 cells (Figure 1C, lane 2), express ERα protein (Figure 1C and data not shown). As shown in Figure 1C, MCF7 cells (lanes 3 and 4) express a greater amount of ERα protein than the RL95-2 cells (Figure 1C, lane 1), confirming the Real-Time RT-PCR results. Additionally, RL95-2 cells lose ERα expression with high passage, which has been previously documented 37. Due to the ability of RL95-2 cells to lose ERα expression with high passage, receptor presence had to be verified prior to experiments.

We have previously demonstrated production of IL-6, IL-8, and the chemokine IP-10 after stimulation of RL95-2 cells with the synthetic dsRNA, Poly I:C26. We have also demonstrated that the response to Poly I:C in the RL95-2 cells is dependent on the expression of TLR326. Since expression of TLR3 mRNA and protein was shown to be cyclically regulated26, we performed experiments to examine whether E₂ and P may influence TLR3 expression and/or function. A dose response was performed for E₂ and P to determine the concentration of hormone to be used. The concentrations chosen, 10^-8 M E2 and 10^-7 M P, resulted in the greatest degree of cytokine and chemokine suppression (data not shown) and are also physiologically relevant335. Cells were stimulated with Poly I:C, PMA/I, or Poly dI:dC in triplicate. PMA/I directly activates intracellular signaling mechanisms leading to cytokine expression and was used as a positive control26. Poly dI:dC, a dsDNA analogue of Poly I:C, does not act as a TLR3 ligand and was used as a negative control to demonstrate the specificity of Poly I:C for TLR326. Supernatants were collected and examined for production of IL-6, IL-8, and IP-10. Experiments were repeated at least three times to ensure reproducibility. RL95-2 cells treated with E₂ showed significant suppression of IL-6, IL-8, and IP-10 protein secretion upon stimulation with Poly I:C in comparison to vehicle-treated controls and P-treated samples (Figure 2A, 2B, and 2C). Treatment with E₂ also significantly suppressed PMA/I-induced IL-6 production, but did not significantly suppress IL-8 (Figure 2A). PMA/I treatment did not induce production of IP-10 (Figure 2C). Suppression of PMA/I response by E₂ has been previously documented1236. The significance of the suppression is notable considering the significant proliferative effect of E₂ on RL95-2 cells as compared to vehicle-treated cells (Figure 2D). P did not induce significant proliferation of RL95-2 cells in comparison to vehicle-treated cells (Figure 2D). The response to the synthetic dsRNA, Poly I:C, was specific because stimulation with the synthetic dsDNA, Poly dI:dC, had no effect. These results suggest that E₂, but not P, significantly suppresses proinflammatory and antiviral cytokine responses upon Poly I:C stimulation of RL95-2 cells.

Previous work in our laboratory has demonstrated cyclic variation of TLR3 mRNA and protein expression in human endometrium26, suggesting the possibility that some of the E₂ suppression of Poly I:C-stimulated cytokine production might be due to E₂ regulation of TLR3 expression. To examine this possibility, RNA collected in triplicate from RL95-2 cells treated with ethanol vehicle, 10^-8 M E₂, or 10^-7 M P was used to examine TLR3 mRNA expression by real time RT-PCR. E₂ and P did not significantly alter TLR3 mRNA expression (P > 0.05) as compared to vehicle-only controls (Figure 3A and 3B). The results in this in vitro model system suggest that neither E₂ nor P directly regulates TLR3 gene transcription.

Protein expression was examined by Fluorescent-Activated Cell Sorter (FACS) analysis of RL95-2 cells treated with ethanol vehicle, 10^-8 M E₂, or 10^-7 M P in triplicate. We had previously shown that RL95-2 cells express TLR3 intracellularly26. In these experiments, the intracellular expression of TLR3 in RL95-2 cells was not altered by treatment with E₂ and P as compared to vehicle-treated controls (Figure 3C). Protein expression remained unaltered in response to E₂ or P treatment for up to 7 days (data not shown). The lack of alteration in TLR3 mRNA and protein expression in response to E₂ suggests that the observed suppression of cytokine production is not due to transcriptional or translational regulation of TLR3.

To determine if similar effects could be observed in other endometrial epithelial cell lines, the cell lines KLE, HEC-1-A, and AN3 CA were used. As expected, the TLR3-negative, hormone receptor-positive cell line, AN3 CA, did not respond to Poly I:C (data not shown). Treatment of KLE cells with E₂ and P resulted in a non-significant decrease in production of IL-6 and a significant decrease in production of IP-10 (Figure 4A), which was expected since KLE cells express TLR3, ER, and PR (Table II). The observed decrease in IL-6 production in the KLE cells treated with E₂ or P was not statistically significant (P = 0.165) and may be due to the described defects in nuclear ER trafficking in the cell line34. The KLE cell line produced background levels of IL-8 too high for observation of a Poly I:C-induced effect (data not shown). A suppressive effect from hormone treatment was not observed in the HEC-1-A cells (Figure 4B), which express TLR3 and ERβ, but do not express ERα, PRA, or PRB (Table II). Our results, therefore, suggest that ERα must be present for suppression of cytokine or chemokine production. Finally, RL95-2 cells have been reported to lose ERα expression with continued passage in culture37. In our study, consistent with previous reports, RL95-2 cells lacking ERα did not exhibit suppressive effects after E₂ treatment (Figure 1C and data not shown) and receptors had to be verified in the RL95-2 cells prior to each experiment to ensure reliable results. Thus, suppression of cytokine and chemokine production by E₂ in endometrial epithelial cells may require ERα.

Since treatment with E₂ and P did not affect TLR3 mRNA or protein expression in the RL95-2 cells (Figure 3A, 3B, and 3C), but did affect TLR3-induced cytokine and chemokine production, and hormone receptor-negative cell lines do not show a suppressive response, we postulated that the suppressive effect may be hormone receptor-dependent. To determine if the suppression of cytokine and chemokine production in RL95-2 cells was hormone receptor-dependent, we utilized the hormone receptor antagonists, ICI 182, 780 and RU486. For the no-antagonist controls, cells were treated with ethanol vehicle, E₂, or P (representative data in Figure 2). Treatment with the ER antagonist, ICI 182, 780, restored cytokine and chemokine levels in E₂-treated samples to those of vehicle-treated controls (Figure 5A). RU486 had no restorative effect on E₂-treated samples, as expected (Figure 5B). Since suppression was not seen in the no-antagonist, P-treated samples, treatment with the PR antagonist, RU486, had no effect (Figure 5B). These results are not due to a suppression of cytokine and chemokine production in the vehicle-treated samples by the antagonists themselves because the antagonists caused a reduction in cell number for all antagonist-treated samples (data not shown). Thus, our results confirm that E₂ suppresses cytokine and chemokine production in Poly I:C-stimulated RL95-2 cells in a hormone receptor-dependent manner.

To determine whether the observed hormone effect was characteristic of only endometrial epithelial cells, or if the suppressive effect occurred in a different epithelial cell line, we utilized the breast epithelial adenocarcinoma, MCF7. MCF7 cells have been shown to express ER and PR and respond to E₂ and P treatment 303132. MCF7 cells were examined for expression of TLR mRNA. We found that MCF7 cells express TLR2, TLR3, TLR5, and TLR6 (Table II, Figure 6A, and data not shown). For detection of TLR3 in the MCF7 cells, the RL95-2 cells were used as a positive control. The amplified band in the RL95-2 and MCF7 cells was of the appropriate size for TLR3 (Figure 6A). To determine if TLR3 expressed in these cells was functional, MCF7 cells were stimulated with Poly I:C, PMA/I, the TLR4 ligand lipopolysaccharide (LPS), or Poly dI:dC in triplicate. Supernatants were collected and examined for production of IL-6, IL-8, and IP-10. MCF7 cells, as expected, did not respond to stimulation with LPS or Poly dI:dC (Figure 6B and data not shown). MCF7 cells did produce significant amounts of IL-8 and IP-10 after stimulation with Poly I:C and IL-8 after stimulation with PMA/I (Figure 6B and data not shown). However, the MCF7 cells did not produce IL-6. Our results demonstrate that MCF7 cells express functional TLR3 and produce cytokines and chemokines upon stimulation with Poly I:C.

Since MCF7 cells express functional TLR3, we examined whether E₂ and/or P suppress production of cytokines and chemokines in Poly I:C-stimulated MCF7 cells. The response to dsRNA was specific, as the MCF7 cells did not respond to stimulation with the synthetic dsDNA, Poly dI:dC. As expected, the cells did not produce IL-6 upon stimulation with Poly I:C. Cells treated with E₂ and P exhibited a significant suppressed response to Poly I:C and PMA/I in comparison to vehicle only controls (Figure 7A). This is in direct contrast to the RL95-2 cells, in which significant cytokine and chemokine suppression when treated with P was not observed (Figure 2A, 2B, and 2C).

MCF7 cells demonstrated suppression of TLR3-induced cytokine and chemokine production in response to E₂ and P treatment (Figure 7A) and we did not observe changes in TLR3 mRNA or protein expression in these cells (data not shown). Therefore, we wanted to determine if the suppressive responses were hormone receptor-dependent, as in the RL95-2 cells. ICI 182, 780 and RU486 have previously been shown to be effective on MCF7 cells by other investigators 383940. Treatment of E₂-treated wells with ICI 182, 780 and P-treated wells with RU486 restored cytokine and chemokine levels to those of vehicle-treated controls upon Poly I:C stimulation (Figure 7B and 7C). Since treatment with antagonists resulted in a decrease in cell number (data not shown), these results are not due to the suppression of cytokine and chemokine levels by the antagonists alone. Had the antagonists caused suppression of cytokine and chemokine production, the levels of cytokine and chemokine protein in the antagonist-, hormone-treated samples would have been decreased as compared to the antagonist-, vehicle-treated samples. Thus, E₂ and P suppress cytokine and chemokine production in Poly I:C-stimulated MCF7 cells in a hormone receptor-dependent manner as was observed in the RL95-2 cells.