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Centre for Reproductive Biology, University of Edinburgh, Edinburgh EH16 4SB, United Kingdom
Address all correspondence and requests for reprints to: Michael T. Rae, Ph.D., Centre for Reproductive Biology, University of Edinburgh, Chancellors Building, 49 Little France Crescent, Edinburgh EH16 4SB, United Kingdom. E-mail: mrae1{at}staffmail.ed.ac.uk.
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Abstract |
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(500 pg/ml) increased mRNA levels of cyclooxygenase-2 (COX-2) (P < 0.01) at 48 h. The COX-2 mRNA response to IL1 was associated with an approximate 18-fold (P < 0.01) increase in mRNA levels of 11ß-hydroxysteroid dehydrogenase type 1 (11ßHSD1), encoding the steroid dehydrogenase that reversibly reduces cortisone to antiinflammatory cortisol. Addition of cortisol to OSE cell culture medium dose-dependently suppressed the COX-2 mRNA response to IL1 (P < 0.01) but reciprocally enhanced the 11ßHSD1 mRNA response (P < 0.05), with both effects strongest at 1 µM cortisol. Presence of glucocorticoid receptor- mRNA and protein was established in OSE cell monolayers and treatment with IL1 shown to significantly up-regulate the glucocorticoid receptor- mRNA level (P < 0.05). Glucocorticoid receptor antagonist (RU486, 10 µM) fully reversed the inhibitory effect of 1 µM cortisol on IL1-stimulated COX-2 mRNA expression. Progesterone also suppressed IL1-induced COX-2 mRNA expression but had no significant effect on IL1-stimulated 11ßHSD1 expression. These data provide direct evidence for antiinflammatory actions of cortisol and progesterone in human OSE cells. |
Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Ovulation can be seen as a natural inflammatory process (5, 6), which if inadequately resolved may give rise to genetic damage of the OSE and predispose to development of ovarian epithelial cancer (7). Experimental evidence exists that OSE cells do indeed suffer inflammation-associated DNA damage during ovulation (8), implying that mechanisms normally exist to minimize this potentially deleterious process. Recently we reported that an ovulation-associated cytokine, IL1 (9, 10), causes increased expression of 11ß-hydroxysteroid dehydrogenase type 1 (11ßHSD1) mRNA expression in human primary OSE cell cultures (11). This translated functionally into increased rates of conversion of cortisone to cortisol by OSE cells in vitro (11), consistent with the intracellular steroidogenic properties of 11ßHSD1 (12). Because of the established role of cortisol as an antiinflammatory agent (13), we postulated a role for ovulation-associated up-regulation of 11ßHSD1 in creating a steroidal milieu that counteracts inflammation at the time of ovulation (14). Here we provide direct support for the hypothesis that cortisol exerts antiinflammatory actions in OSE cells. These actions include up-regulation of cytokine-induced 11ßHSD1 itself, providing a means to sustain the regeneration of antiinflammatory cortisol at its site of action on the ovarian surface.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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OSE cells were obtained from the ovaries of 16 premenopausal women undergoing surgery for nonmalignant gynecological conditions. All patients had given informed consent, and the study had local ethics committee approval. Details of the patients and the analyses done on their OSE cells are given in Table 1
. None of these subjects were receiving medications that would have been likely to influence ovarian function. Cells were collected at laparotomy by gentle scraping of the ovarian surface with a sterile wooden spatula, which was then rinsed into sterile, warmed OSE-1 culture media, as described previously (15). Cells were collected as near to the beginning of the surgical procedure as practicable to avoid any contamination with blood cells. Collections were then examined by phase-contrast microscopy to verify that a representative biopsy of the OSE had been recovered.
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All tissue culture reagents were obtained from Life Technologies, Inc. (Renfrewshire, UK) and Sigma Chemical Co. (Poole, Dorset, UK). OSE scrapings were cultured in donor calf serum-precoated flasks (75 cm2, Corning Inc. Glass Works, Corning, NY). Culture media (OSE 1) consisted of Medium199:MCDB105 (1:1 vol/vol) supplemented with fetal calf serum (15% vol/vol), streptomycin (50 µg/ml), penicillin (50 IU/ml), and L-glutamine (2 mmol/liter). Cells were incubated at 37 C in a humidified incubator under an atmosphere of 95% air, 5% CO2 for up to 28 d, with media renewed every 7 d. Confluent cell monolayers are usually obtained within 21 d using this system (15). Monolayers were routinely examined by phase-contrast microscopy for contaminating cells such as fibroblasts. Confirmation of cell purity was obtained in selected cases by immunocytochemical staining for cytokeratin 5, 6, 8, and 17 (11) using a commercially available monoclonal antihuman cytokeratin antibody (Dako, Ely, Cambridgeshire, UK).
Experimental treatment of OSE cell cultures
Confluent OSE cell monolayers were treated with trypsin-EDTA in Hanks’ balanced salt solution (0.05% wt/vol trypsin, 0.5 mM EDTA, Invitrogen, Paisley, UK) at 37 C for 5 min. Cells were then collected by centrifugation at 800 x g for 5 min. This pellet was washed in fresh OSE1 media and then resuspended in fresh media. Cell numbers and viability were determined by trypan-blue (Sigma) exclusion counting in a hemocytometer; viability ranged from 75 to 95%. Cell suspension volume was adjusted to yield 50,000 cells per 500 µl medium. Equal portions (500 µl) of the cell suspension were distributed in 24-well polystyrene culture plates (Corning) and incubated for 24 h. The medium was then aspirated and replaced with serum-free OSE1 media, containing 0.01% BSA (Sigma) (OSE2). After 24 h, experimental treatments were added to wells and the culture routinely continued for a further 48 h. Time-course studies included, 6-, 12-, 24-, and 48-h treatments, with untreated controls at each time point. IL1 (R&D Systems Europe Ltd., Abingdon, Oxon, UK) at a final (well) concentration of 500 pg/ml was predissolved in OSE2. The rationale for choosing this fixed dose of IL1 is based on our previously published data showing 500 pg/ml to be maximally effective in induction of 11ßHSD1 expression (11). Cortisol or progesterone was dissolved in ethanol and added to cultures such that a concentration of ethanol in culture wells was kept constant at 0.02% (control wells received ethanol alone). The steroid concentration range (0.01–1 µM) was based on published ranges of cortisol concentrations in serum and follicular fluid in women undergoing in vitro fertilization with exogenous gonadotrophins (16). Similar concentrations of progesterone were selected for comparison, bearing in mind that ovarian progesterone concentrations exceed these doses significantly during the luteal phase. After the 48-h treatment period, total RNA was extracted from OSE cell monolayers for RT-PCR analysis as described below. To delineate glucocorticoid effects mediated by the glucocorticoid receptor (GR) from those mediated by the mineralocorticoid receptor, selected experiments were performed in the presence of a 10-fold molar excess of the GR antagonist RU486. Each set of experiments was performed a minimum of three times using cells donated from three different patients. The actual number of times each experiment was replicated is detailed in the figure legends.
RNA extraction and quality analysis
RNA was extracted from OSE cells using RNeasy minispin columns (Qiagen, Crawley, West Sussex, UK) as per manufacturer’s protocols. Aliquots (1 µl) of purified RNA were removed for quantification and quality assessment. RNA was quantified and quality assessed using the Agilent 2100 bioanalyzer system for total RNA in combination with RNA6000nano chips (Agilent Technologies, Cheshire, UK). Only RNA that displayed intact 18S and 28S ribosomal RNA peaks was reverse transcribed to cDNA for real-time PCR analysis. This quality control step was included for each experimental run to avoid generation of false-negative results due to RNA degradation before and during extraction steps, and also as a quantification method to ensure equal amounts of RNA were reverse transcribed in each RT reaction.
Real-time PCR analysis
Total RNA (1 µg) was DNAase treated (1 U) (Invitrogen) before analysis. DNAase-treated RNA (200 ng) was reverse transcribed (random hexamer kit; Applied Biosystems, Warrington, Cheshire, UK), and 2 µl of the reverse transcription (RT) mix was analyzed. cDNA was analyzed in a reaction mixture (final volume 25 µl) containing 300 nmol/liter primers and 200 nmol/liter TaqMan hybridization probe (Biosource UK Ltd., Nivelles, Belgium). Primers and probes were designed using Primer-Express software (Perkin-Elmer, Beaconsfield, Bucks, UK). Target mRNA was quantified in relation to the abundance of 18S rRNA in each sample. The positive controls were human liver total RNA (Ambion, Huntingdon, Cambridgeshire, UK) and in-house prepared human placental mRNA. The negative controls consisted of RT-negative (RNA template with no reverse transcriptase enzyme) and RT-H2O (water in place of RNA template). A Taqman reaction-negative control (water in place of cDNA) was also included. The sequences of all primer and probe sets used are shown in Table 2. All primer/probe sets were validated before use.
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OSE cell RNA from unstimulated cultures was reverse transcribed (Superscript RT, Invitrogen) with Oligo-dT priming (Invitrogen) as per the manufacturer’s protocols. Resulting cDNA was PCR amplified with primers specific to GR and GRß isoforms (17, 18). Promega complete PCR mix was used in a total reaction volume of 50 µl. PCR products were visualized on a 1.5% agarose gel containing 0.002% ethidium bromide (Sigma) under UV light.
Immunohistochemistry
Cultured OSE cells were fixed in neutral buffered formalin for 10 min at room temperature followed by washing in 0.05% triton-x-100 (Sigma) for 20 min at 37 C. After triplicate 3-min washes in PBS, cells were sequentially blocked with avidin (Vector, Peterborough, UK), biotin (Vector), and nonimmune serum (horse, Vector). Mouse-antihuman GR (Novocastra, Newcastle upon Tyne, UK) serum was then applied at 1:100 dilution for 1 h at 37 C. After three washes with PBS+0.01% Tween 20 (Sigma) (3 min each), secondary antibody (horse-antimouse) diluted in nonimmune horse serum was applied for 1 h at room temperature. Sequential washes with PBS+0.01% Tween 20 (three x 3 min) were then performed before incubating with Vector-ABC Elite reagents and visualization via chromagen (diaminobenzidine) staining. Negative controls consisting of nonimmune mouse IgG2 substituted for primary antiserum were run routinely.
Statistical analyses
Treatment differences were analyzed by ANOVA. Specific treatment effects were analyzed by Fisher’s protected least significant difference and Student’s t test after logarithmic (base 10) transformation. Contrasts between treatments and untreated controls were considered significant at P < 0.05.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Ovarian surface epithelial cell cultures from three patients were used to establish the time dependence of inflammation-associated gene expression in response to treatment with IL1 (500 pg/ml). Results showed consistent trends toward increased expression of both cyclooxygenase (COX)-2 and 11ßHSD1 mRNA with duration of treatment (Fig. 1). Expression of COX-2 mRNA was maximal at 6 h (P < 0.01), falling thereafter but remaining significantly elevated (
18-fold) at 48 h (P < 0.05) (Fig. 1A). Levels of 11ßHSD1 mRNA were significantly increased (P < 0.05) at 12 and 24 h and maximally stimulated (16-fold) at 48 h (P < 0.01) (Fig. 1B). Cultured OSE cells contained low levels of 11ßHSD2 that were unaltered by any treatment with IL1 (data not shown). Because 11ßHSD1 mRNA expression is the reference response to IL1 in this OSE cell culture system (11 11A ), treatment duration was fixed at 48 h for all subsequent experiments.
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and cortisol on COX-2, 11ßHSD2 mRNA expression in OSE cell cultures
OSE cultures from six patients were used to investigate interactions between IL1 (500 pg/ml) and cortisol (0.01–1.0 µM) on COX-2 and 11ßHSD mRNA expression (Fig. 2). Treatment with cytokine alone for 48 h induced an average 16-fold (P < 0.01) increase in COX-2 mRNA. Cortisol alone had no effect but when combined with IL1 caused concentration-dependent suppression of COX-2 mRNA to a level not significantly different from nonstimulated control values at all doses tested (Fig. 2A).
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alone also induced substantial (average 20-fold, P < 0.01) increases in 11ßHSD1 mRNA expression at 48 h (Fig. 2B
). Cortisol alone had no effect but caused striking, dose-dependent enhancement of 11ßHSD1 in the presence of IL1, which was maximal at 1 µM cortisol (P < 0.01).
There was no measurable effect of cortisol or any interaction between cortisol and IL1 on expression of 11ßHSD2 mRNA (data not shown).
Inhibitory action of GR antagonist RU486 on antiinflammatory cortisol action in OSE cell cultures
The mode of antiinflammatory cortisol action in OSE cells was explored using OSE cells from a further three patients (Fig. 3). Presence of a 10-fold molar excess of the GR antagonist RU486 (10 µM) in medium containing 1 µM cortisol + 500 pg/ml IL1 totally reversed the inhibitory effect of cortisol on IL1-induced COX-2 mRNA expression at 48 h in all three patients (P < 0.05, compared with IL1 alone).
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In five of five patients, cultured OSE cells tested by RT-PCR were positive for GR mRNA (Fig. 4, upper panel) and negative for GRß mRNA (data not shown). Immunohistochemical staining with antihuman GR serum confirmed the expression of GR protein (Fig. 4, lower panel).
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and cortisol on GR mRNA expression in OSE cell cultures
Treatment of OSE cell cultures from four of four patients with IL1 caused an average 6-fold (P < 0.05) increase in GR mRNA expression at 48 h (Fig. 5). Cortisol (0.01–1.0 µM) had no significant effect on basal or IL1-stimulated levels of GR mRNA.
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Progesterone dose-dependently suppressed IL1-stimulated COX-2 mRNA levels in five of five patients (Fig. 6A); albeit inhibition remained incomplete at the highest (1 µM) progesterone concentration tested, this was significantly reduced, compared with IL1 alone (P < 0.05). Progesterone had no measurable effect on COX-2 or 11ßHSD1 mRNA expression in the absence of IL1, nor did it significantly affect IL1-stimulated 11ßHSD1 mRNA expression (Fig. 6B).
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, had no significant effect on GR or progesterone receptor (PR) mRNA expression; in the case of PR, there was no regulation by cortisol either alone or in combination with IL1 (data not shown). |
Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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increases mRNA expression of 11ßHSD1 in cultured OSE cells, consistent with increased regeneration of cortisol from exogenous cortisone in vitro (11). We now extend these observations to demonstrate that treatment with IL1 induces expression of the inflammation-associated gene COX-2. Furthermore, we show that cortisol acts in a direct antiinflammatory manner by down-regulating cytokine- induced COX-2 and indirectly by up-regulating cytokine- induced 11ßHSD1. This suggests a feed-forward control mechanism through which cortisol is able to regenerate itself at its site of action on the inflamed ovarian surface.
A strength of these data is that they are based on replicate analyses of OSE cell function in multiple individuals. Although OSE cells were obtained at various stages of the menstrual cycle (Table 1), their initial propagation in serum-containing medium for a minimum of 3 wk before analysis should have nullified any contribution of the steroidal milieu existing at the time of cell collection, as previously demonstrated (11). The relative paucity of OSE cells that can be collected from a single patient imposes limits on the number of replicated analyses that can be performed on any particular set of cells. This constraint was offset by repeating all experiments on tissue from at least three patients, allowing a considerable measure of confidence in the conclusions drawn. However, it remains to be determined whether this pattern of response to IL1 truly reflects that shown in vivo.
Increased expression of COX-2 mRNA by the OSE upon exposure to IL1 is consistent with the postulated inflammatory basis of ovulation. During ovulation the OSE is exposed to multiple inflammatory mediators including cytokines (IL-1 and IL-6) that up-regulate COX-2 (19). Prostaglandin E2 formed via COX-2 initiates the acute cellular events associated with inflammation (20). The subsequent biochemical cascade leads to collagen breakdown, apoptotic cell death at the ovarian surface, and follicular rupture (21). Glucocorticoids acting via GR have been previously demonstrated to reduce inflammation through inhibition of phospholipase A2 and COX-2 (22). In ovine OSE cells, expression of prostaglandin endoperoxide synthase undergoes up-regulation in OSE cells of the follicular apex just before ovulation (23), and prostaglandin synthesis in these cells is thought to be a causative agent of apoptotic death (24). Collectively, these observations are consistent with the hypothesis that ovulation is effectively a natural wound, with attendant inflammation, and that serial wounding and repair occurs at the ovarian surface as a consequence of the ovarian cycle (5, 6, 14).
We previously proposed that intracrine formation of cortisol in granulosa and OSE cells limits inflammation and associated tissue injury at the time of ovulation (11, 14). The present study provides direct evidence for an antiinflammatory action of cortisol in OSE cells through down-regulation of cytokine-induced COX-2 expression. Glucocorticoids generally exert antiinflammatory effects via the ligand-activated GR signaling pathway in inflamed tissues to antagonize the induction of the proinflammatory transcription factor nuclear factor-
B (13, 25). Based on the present ability of the GR antagonist RU486 to blockade cortisol action, it would appear that the same mode of antiinflammatory glucocorticoid action operates in OSE cells. Detection of GR expression in the absence of GRß reinforces the premise that the OSE is a glucocorticoid target because GRß appears to be involved in inhibition of GR (17, 18). Additionally, the finding that IL1 treatment stimulates mRNA expression of GR suggests a novel mechanism through which inflammatory cytokines might sensitize OSE cells to glucocorticoids.
The IL1-induced increase in expression of 11ßHSD1 by OSE cells was further augmented by cortisol, indicating a feed-forward intracrine mechanism whereby cortisol can promote its own synthesis from cortisone. Both the level and type of 11ßHSD isoform expressed, along with GR, are critical determinants of cellular responses to glucocorticoids (12). Cytokine-stimulated cells from sites of inflammation throughout the body have been shown to undertake increased 11ßHSD1 expression with concomitantly decreased 11ßHSD2 expression in vitro (11, 26, 27, 28, 29, 30). Because 11ßHSD1 reversibly converts inactive cortisone to active cortisol whereas 11ßHSD2 inactivates cortisol to cortisone, this pattern of 11ßHSD isoform expression predisposes to increased intracellular formation of cortisol from cortisone (12). From the present analyses of 11ßHSD1 and -2 mRNA expression in OSE cells, we deduced absence of induction of 11ßHSD2 but presence of l1ßHSD1 positively coupled to IL receptor signaling. This is in agreement with previous evidence that IL1 stimulates metabolism of cortisone to cortisol in OSE cultures via an IL receptor-mediated postreceptor signaling pathway (11).
OSE cells are established sites of progesterone action mediated by PR (31, 32). Consistent with its established immunosuppressive and antiinflammatory properties (13), the present results demonstrate that progesterone, like cortisol, also prevents IL1-stimulated increases in COX-2 expression by OSE cells. Unlike cortisol, progesterone had no effect on IL1-stimulated 11ßHSD1 expression. The mode of progesterone action on COX-2 mRNA expression was not investigated in the present study. However, because the progesterone concentration rises to around 10 µM in preovulatory follicular fluid (16), a physiologically significant antiinflammatory action of progesterone on the OSE mediated by PR seems likely.
A link between ovulation and development of ovarian cancer has been postulated for many years (33). The initial trigger for ovarian cancer formation has been suggested to arise from ovulatory damage to the ovarian surface (34, 35) associated with inflammatory assault (4). Given that prostaglandins are not only proinflammatory but are also directly implicated in tumorogenesis (4), negative regulation of inflammatory processes involving prostaglandin formation has obvious potential relevance to the prevention of ovarian cancers. It is noteworthy in this regard that loss of PR has previously been associated with neoplastic transformation in ovarian cancer cells (31).
Based on the divergent effects of 11ßHSD1 and -2 on cell proliferation observed in vitro, Rabbitt et al. (36) previously suggested that the ability of 11ßHSD1 to generate cortisol might act as an autocrine antiproliferative, prodifferentiation stimulus in normal adult tissues. In contrast, the cortisol-inactivating properties of 11ßHSD2 might lead to proproliferative effects, particularly in tumors. Our finding that normal OSE cells are essentially devoid of 11ßHSD2 mRNA lends weight to the concept that the ovulation-associated increase in 11ßHSD1 expression leading to locally increased formation of cortisol promotes healthy functioning of the OSE.
In conclusion, these data provide direct evidence for antiinflammatory actions of glucocorticoids and progesterone mediated by interaction with GR and PR, respectively, in OSE cells. Antiinflammatory actions of cortisol in OSE cells include up-regulation of cytokine-induced 11ßHSD1, indicating a means for regenerating cortisol at its site of action on the ovarian surface. The discovery of this antiinflammatory intracrine mechanism in OSE cells has implications for the diagnosis and treatment of reproductive disease states that involve chronic or excessive ovarian inflammation, including endometriosis and ovarian cancer.
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Acknowledgments |
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Footnotes |
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Abbreviations: COX, Cyclooxygenase; GR, glucocorticoid receptor; 11ßHSD1, 11ß-hydroxysteroid dehydrogenase type 1; OSE, ovarian surface epithelium; PR, progesterone receptor; RT, reverse transcription.
Received December 30, 2003.
Accepted April 30, 2004.
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References |
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determined by microarray analysis. J Endocrinol, in press
-B/steroid hormone receptor interactions as a functional basis of anti-inflammatory action of steroids in reproductive organs. Mol Hum Reprod 2:433–438
and ß in cultured human ovarian surface epithelial cells. Mol Hum Reprod 4:811–815B and steroid receptorsignaling pathways. Endocr Rev 20:435–459 and interleukin 1ß enhance the cortisone/cortisol shuttle. J Exp Med 186:189–198 and -ß, progesterone receptor, and androgen receptor mRNA in normal and malignant ovarian epithelial cells. Proc Natl Acad Sci USA 96:5722–5727This article has been cited by other articles:
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  D. A. Symonds, I. Merchenthaler, and J. A. Flaws Methoxychlor and Estradiol Induce Oxidative Stress DNA Damage in the Mouse Ovarian Surface Epithelium Toxicol. Sci., September 1, 2008; 105(1): 182 - 187. [Abstract] [Full Text] [PDF]
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 S. van den Driesche, V. M Smith, M. Myers, and W C. Duncan Expression and regulation of oestrogen receptors in the human corpus luteum Reproduction, April 1, 2008; 135(4): 509 - 517. [Abstract] [Full Text] [PDF]
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 K S Fegan, M T Rae, H O D Critchley, and S G Hillier Anti-inflammatory steroid signalling in the human peritoneum J. Endocrinol., February 1, 2008; 196(2): 369 - 376. [Abstract] [Full Text] [PDF]
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 M. Myers, M. C. Lamont, S. van den Driesche, N. Mary, K. J. Thong, S. G. Hillier, and W. C. Duncan Role of Luteal Glucocorticoid Metabolism during Maternal Recognition of Pregnancy in Women Endocrinology, December 1, 2007; 148(12): 5769 - 5779. [Abstract] [Full Text] [PDF]
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S. G Hillier Diamonds are forever: the cortisone legacy J. Endocrinol., October 1, 2007; 195(1): 1 - 6. [Abstract] [Full Text] [PDF]
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R. G Lea and O. Sandra Immunoendocrine aspects of endometrial function and implantation Reproduction, September 1, 2007; 134(3): 389 - 404. [Abstract] [Full Text] [PDF]
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 M. T. Rae, O. Gubbay, A. Kostogiannou, D. Price, H. O. D. Critchley, and S. G. Hillier Thyroid Hormone Signaling in Human Ovarian Surface Epithelial Cells J. Clin. Endocrinol. Metab., January 1, 2007; 92(1): 322 - 327. [Abstract] [Full Text] [PDF]
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K. C. Jonas, C. Chandras, D. R. E. Abayasekara, and A. E. Michael Role for Prostaglandins in the Regulation of Type 1 11{beta}-Hydroxysteroid Dehydrogenase in Human Granulosa-Lutein Cells Endocrinology, December 1, 2006; 147(12): 5865 - 5872. [Abstract] [Full Text] [PDF]
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W. C. Duncan, S. G. Hillier, E. Gay, J. Bell, and H. M. Fraser Connective Tissue Growth Factor Expression in the Human Corpus Luteum: Paracrine Regulation by Human Chorionic Gonadotropin J. Clin. Endocrinol. Metab., September 1, 2005; 90(9): 5366 - 5376. [Abstract] [Full Text] [PDF]
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