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Endocrinology Vol. 139, No. 8 3597-3605
Copyright © 1998 by The Endocrine Society

ARTICLES

The Expression of Interleukin-6 in the Pregnant Rat Corpus Luteum and Its Regulation by Progesterone and Glucocorticoid1

C. M. Telleria, J. Ou, N. Sugino, S. Ferguson and G. Gibori2

Department of Physiology and Biophysics, College of Medicine, University of Illinois, Chicago, Illinois 60612

Address all correspondence and requests for reprints to: Dr. Geula Gibori, Department of Physiology and Biophysics (M/C 901), University of Illinois, 835 South Wolcott Avenue, Chicago, Illinois 60612-7342. E-mail: ggibori{at}uic.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Interleukin (IL)-6, a multifunctional cytokine originally described as a T cell-derived factor, is also produced by different cell types, and it influences a wide variety of physiological and pathophysiological processes. Recent studies further suggest that IL-6 has a role in down-regulating hormone production by endocrine organs and can negatively affect the steroidogenic capacity of both ovaries and testes. Thus, the aims of this investigation were to examine whether IL-6 plays a role in luteolysis and, more specifically, to determine whether luteal cells express the IL-6 gene, whether this expression is developmentally and hormonally regulated in pregnancy, and whether the corpus luteum could be a target for IL-6 action. Using semiquantitative RT-PCR, messenger RNA (mRNA) encoding both components of the IL-6 receptor [the ligand-binding subunit (IL-6 R) and the IL-6 R-associated signal transducer (gp130)] were found to be highly expressed in corpora lutea throughout pregnancy. In contrast, IL-6 mRNA expression was barely detectable from day 4 through the end of pregnancy, whereas a sharp and abrupt expression of IL-6 mRNA occurred immediately after parturition. Although the corpus luteum does not express IL-6 mRNA during most of pregnancy, it could be induced to express this gene with an in vivo injection of the bacterial endotoxin, lipopolysaccharide. In addition, when corpora lutea from day-15 pregnant rats were isolated and maintained in culture, IL-6 mRNA that was undetectable at 0 h increased in a time-related manner and reached significant levels after 4 h of incubation, followed by a similar increase in IL-6 protein secreted in the culture media. Isolation of the small and large luteal cells by elutriation indicated that both cell populations can secrete IL-6 in culture. The apparent ability of luteal cells to spontaneously express IL-6 in vitro, together with the lack of IL-6 expression during most of pregnancy, led us to examine whether the IL-6 gene is silenced throughout pregnancy by luteotropic hormones. Corpora lutea from day-15 pregnant rats were cultured in the presence of different doses of progesterone; the synthetic glucocorticoid, dexamethasone; 17ß-estradiol; and PRL. Progesterone and dexamethasone markedly inhibited IL-6 mRNA expression, whereas 17ß-estradiol had a minimal inhibitory effect, and PRL did not affect IL-6 mRNA expression. In summary, results of this investigation have revealed that the rat corpus luteum expresses the IL-6 receptor system and that luteal cells are able to secrete IL-6. However, IL-6 gene expression is silenced during most of pregnancy, probably by the high levels of progesterone locally produced in the corpus luteum. The salient finding that progesterone and glucocorticoid strongly inhibit the expression of IL-6 in the corpus luteum suggests that one important luteotropic role of progesterone and glucocorticoids could be to prevent the expression of IL-6, which might have a deleterious effect on luteal function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INTERLEUKIN (IL)-6 is a multifunctional cytokine, not only produced by cells of the immune system, but also produced by a variety of cells (including fibroblasts, epithelial cells, and endothelial cells) (1). IL-6 was originally identified as a B-cell differentiation factor that induces the final maturation of B cells into antibody-producing cells (2, 3). However, subsequent studies revealed that IL-6 acts not only on B cells but also on T cells (inducing cell activation), on hepatocytes (inducing the expression of proteins involved in the acute-phase reaction), and on hematopoietic progenitor cells (stimulating cell growth and maturation) (4, 5, 6). Besides its effects in the immune system, recent evidences have revealed a role for IL-6 in the endocrine system. IL-6 has been shown to be involved in bone formation (7), in endometrial growth (8), in glucocorticoid production by adrenals (9), and in the release of anterior pituitary hormones, such as ACTH, PRL, GH, and LH (10, 11). Accumulated evidence demonstrates that IL-6 can directly modulate gonadal processes. In the testis, Sertoli (as well as Leydig) cells are able to produce IL-6 in culture (12, 13, 14, 15, 16); in turn, IL-6 affects Sertoli cell function by regulating the expression of immediate early genes (17, 18), and it down-regulates steroid production by Leydig cells (19). In the ovary, IL-6 is produced by rat granulosa cells in vitro (20, 21), can inhibit progesterone secretion in both rat and porcine granulosa cells (20, 22, 23), and can stimulate apoptosis in FSH-treated rat granulosa cells (23). Taken together, these evidences clearly support a detrimental effect of IL-6 in the normal gonadal function. Because cytokines have been shown to negatively regulate steroidogenesis, their role in luteolysis has been suggested. Therefore, the aims of the present investigation were to determine: 1) whether the rat luteal cells express the IL-6 gene; 2) whether this expression is developmentally and hormonally regulated in pregnancy and becomes expressed in association with luteolysis; and 3) whether the corpus luteum could be a target for IL-6 action by expressing the IL-6 receptor system formed by the IL-6-binding subunit (IL-6 R) and the IL-6 R-associated signal transducer (gp130).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
Acrylamide and bis-acrylamide were obtained from Accurate Chemical Inc. (Westbury, NY) and Eastman Kodak (Rochester, NY), respectively; Taq DNA polymerase was purchased from Perkin-Elmer Corp. (Foster City, CA); [32P]-deoxycytidine triphosphate was from Amersham (Arlington Heights, IL); the oligonucleotides used as primers in the RT-PCR analysis were obtained from Life Technologies (Grand Island, NY); antibiotic-antimycotic solution was from Mediatech (Washington, DC); FBS was from HyClone (Logan, UT); collagenase (type IV, 150 U/mg) was from Worthington Biochemicals (Freehold, NJ); Dispase (Type II, 0.5 U/mg) and deoxyribonuclease (2000 U/mg) were from Boehringer-Mannheim Biochemicals (Indianapolis, IN); 17ß-estradiol was from Steraloids Inc. (Wilton, NH); HBSS (without Ca2+ and Mg2+), DMEM: Ham’s F12 (DMEM/F12), McCoy’s 5A:Ham’s F12 (1:1), lipopolysaccharide (LPS), human CG (hCG), dexamethasone, progesterone, and all other reagent grade chemicals were purchased from Sigma Chemical Co. (St. Louis, MO); ovine PRL (oPRL, PRL-18, 30 IU/mg) was a gift from NIDDK (NIH, Bethesda, MD); recombinant murine IL-1{alpha} and recombinant murine IL-1ß were from R&D Systems (Minneapolis, MS); the IL-6 receptor complementary DNA (cDNA) was obtained from the American Type Culture Collection (Rockville, MD).

Animal model and tissue preparation
Pregnant (day 1 = sperm positive) and immature female (day 26 of age) Sprague-Dawley rats were obtained from Sasco Animal Labs. (Madison, WI). Pseudopregnant Sprague-Dawley rats were obtained from Harlan Labs. (Madison, WI). The animals were kept under controlled conditions of light (lights on 0500–1900 h) and temperature (22–24 C) with free access to standard rat chow and water. All experiments were conducted in accordance with the principles and procedures of the NIH Guide for the Care and Use of Laboratory Animals and were approved by the University of Illinois Animal Care and Use Committee.

For the developmental studies, rats were killed with an overdose of ether at various stages of pregnancy, from days 4–22 (day of parturition), and at the day after parturition. Corpora lutea were dissected from the ovaries under a stereoscopic microscope. Liver, spleen, kidney, and lung obtained from pregnant rats, as well as decidual tissue obtained from rats on day 10 of pseudopregnancy, were used to study the tissue distribution of the messenger RNA (mRNA) encoding the IL-6 receptor. All tissues were frozen in liquid nitrogen and stored at -80 C until processed for RNA.

To determine the effect of LPS in vivo on IL-6 mRNA expression, animals were injected with either LPS (5 mg ip) or vehicle, on day 15 of pregnancy, and were killed 2 h later. The spleen, adrenals, and corpora lutea were obtained, frozen in liquid nitrogen, and stored at -80 C until RNA isolation.

Tissue culture
Corpora lutea (5 corpora lutea/ml in a 24-well tissue culture plate) were cultured in serum-free medium (McCoy’s 5A:Ham’s F12, 1:1), containing 25 mM HEPES and 2% antibiotic-antimycotic solution at 37 C for a maximum of 4 h in an atmosphere of 100% oxygen. After incubation with either vehicle (or with potent inducers of IL-6 expression, such as LPS, IL-1{alpha}, or IL-1ß), corpora lutea were immediately frozen in liquid nitrogen and stored at -80 C until RNA isolation.

Luteal cell dispersion, separation, and culture
Corpora lutea from day-15 pregnant rats were dissected and incubated at 37 C during 2 h under an atmosphere of 100% oxygen, in the presence of collagenase (50 U/ml), dispase (2.4 U/ml), and deoxyribonuclease (200 U/ml). The dispersed cells were separated into populations based on size, by elutriation, as previously described (24). After elutriation, cells were plated during 2 h to remove macrophages. The unattached cells were then placed into new culture wells and cultured for 2 days in the presence of 10% FBS to allow optimal attachment, and then incubated in serum-free medium (McCoy’s 5A:Ham’s F12, 1:1) containing 25 mM HEPES and 2% antibiotic-antimycotic solution at 37 C under a humidified atmosphere of 5% CO2/95% air during an additional 24-h period.

Granulosa cell culture
Highly luteinized granulosa cells were obtained, according to the protocol previously described (25). In brief, granulosa cells were isolated from 28-day-old immature rats, treated with 0.15 IU hCG sc twice daily for 2 days, followed by 10 IU hCG via the tail vein on the third day. Seven hours later, granulosa cells were harvested and cultured for 3 days at 37 C in an atmosphere consisting of 5% CO2/95% air in DMEM/F12 (1:1) with 15 mM HEPES, 1% FBS, and 2% antibiotic-antimycotic solution. Cells were washed with PBS several times after incubation and stored at -80 C until RNA extraction.

RNA isolation and RT-PCR analysis
Total RNA from frozen corpora lutea was purified by homogenization in guanidinium thiocyanate and centrifugation through a cesium chloride cushion (26), whereas total RNA from cultured cells was isolated by a one-step guanidinium-thiocyanate-phenol-chloroform extraction procedure (27).

Oligonucleotide primer pairs were based on the sequence of the rat IL-6 gene (28) (5'-GACTGATGTTGTTGACAGCCACTGC-3' and 5'-TAGCCACTCCTTCTGTGACTCTAACT-3'), rat IL-6 receptor gene (29) (5'-TCACAGAGCAGAGAATGGACT-3' and 5'-GTATGGCTGATAC CACAAGGT-3'), and rat gp130 gene (30) (5'-TCAACTTGTGGAACCATGTGG-3' and 5'-TCCAACTGACACAGCATGTTC-3'). In each reaction, an additional pair of oligonucleotides specific for either the rat ribosomal protein S16 (31) (5'-TCCAAGGGTCCGCTGCAGTC-3' and 5'-CGTTCACCTTGATGAGCCCATT-3') or the rat ribosomal protein L19 (32) (5'-CTGAAGG TCAAAGGGAATGTG-3' and 5'-GGACAGAGTCTTGATGATCTC-3') was included for use as an internal control. The predicted sizes of the PCR-amplified products were 509 bp for IL-6, 480 bp for IL-6 receptor, 375 bp for gp130, 194 bp for L19, and 100 bp for S16. One to 3 µg of total RNA were reverse-transcribed at 42 C using random hexamer primers (Pharmacia, Piscataway, NJ) and Maloney murine leukemia virus reverse transcriptase (Life Technologies, Gaithersburg, MD) in a 20-µl reaction mixture. The reaction mixture was added to tubes containing specific oligonucleotide primers (50 pmol each) for amplification of either form of the complementary DNAs. A mix containing the oligonucleotide primers for either L19 or S16 mRNA (50 pmol each), Taq DNA polymerase (2.5 U), and [32P]-deoxycytidine triphosphate (2 µCi of 3000 Ci/mmol) was added to each tube; and the final vol was increased to 90 µl with 1 x PCR buffer [20 mM Tris-HCl (pH 8.4), 50 mM KCl, and 2.5 mM MgCl2]. The samples were overlaid with light mineral oil, and PCR was carried out for 30 cycles using 95 C for denaturing, 65 C for annealing, and 72 C for extension in a Perkin-Elmer/Cetus Thermal Cycler (Perkin-Elmer, Norwalk, CT). The conditions were such that the amplification of the products was in the exponential phase, and the assay was linear, with respect to the amount of input RNA. Reaction products were electrophoresed on a 8% polyacrylamide nondenaturing gel. After autoradiography data were analyzed using a Molecular Dynamics PhosphorImager and ImageQuant version 3 software (Molecular Dynamic, Sunnyvale, CA), the intensities of the signals were normalized to that of the ribosomal protein internal control.

Southern blot analysis
One microgram of total RNA was reverse-transcribed and subjected to PCR using the specific oligonucleotides for the rat IL-6 receptor. At the end of the reaction, the PCR mixture was fractionated by electrophoresis through a 1% agarose gel and blotted to GeneScreen nylon membranes (New England Nuclear, Boston, MA) by capillary transfer. DNA was fixed to the membrane by baking in vacuo at 80 C for 2 h. A full-length (4.6 kb) rat IL-6 receptor cDNA (29) was labeled with [32P]-deoxycytidine triphosphate using the random primer DNA-labeling method, according to the instructions of the manufacturer (Boehringer-Mannheim Biochemicals). Blots were prehybridized for 3 h at 65 C in a solution containing 50 mM [piperazine-N,N'-bis(2-ethanesulfonic acid)], 100 mM NaCl, 50 mM sodium phosphate, 1 mM EDTA, and 5% SDS wt/vol, pH 6.8. Hybridization was completed in the same solution, containing 32P-labeled cDNA probe (1 x 106 cpm/ml) and 50 µg/ml salmon sperm DNA, for 16 h at 65 C. Blots were washed four times in 2 x SSC and 0.1% SDS for 5 min at room temperature and twice with 0.1 x SSC and 0.1% SDS for 15 min at 50 C. Radioactivity was monitored after each wash. The blots were then exposed to Kodak X-Omat films (Eastman Kodak) with an intensifying screen at -80 C.

IL-6 assay
IL-6 was measured in the conditioned medium after incubation of the corpora lutea or luteal cells, using a mouse IL-6 enzyme-linked immunosorbent assay (ELISA) kit, according to the instructions of the manufacturer (R&D Systems). The sensitivity of the assay was 3.1 pg/ml, and the inter- and intraassay coefficients of variation were 7.1% and 4.5%, respectively.

Statistics
Data were examined by one-way ANOVA, followed by Duncan’s multiple-range test. A level of P < 0.05 was accepted as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Developmental expression of IL-6 R, gp130, and IL-6 mRNA in the rat corpus luteum during pregnancy and after parturition
All the diverse biological activities of IL-6 are mediated by a specific cell surface receptor, IL-6 R, which (upon binding to IL-6) triggers the dimerization of gp130 to transduce IL-6 signal (33). To determine whether the rat corpus luteum could be a target for IL-6, either locally produced or provided through the circulation, we examined for both IL-6 R and gp130 mRNA expression in the corpus luteum throughout pregnancy. As shown in Fig. 1AGo (upper and middle panels), IL-6 R mRNA was expressed throughout pregnancy, with an increase in mRNA levels between days 20 and 22. Hybridization of the IL-6 R RT-PCR products, with a specific IL-6 R cDNA probe cloned from rat liver (29), indicated that the IL-6 R mRNA species expressed in ovarian luteal and granulosa cells corresponds to that detected in liver, spleen, lung, and decidua (Fig. 1AGo, lower panel). The developmental studies represented in Fig. 1BGo revealed that the gp130 mRNA is also expressed throughout pregnancy in corpora lutea with maximum levels at midpregnancy. Interestingly, whereas both elements of the IL-6 R system were expressed throughout pregnancy in the corpus luteum, IL-6 mRNA was barely detectable, except for day 4 of pregnancy and at parturition, when it became abruptly and markedly expressed (Fig. 1CGo).



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  		Figure 1. Developmental expression of IL-6 R, gp130, and IL-6 mRNA in the rat corpus luteum during pregnancy and after parturition. Corpora lutea were dissected from ovaries of rats at different stages of pregnancy, and total RNA was isolated and analyzed by RT-PCR using specific primers for the IL-6 R (A, upper), gp130 (B, upper), and IL-6 (C, upper), as described in Materials and Methods. PP0 and PP1 are postpartum day 0 and day 1, respectively. Data were quantified by densitometry and corrected using L19 or S16 as internal standards. The mRNA levels are graphically represented in A (middle) for IL-6 R, B (lower) for gp130, and C (lower) for IL-6 as the mean ± SEM (n = 3); to compare results from separate experiments, values were also normalized within each experiment to the maximum ratios of IL-6 R/L19, gp130/S16, or IL-6/L19 products, which were considered 100%. A (lower) depicts a representative Southern blot showing the specificity of the RT-PCR signals in corpora lutea and granulosa cells, as compared with that obtained in decidua, spleen, and liver used as positive controls. In panel A, a, P < 0.05, compared with days 6, 12, 15, 17, 18, and 19 of pregnancy. In panel B, a, P < 0.05, compared with the other days of pregnancy studied. In panel C, a, P < 0.01, compared with all the days of pregnancy studied; b, P < 0.01, compared with the other days of pregnancy studied; the arrow indicates the time of parturition that occurred on day 22 of pregnancy between 1200 and 1800 h.

 
LPS-induced expression of IL-6 mRNA in the rat corpus luteum
To examine whether IL-6 mRNA can be induced in the corpora lutea that do not express the gene in physiological conditions, the bacterial endotoxin LPS, a potent stimulator of IL-6 expression in a wide variety of cell types (34, 35), was injected into rats on day 15 of pregnancy, and the expression of IL-6 mRNA was examined in the corpora lutea 2 h after treatment. Spleen and adrenals were used as control tissues (36). Results in Fig. 2Go show that IL-6 mRNA was expressed in spleen and adrenal of control animals, whereas no message could be detected in the corpus luteum. However, 2 h upon LPS administration, the IL-6 mRNA was up-regulated in the spleen and adrenal and was strongly induced in the corpus luteum.



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  		Figure 2. Comparison of IL-6 mRNA levels in the corpus luteum, adrenal gland, and spleen 2 h after ip administration of either vehicle or LPS to rats on day 15 of pregnancy. The ribosomal L19 mRNA was used as an internal standard. Shown in the figure is a representative autoradiograph performed using total RNA of the organs obtained from five animals per experimental group.

 
IL-6 mRNA expression and IL-6 secretion by corpora lutea maintained in culture: effect of LPS, IL-1{alpha}, and IL-1ß
Because systemic administration of LPS induces stress immune response (35), with a subsequent activation of the hypothalamic-pituitary-adrenal axis (37), we examined whether the LPS-mediated induction of IL-6 mRNA is direct on the corpus luteum or is the consequence of a systemic inflammatory reaction triggered by the endotoxin. Corpora lutea obtained from intact animals on day 15 of pregnancy were cultured in serum-free conditions, in the presence of either vehicle or potent inducers of the IL-6 expression, such as LPS, IL-1{alpha}, or IL-1ß (4, 6). After 4-h incubation, total RNA was obtained from the tissues and subjected to RT-PCR for IL-6 mRNA. Also, because the induction of IL-6 mRNA expression generally follows the release of IL-6 (15), the IL-6 accumulated in the culture media was measured. Results shown in Fig. 3AGo indicate that IL-6 mRNA was strongly expressed in corpora lutea incubated with LPS, IL-1{alpha}, or IL-1ß. Nonetheless, IL-6 mRNA was also induced in corpora lutea incubated with vehicle alone (Fig. 3AGo, line 2). In addition, IL-6 protein was secreted in the culture media after 4-h incubation of corpora lutea treated with vehicle, whereas a further stimulation of IL-6 secretion was obtained after treatment with LPS, IL-1{alpha}, or IL-1ß (Fig. 3BGo).



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  		Figure 3. Effects of LPS, IL-1{alpha}, and IL-1ß on IL-6 mRNA expression and IL-6 secretion by corpora lutea dissected from day-15 pregnant rats and maintained in culture. A, IL-6 mRNA was analyzed by RT-PCR, either immediately after isolation of the corpora lutea from the animals (CL15) or 4 h after incubation of the corpora lutea, in the presence of vehicle (veh), LPS (10 µg/ml), recombinant murine (rm) IL-1{alpha} (100 ng/ml), or rmIL-1ß (100 ng/ml). The data are representative of three different experiments. B, IL-6 accumulated in the conditioned media after 4-h incubation, as measured by ELISA. Results are mean ± SEM (n = 4). a, P < 0.01; b, P < 0.05 (compared with veh).

 
Time-course of the spontaneous expression of IL-6 mRNA and protein in cultured corpora lutea
To investigate further the spontaneous expression of IL-6 mRNA and IL-6 secretion by corpora lutea maintained in culture, a time-course experiment was performed. As shown in Fig. 4AGo, IL-6 mRNA was induced within 30 min of incubation and was further increased with time of incubation. IL-6 secreted in the culture media also increased in a time-dependent manner, in parallel with the enhanced expression of IL-6 mRNA (Fig. 4BGo).



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  		Figure 4. Time-course of IL-6 mRNA expression and IL-6 secretion by corpora lutea obtained from rats on day 15 of pregnancy and maintained in culture in serum-free conditions. A, IL-6 mRNA was analyzed by RT-PCR immediately after isolation of the corpora lutea from the animals (time = 0) or after 15, 30, 60, 120, or 240 min of incubation. The data are representative of three different experiments. B, IL-6, accumulated in the conditioned media at the different time-points, as measured by ELISA. Results are mean ± SEM (n = 4). a, P < 0.01, compared with values measured after 15-min incubation.

 
IL-6 production by the different luteal cell populations forming the rat corpus luteum
The rat corpus luteum is formed by two steroidogenically active luteal cell populations that differ by their sizes, and is formed also by nonsteroidogenic cells, such as fibroblasts, endothelial cells, and blood cells (24). To examine whether luteal cells produce IL-6 in culture, corpora lutea from day-15 pregnant rats were subjected to cell dispersion and elutriation. The large and small luteal cell populations were isolated from the nonluteal cells, as described previously (24). Because resident macrophages have been identified in the pregnant rat corpus luteum (38) and because they are an important source of IL-6 when activated (28), the luteal cell preparations obtained after elutriation were depleted of macrophages by first 2 h-incubation in serum-containing media that allows macrophages to adhere to the culture plates (39). Interestingly, only a few cells attached during this 2-h incubation period, indicating that few macrophages reside within the corpus luteum on day 15 of pregnancy, confirming results presented by others (38). Results shown in Fig. 5Go revealed that both luteal cell populations secrete IL-6 in culture at levels much higher than that produced by nonluteal cells.



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  		Figure 5. IL-6 production by the different rat luteal cell populations in culture. Corpora lutea from animals on day 15 of pregnancy were dissected and subjected to cell dispersion. Dispersed cells were separated into populations, based on size, by elutriation. Cells were then plated during 2 h in serum-containing medium for allowing macrophage adherence to the dishes. The unattached cells were cultured for 2 days in 10% FBS and then with serum-free medium during an additional 24 h. The IL-6 accumulated in the conditioned media was measured by ELISA. Results are mean ± SEM (n = 6). a, P < 0.01, compared with nonluteal cells.

 
Effects of progesterone, dexamethasone, 17ß-estradiol, and PRL on the spontaneous expression of IL-6 mRNA in cultured corpora lutea
The apparent ability of the corpora lutea to spontaneously express IL-6 when maintained in culture (Figs. 3Go and 4Go), together with the lack of expression of IL-6 mRNA during most of pregnancy (Fig. 1CGo), prompted us to examine whether the expression of the IL-6 gene is silenced by hormones known to affect corpus luteum function, such as PRL, progesterone, glucocorticoids, and estradiol (40, 41, 42, 43, 44, 45). Fig. 6Go shows the effects of PRL, progesterone, 17ß-estradiol, and the synthetic glucocorticoid, dexamethasone, on IL-6 mRNA expressed in corpora lutea maintained in culture for 4 h in serum-free conditions. Progesterone and dexamethasone caused a concentration-dependent inhibition of IL-6 mRNA levels. Estradiol only inhibited IL-6 mRNA expression at the higher concentration used, being less potent than the other two steroids, whereas PRL did not affect IL-6 mRNA levels at any of the concentrations tested.



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  		Figure 6. Effects of progesterone (P4), dexamethasone (Dex), 17ß-estradiol, and PRL on IL-6 mRNA expression in corpora lutea dissected from day-15 pregnant rats and maintained in culture. Total RNA was isolated, reverse-transcribed into single-stranded complementary DNA, and amplified with specific oligonucleotide pairs for IL-6 mRNA, as described in Materials and Methods. Included in each reaction was a pair of oligonucleotide primers for the S16 ribosomal mRNA used as an internal standard. IL-6 mRNA was analyzed 4 h after incubation of the corpora lutea in the presence of either P4, Dex, 17ß-estradiol (E2) (A), or PRL (B). C depicts the densitometric analysis from three independent experiments (mean ± SEM of values expressed as percentage of control which was considered 100%). a, P < 0.01; b, P < 0.05 [compared with controls (indicated as 0)].

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Activation and maintenance of the corpus luteum in the rat involve the complex interplay of pituitary, placental, and ovarian hormones. It is well known that PRL and PRL-like hormones from placental origin are the primary regulators of corpus luteum function (40, 45), whereas locally produced steroids (such as estradiol, progesterone, and androgens) are important intraluteal modulators of steroidogenesis (40, 41, 42, 43, 45, 46, 47). Recently, accumulating evidence supports the concept that cytokines also may be normal components of the corpus luteum and serve essential roles in the regulation of its function. IL-1 expression has been documented in human granulosa lutein cells (48) and in mouse corpus luteum (49), and IL-1 was shown to inhibit progesterone production in cultured rat luteal cells (50). Tumor necrosis factor-{alpha} (TNF-{alpha}), produced by macrophages that invade the regressing corpus luteum, seems to participate in the later stages of decline in luteal progesterone (51). In the present investigation, we report that the luteal cells themselves are a source of another inflammatory cytokine, IL-6, and that the pregnant rat corpus luteum could be a target of IL-6 actions because it expresses the two components of the IL-6 receptor system.

IL-6 was barely detectable in the corpora lutea during most of the period of pregnancy studied. However, the mRNA encoding IL-6 could be highly induced by administration of the bacterial endotoxin LPS at midpregnancy. LPS is a potent inducer of IL-6 production, not only from cells of the immune system (6), but it also can stimulate the production of IL-6 from steroidogenic cells, as demonstrated in Sertoli and granulosa cells of the gonads (13, 16, 21, 52). The end point of the action of LPS in steroidogenic tissues results in the inhibition of steroidogenesis (52, 53). Thus, luteal expression of IL-6, induced by LPS, may account for pregnancy loss during pelvic infection by inducing luteal regression, as proposed by Terranova and Mongomery-Rice (54). The expression of IL-6 mRNA and protein could be induced also by culturing the corpora lutea in serum-free medium. As expected, a further increase in IL-6 secretion could be measured when corpora lutea were incubated in the presence of LPS, IL-1{alpha}, or IL-1ß. The same phenomenon was observed when either Sertoli and Leydig cells obtained from testes of immature rats (14), or granulosa cells from ovaries of estrogen-treated immature rats (21), were incubated in vitro in the presence of LPS and IL-1. Macrophages, a source of cytokine production within the ovary (50), were not present in the highly enriched luteal cell preparation containing steroidogenic small and large luteal cells (24). Therefore, the secretion of IL-6 by the luteal cells indicates that this cell type represents an ovarian source of IL-6, as demonstrated previously for granulosa cells (20). The role of the luteal cell-derived IL-6 remains to be investigated; the synthesis and release of IL-6 by luteal cells may provide an important means of communication between the resident endocrine cells and immune cells that invade this gland at parturition (55, 56). On the basis of the activities exerted by IL-6 on granulosa cells, such as the inhibition of progesterone production (20) and the induction of apoptosis (23), we originally examined for IL-6 expression in the corpus luteum, expecting IL-6 to be involved in the luteolytic process. However, the fact that the IL-6 gene is expressed in this tissue at very low levels during days 20–22, when the luteolytic cascade is triggered (41, 57), indicates that a physiological role for the luteal IL-6 in the functional luteolysis is rather remote. However, because IL-6 becomes markedly expressed on the day of parturition, it may be involved in the structural involution of the corpus luteum that takes place after delivery.

The ability of the corpus luteum to spontaneously express IL-6 in vitro, together with the lack of IL-6 expression in the corpus luteum during most of pregnancy, seems to be caused by local inhibitors expressed at high levels within the corpus luteum. Whereas multiple agents activate IL-6 gene transcription (6), steroid hormones that bind estrogen, progesterone, and glucocorticoid receptors have been described as potent repressors of the expression of the IL-6 gene (58, 59, 60, 61). Our results revealed that progesterone and the glucocorticoid analog, dexamethasone, caused a strong inhibition of the spontaneous expression of luteal IL-6 mRNA. The rat corpus luteum does not express the progesterone receptor (62); however it expresses the glucocorticoid receptor that was shown previously to transduce progesterone signaling in this endocrine gland (46). Despite the fact that dexamethasone is, at equal concentration, a more potent inhibitor of luteal IL-6, progesterone probably plays a more important physiological role because its concentration within the corpus luteum far exceeds that of glucocorticoids. Indeed, the highest concentration of progesterone found in any tissue is in the corpus luteum, and this steroid has relatively high affinity for the glucocorticoid receptor (46). Although the rat corpus luteum does express both forms of estrogen receptor (63), estradiol-mediated inhibition of IL-6 mRNA was rather limited. It is amply known that IL-6, together with TNF-{alpha} and IL-1, constitute a group of cytokines whose expression is rapidly induced under situations of tissue damage (64). Therefore, most probably under the physiological conditions of pregnancy, the elevated progestational milieu within the corpora lutea, together with the high levels of corticosterone in circulation (65), may prevent the development of localized inflammation by down-regulating the expression of the IL-6 gene.

Interestingly, the DNA elements in the IL-6 5'-flanking region that respond to inhibition by steroid hormones in functional assays do not bind the steroid receptor (58, 61); however, a growing body of evidences indicate that the inhibition of IL-6 gene expression by steroid hormones involves a physical association and functional antagonism between the steroid hormone receptors and the transcription factor known as nuclear factor kappa B (NF-kB), a potent enhancer of the transcription of the IL-6 promoter (58, 59, 60, 61). Whether the mechanism by which progesterone and glucocorticoid inhibit IL-6 gene expression in the corpus luteum involves the interference with the action of NF-kB family of transcription factors remains to be investigated.

The biological activities of IL-6 are mediated by a receptor system consisting of a ligand binding protein, the IL-6 R, and a signal transducer molecule known as gp130 (33). The binding of IL-6 to the IL-6 R triggers the dimerization of gp130 and activation of cytoplasmic protein tyrosine kinases in the Janus kinase family (JAKs) that are already associated with the membrane-proximal portion of gp130 (33). Tyrosine residues in the cytoplasmic region of gp130 are then phosphorylated and the signal transducer and activator of transcription-3 (STAT-3) recruited to gp130 and tyrosine-phosphorylated by the JAKs. Tyrosine-phosphorylated STAT-3 proteins then form homodimers, translocate to the nucleus, and regulate the transcription of target genes for IL-6. The constitutive expression of the IL-6 R in the corpus luteum during pregnancy reported in this investigation raises the possibility of a role for the circulating IL-6 in the normal function of the corpora lutea, in the absence of locally produced IL-6. On the other hand, the signal transduction mechanism involving the gp130 protein is shared by all the members of the IL-6 family of cytokines (i.e. IL-11, leukemia inhibitory factor, oncostatin M, ciliary neurotropic factor, and cardiotropin-1) (1, 5, 33). Because IL-6 R and gp130 mRNAs are not regulated in tandem during pregnancy, it is possible that other members of the cytokine family that signals through gp130, play a role in luteal function.

In conclusion, results of this investigation have revealed that the rat corpus luteum expresses the IL-6 receptor system and, therefore, could be a target of IL-6 action. The fact that the IL-6 gene is barely expressed in the rat corpus luteum before parturition indicates that a physiological role of the locally produced IL-6 in functional luteolysis is rather remote; however, IL-6 may be involved in the structural involution of the corpus luteum that occurs after delivery. The salient finding that progesterone and glucocorticoid are able to prevent the expression of IL-6 in the corpus luteum suggests that one important luteotropic role of progesterone and glucocorticoids could be to prevent the expression of IL-6, which might be detrimental for luteal function.


   Acknowledgments
 
We are grateful to the NIDDK and National Hormone and Pituitary Program (NIH) for the oPRL, to R. Clepper for animal care, to L. Alaniz-Avila for photography, and to V. Rogala for the preparation of the manuscript.


   Footnotes
 
1 This work was supported by NIH Grants HD-11119 (to G.G.) and FIC-1F-05TW-05241 (to C.M.T.) Back

2 NIH Merit Awardee (HD-11119). Back

Received January 21, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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