Mol. Hum. Reprod. Advance Access originally published online on October 27, 2005
Molecular Human Reproduction 2005 11(10):751-760; doi:10.1093/molehr/gah233
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Differential regulation of interleukins IL-13 and IL-15 by ovarian steroids, TNF-
and TGF-ß in human endometrial epithelial and stromal cells
Department of Obstetrics & Gynecology, University of Florida College of Medicine, Gainesville, FL, USA
1 To whom correspondence should be addressed at: Department of Obstetrics & Gynecology, University of Florida College of Medicine, P.O. Box 100294, Gainesville, FL 32610-0294, USA. E-mail: cheginin{at}obgyn.ufl.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Based on the endometrial spatial and temporal expression of interleukins (ILs) IL-13 and IL-15 during the normal menstrual cycle, we hypothesized that ovarian steroids and non-steroidal factors regulate their expression in a cell-specific manner. To test this hypothesis and determine IL-13/IL-15 actions, we used endometrial epithelial (EEC) and stromal (ESC) cells isolated and cultured under defined conditions. We confirmed the expression of IL-13 and IL-15 in these cells and further demonstrated that 17ß estradiol (E2), medroxyprogesterone acetate (MPA) and their combination differentially regulated their mRNA expression and protein production in a time- and cell-specific manner (P < 0.05). We also showed that tumour necrosis factor-
(TNF-; 10 and 25 ng/ml) and transforming growth factor-ß (TGF-ß; 1 and 5 ng/ml), cytokines with inflammatory and immune regulatory functions in a cell- and dose-dependent manner regulate the expression of IL-13 and IL-15 (P < 0.05). Functionally, IL-13 and IL-15 1-100 ng/ml displayed a limited mitogenic activity towards EEC and ESC; however, they regulated the expression of TNF receptor type 1 (TNFR) mRNA and soluble protein in a cell-specific manner (P < 0.05). We conclude that ovarian steroids, TNF- and TGF-ß act as key regulators of endometrial IL-13 and IL-15 expression which act locally regulating TNFR expression in a cell-specific manner. Based on these findings, we conclude that IL-13/IL-15, either alone or through their interactions with other cytokines, influence the outcome of endometrial inflammatory/immune responses during the normal menstrual cycle, and due to their altered expression may extend these processes in dysfunctional bleeding and endometriosis.
Key words:
endometrium/expression/IL-13/IL-15/interleukins/regulation/steroids/TGF-ß/TNF-
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The infiltration of inflammatory and immune-related cells into endometrial tissue is an integrated event and central to various endometrial activities throughout the menstrual cycle. The endometrial recruitment of these cells is menstrual cycle dependant, and their maintenance and activation most likely regulated by local expression of various cytokines and chemokines, with the ability to direct the extent and duration of endometrial inflammatory and immune responses. The expression of many of these cytokines and chemokines has been documented in the endometrium throughout the menstrual cycle, and their autocrine/paracrine actions to a lesser extent have been associated with several endometrial cellular activities (Chegini and Williams, 2000
; Critchley et al., 2001; Kelly et al., 2001). Altered endometrial expression of some of these cytokines/chemokines has also been correlated with several disorders including failure of embryo implantation, recurrent pregnancy loss, dysfunctional uterine bleeding and endometriosis (Salamonsen and Lathbury, 2000; Critchley et al., 2001; Kharfi et al., 2003; Laird et al., 2003; Rhoton-Vlasak et al., 2005).
Among the cytokines, interleukins (ILs) represent a group of multifunctional molecules recognized for their ability to regulate inflammatory and immune responses under both normal and pathological settings. Several members of the IL family, including IL-13 and IL-15, are expressed in human endometrium throughout the menstrual cycle (Kitaya et al., 2000; Kelly et al., 2001; Chegini et al., 2002, 2003; Okada et al., 2000, 2002; Riesewijk et al., 2003). IL-13 and IL-15 are expressed by a variety of cells and tissues, with inflammatory and immune-related cells serving as their major source; however, many cells release low levels of IL-15, with the exception of a few cell types such as activated macrophages and epithelial cells in response to inflammation (Chomarat and Bannchereau, 1998; Brombacher, 2000; Kurys et al., 2000; Fehniger and Caligiuri, 2001; Lodolce et al., 2002; Waldmann, 2002; McInnes and Gracie, 2004). Among specific biological activities ascribed to IL-13 are regulation of mast cell and B-cell proliferation, IgG, IgE and MHC class II antigen expression and inhibition of cytokine production by Th1-related cells (Chomarat and Bannchereau, 1998; Brombacher, 2000). In addition, IL-13 through differential regulation of IL-1ß, tumour necrosis factor- (TNF-), IL-1 receptor antagonist and IL-1 type II receptor, acts as an anti-inflammatory cytokine, whereas through the induction of transforming growth factor-ß (TGF-ß) enhances tissue fibrosis (Brombacher, 2000; Wynn, 2004). Owing to shared receptor components, some IL-13 biological activities are in common with IL-4, although their target cells may display variable response (Hershey, 2003; Wynn, 2004; Wynes and Riches 2003; Wynes et al., 2004). IL-15 is a key regulator of local innate tissue inflammatory infiltrate and adaptive immunity, and more specifically natural killer (NK) cell proliferation and survivor, and interferon-
(IFN-) and TNF- production (Fehniger and Caligiuri, 2001; Lodolce et al., 2002; McInnes and Gracie, 2004; Nishimura et al., 2005). Several biological activities of IL-15 are identical to IL-2 because of their interactions with shared receptor components that are expressed in various cell types (Fehniger and Caligiuri, 2001).
In the endometrium, the spatial and temporal expression of IL-13 and IL-15 is characterized by elevated levels before and during menses and during proliferative and secretory phases of normal menstrual cycle (Chegini et al., 2002, 2003). Further increases in endometrial expression of IL-13 and IL-15 have been reported in ectopic compared with eutopic endometrium of women with endometriosis, women experiencing recurrent pregnancy loss and in dysfunctional uterine bleeding because of progesterone-only contraceptive as compared with endometrium of women with regular menstrual cycle (Chegini et al., 2002, 2003; Rhoton-Vlasak et al., 2005). Recent reports have also indicated that progesterone (P4), as well as PGE2 and IL-1ß regulate the expression of IL-15 in endometrial stromal, decidual and decidual NK cells (Dunn et al., 2002; Okada et al., 2002, 2004). In addition to IL-1b, the endometrial expression of TNF-, TGF-ß and their receptors throughout the normal menstrual cycle display a considerable overlap in their peak expression with IL-13 and IL-15, suggesting potential regulatory interactions among these cytokines (Chegini et al., 1994, 1999a,b; Tabibzadeh et al., 1999; von Wolff et al., 1999; Verma et al., 2000; Luo et al., 2004). TNF- and TGF-ß also act as key regulators of local immune and inflammatory responses, and their altered expression have been associated with several endometrial abnormalities including endometriosis and dysfunctional uterine bleeding. The expression of TNF- and TGF-ß is highly regulated, and their biological activities at the tissue level are controlled through interactions with their soluble receptors and, in the case of TGF-ß, through production as latent or inactive forms (Aggarwal, 2003; Chegini et al., 1999b). With respect to TNF-, it interacts with TNF receptor type 1 (TNFR1, p55) and type 2 (TNFR2, p75) to mediate its apoptotic and mitogenic activities, respectively, with TNFR2 preferentially interacting with the membrane-bound ligand, with TNFR1 also serving as a soluble receptor (Aggarwal, 2003).
Based on these observations and our previous studies with IL-13 and IL-15, we designed this study to test the hypothesis that ovarian steroids and non-steroidal factors such as TNF- and TGF-ß differentially regulate the endometrial expression of IL-13 and IL-15. For this purpose, we isolated endometrial stromal and epithelial cells and under a defined culture conditions evaluated the regulatory function of 17ß estradiol (E2), medroxyprogesterone acetate (MPA), TNF- and TGF-ß on the expression of IL-13 and IL-15 mRNA and protein. We also determined the biological activity of IL-13 and IL-15 on these cells by examining their mitogenic potential and regulation of TNFR1 expression as a mechanism influencing the outcome of endometrial inflammatory and immune response, under normal physiological and uterine abnormalities owing to the altered expression of these cytokines.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
All the materials for quantitative and real-time PCR, enzyme-linked immunosorbent assay (ELISA), DNA synthesis and cell proliferation assays were purchased from Applied Biosystems (Foster City, CA, USA), R&D system (Minneapolis, MN, USA), Amersham-Pharmacia Biotech (Piscataway, NJ, USA) and Sigma Chemical Company (St. Louis, MO, USA), respectively, or other commercial sources, as previously described (Chegini et al., 1992
, 1999b, 2002, 2003). The endometrial epithelial (EEC) and stromal (ESC) cells were isolated from endometrial tissues obtained from premenopausal women ages ranging from 21 to 39, who were undergoing hysterectomy for medically indicated reasons, excluding endometrial cancer and leiomyoma. These patients were not under any hormonal treatments at the time of surgery. The tissues were collected following approval from the University of Florida Institutional Review Board. The EEC and ESC were isolated and cultured in DMEM-Ham’s F-12 supplemented with 10% fetal bovine serum (FBS), as previously described (Chegini et al., 1992, 1999b). The purity of the EEC and ESC preparations was assessed by cytokeratin and vimentin immunostaining, respectively, typically displaying a greater than 95% purity (Chegini et al., 1992). Following characterization, the cells were cultured in 75 mm flask in the presence of 10% FBS until reaching visible confluence and used in the following experiments.
Regulation of IL-13 and IL-15 expression in EEC and ESC by ovarian steroids
To determine the effect of ovarian steroids on IL-13 and IL-15 expression, EEC and ESC were cultured at a density of 1 x 106 or 2.5 x 105 cells in 6- and 24-well dishes, respectively, in phenol-free media containing 10% charcoal-stripped fetal calf serum (FCS) for 48 h and phenol-red/serum-free conditions for 24 h. The cells were then treated with E2, MPA or E2 + MPA (Sigma) at a concentration of 10–8 M in phenol-free medium containing 2% charcoal-stripped FCS for 2, 4 and 6 h to assess mRNA expression and 18, 24 and 36 h for protein production. After incubations, the conditioned media were collected, the cells were washed, scraped and used either for isolation of total cellular RNA or lysed to obtain the total cellular proteins, as previously described (Chegini et al., 2002, 2003). The levels of IL-13 and IL-15 mRNA and protein expression were determined by real-time PCR and ELISA, respectively.
Regulation of IL-13 and IL-15 expression by TNF- and TGF-ß1
To examine whether TNF- and TGF-ß1 regulate the endometrial expression of IL-13 and IL-15, EEC and ESC were cultured in 48-well dishes and treated with TNF- (10 and 25 ng/ml) and TGF-ß1 (1 and 5 ng/ml) added to phenol-free medium containing 2% charcoal-stripped FCS for 24 h. Their culture- conditioned media were collected and assayed for IL-13 and IL-15 using ELISA. The cells cultured in 6-well dishes were treated with TNF- (25 ng/ml) and TGF-ß1 (5 ng/ml) for 2 h, and their total RNA isolated and subjected to real-time PCR determining IL-13 and IL-15 mRNA expression. The concentrations of TNF- and TGF-ß1 used in this study were selected based on our previous experience and related literature with these cytokines (Chegini et al., 1999b; Tabibzadeh et al., 1999; Laird et al., 2003; Luo et al., 2004).
The effect of IL-13 and IL-15 on 3H-thymidine and cell proliferation
To determine the effect of IL-13 and IL-15 on the rate of DNA synthesis and cell proliferation, ESC and EEC were cultured in 48- or 96-well dishes at an approximate density of 2.5 x 104 and 2.5 x 103 cells/well, respectively, and incubated with phenol-free media containing 10% charcoal-stripped FBS for 48 h and serum-free condition for 24 h. The cells were then treated with several concentrations of IL-13 and IL-15, added to phenol-free medium containing 2% charcoal-stripped FCS and 2 µCi/ml of [3H]-thymidine (Amersham-Pharmacia Biotech), and the rate of incorporation into DNA was determined after 24 h (Chegini et al., 1992, 1999b). The rate of cell proliferation was determined after similar treatments following 48, 72 and 96 h of incubation using cell-proliferation assay (MTT), as previously described (Chegini et al., 1992, 1999b).
Regulation of TNFR1 by IL-13 and IL-15
EEC and ESC were cultured in 6- and 48-well dishes, as described above, and treated with IL-13 and IL-15 (50 ng/ml) added to phenol-free medium containing 2% charcoal-stripped FCS for 2, 4 and 6 h for mRNA expression and 18, 24 and 36 h for protein production. IL-13 and IL-15 doses used in this assay were selected from the dose–response curve generated from DNA synthesis and proliferation assays. The level of TNFR1 mRNA and soluble TNFR1 protein was determined using real-time PCR and ELISA, respectively.
For quantitative real-time PCR, total cellular RNA was isolated from the above treated and untreated EEC and ESC using Trizol Reagent (Invitrogen, Carlsbad, CA, USA), as previously described (Umekawa et al., 2002; Luo et al., 2004). Two micrograms of total RNA was reverse transcribed to complementary DNA (cDNA) in a 50 µL reaction containing 3 µL of 100 mM MgCl, 1.25 µl of RNase inhibitor, 5 µl of 10 x PCR buffer, 10 µl of 10 mM dNTP mix, 1.3 µl of Oligo d (T), 1.5 µl of reverse transcriptase (Invitrogen), mixture was incubated for 60 min at 37°C and the reaction was stopped by heating to 94°C for 5 min. Primer/probe sets for IL-13, IL-15, TNFR1 and glyceraldehyde-3-phosphatedehydrogenase (GAPDH) were purchased and designed by Assays-on-Demand (Applied Biosystems, Foster City, CA, USA). For the experiments presented in Figure 1, 5 µl of 1:20 diluted cDNA was added to 45 µl of the PCR master mix containing: 1 x SYBR PCR buffer, 3 mM MgCl2, 200 µM dATP, dCTP and dGTP, 400 µM dUTP, 300 µM primer set for cytokines and GAPDH, and 1.25 U of AmpliTaq Gold DNA polymerase (Umekawa et al., 2002). After an initial 10 min denaturation at 95°C, the thermal cycling comprised 40 cycles of denaturation at 95°C for 15 s and annealing and extension at 60°C for 1 min. The relative expression of IL-13 and IL-15 mRNA was compared with GAPDH mRNA expression as internal control. The parameter of threshold cycle (CT) was defined as the fractional cycle number at which fluorescence exceeds a threshold level. The comparative CT method quantifying the amount of mRNA relative to that of a reference sample (calibrator) was used to determine the expression level of unknown sample (Umekawa et al., 2002). For other experiments, 2 µg of total RNA was subjected to real-time PCR using Taqman and ABI-Prism 7700 and Sequence Detection System 1.6 software (Applied Biosystems). Results were analysed using comparative method following normalization of expression values to 18S rRNA expression (Luo et al., 2004).
|
The level of IL-13, IL-15 and soluble TNFR1 released into the culture-condition media and present in the cell lysates was determined using human-specific ELISA kits (R&D) with detection limits of 32, 2 and 1 pg/ml, respectively, as previously described (Chegini et al., 2002). For preparation of cell lysate, the cells were homogenized in buffer consisting of 25 mM Tris–HCl, pH 8.0, 1 mM EDTA, 150 mM NaCl, 1% triton X-100, 5 mM NaF and protease inhibitor cocktail, centrifuged at 10 000 x g for 15 min at 4°C, and supernatants were collected and their protein content determined using standard method (Chegini et al., 1999b, 2002).
All the experiments were performed with three individual endometrial and stromal cell cultures except otherwise stated. The results are expressed as mean ± SEM and where appropriate statistically analysed using unpaired Student’s t-test and analysis of variance (ANOVA), Tukey test. A probability level of P < 0.05 was considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Expression of IL-13 and IL-15 by endometrial cells
Isolated EEC and ESC cells express IL-13 and IL-15 mRNA and protein. The level of IL-13 and IL-15 mRNA expression and protein production either released into the culture-conditioned media or detected in the cell lysate (cell-associated or non-secreted) was higher in EEC as compared with ESC, with higher IL-13 expression compared with IL-15 (Figures 1 and 2). These results are consistent with and support our previous observations that the EEC are the major sites of IL-13 and IL-15 expression (Chegini et al., 2002
, 2003). Low production of IL-15 by endometrial cells is also consistent with results obtained in other cells types because of predominant regulation of IL-15 at the posttranscriptional level and trafficking (Kurys et al., 2000; Nishimura et al., 2005).
|
Regulation of IL-13 and IL-15 expression by ovarian steroids
As shown in Figures 1 and 2, E2, MPA or E2 + MPA in a time- and cell-specific manner differentially regulated the expression of IL-13 and IL-15 mRNA and their protein production in EEC and ESC. The effect of E2 on the expression of IL-13 and IL-15 mRNA expression occurred rapidly, with sustained induction of IL-13 expression as compared with IL-15 whose expression was increased only after 4 and 2 h of treatment of ESC (Figure 1A and C) and EEC (Figure 1B and D), respectively (P < 0.05). MPA moderately increased the expression of IL-13 mRNA in EEC and ESC; however, it induced IL-15 expression within 2–4 h reaching control values after 6 h of treatment in EEC (Figure 1B and D; P < 0.05). Cotreatment with E2 + MPA also increased IL-13 and IL-15 expression in EEC and ESC in a time-dependent manner; however, their actions were either similar or reduced compared with E2 or MPA actions (Figure 1).
IL-13 and IL-15 protein production either released into the EEC and ESC culture-conditioned media or detected in their cell lysates (cell-associated or non-secreted) was also regulated by E2 and MPA in cell- and time-dependent manners (Figure 2). E2 was more effective in regulating the production of IL-13 as compared with MPA, whereas MPA increased secreted and cell-associated IL-15 in both EES and ESC (Figure 2; P < 0.05). The effect of E2 + MPA on IL-13 and IL-15 production by EEC and ESC was additive as compared with E2 or MPA alone (Figure 2). Since previous reports have demonstrated an increase in IL-15 production by experimentally decidulalized endometrial cells because of prolonged exposure to P4 (Okada et al., 2002, 2004), we exposed ESC and EEC to E2, MPA or E2 + MPA for several days. The results indicated that treatment with E2, MPA or E2 + MPA resulted in a trend towards lower production of IL-13 and IL-15 by ESC and EEC following 10 days of treatment as compared with shorter (18, 24 and 36 h) treatment periods; however, these values did not reach statistical significance (data not shown).
Regulation of IL-13 and IL-15 expression by TNF- and TGF-ß
TNF- at 10 ng/ml significantly increased IL-13 production by EEC and ESC, with a moderate increase in cell-associated IL-13 in ESC, whereas at TNF- at 25 ng/ml concentration reduced IL-13 production in ESC (Figure 3; P < 0.05). TNF- at 10 and 25 ng/ml increased the level of secreted and cell-associated IL-15 in EEC and ESC, with significantly higher cell-associated IL-15 (Figure 3; P = 0.01). Treatment of EEC and ESC with TGF-ß at doses of 1 and 5 ng/ml significantly increased IL-13, and secreted and cell-associated IL-15 production, with a trend towards lower IL-15 production, specifically in ESC following treatment with TGF-ß at 5 ng/ml (Figure 3; P < 0.05). TNF- and TGF-ß at doses of 10 and 1 ng/ml, respectively, also increased IL-13 and IL-15 mRNA expression in EEC and ESC (Figure 4; P = 0.05). However, TNF- induced a higher level of IL-15 mRNA expression (Figure 4A), whereas TGF-ß induced more IL-13 expression in both EEC and ESC (Figure 4B; P = 0.05).
|
|
The effect of IL-13 and IL-15 on 3H-thymidine incorporation and cell proliferation
The rate of 3H-thymidine incorporation into EEC and ESC was determined following treatment with IL-13 and IL-15 at doses ranging from 0.5 to 100 ng/ml (Figure 1A and B). The results indicated that IL-15 (Figure 5B), but not IL-13 (Figure 1A), increased the rate of 3H-thymidine incorporation into ESC, except a reduction at doses lower than 1 ng/ml (P < 0.05). Similarly, IL-15 increased the rate of 3H-thymidine incorporation into EEC, while IL-13 decreased that at doses above 5 ng/ml (P 
0.05). Both IL-13 and IL-15 had a limited proliferative activity in EEC and ESC after 48 h of treatment, with a moderate increase in the rate of proliferation after 72 and 96 h of treatment with IL-15 (Figure 5C–F; P 0.05).
|
) and IL-15 (
) at various concentrations on rate of 3H-thymidine incorporation (Figs. A & B) and cell proliferation (Figs. C-F) on endometrial stromal (ESC) and epithelial (EEC) cells incubated in the presence of 2% fetal bovine serum (FBS) for 24 h (3H-thymidine incorporation) or 48–96 h (cell proliferation), respectively. Note a gradual increase in the rate of 3H-thymidine incorporation into these cells following IL-13 and IL-15 treatments of ESC, with the exception of an inhibitory effect at a lower dose, and inhibition in EEC by IL-13. Points represent mean ± SEM of triplicate experiments using cells isolated from three different tissues. * is different from untreated control (Ctrl), P < 0.05.
Regulation of TNFR1 expression by IL-13 and IL-15
IL-13 and IL-15 (50 ng/ml) in time- and cell-dependent manners increased the expression of TNFR1 mRNA (Figure 6A and B) and production of soluble TNFR1 (sTNFR1) protein (Figure 6A and B). EEC expressed more TNFR1 mRNA and sTNFR1 protein as compared with ESC; however, they responded differently to IL-13 and IL-15 treatments, with a rapid induction with a gradual decrease in TNFR1 expression in ESC by IL-13 and a delayed and lowered expression by IL-15 (Figure 6; P < 0.05). IL-13 and IL-15 increased the expression of TNFR1 in EEC reaching maximal after 4 and 2 h of treatment, respectively, with a steady decline in expression in IL-15-treated cells (P < 0.05). EEC also released a significantly higher level of sTNFR1 protein as compared with ESC, with increased production following treatment with IL-13 and IL-15 for 18, 24 and 36 h, reaching maximal after 24 h as compared with untreated control (Figure 6C and D; P < 0.05).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Using EEC and ESC maintained in defined culture conditions, this study provided further evidence for the endometrial expression of IL-13 and IL-15 and additional evidence for their regulation and actions in these cells. We found that EEC express a higher level of IL-13 and IL-15 supporting our previous observations that endometrial surface and glandular epithelial cells are the major sites of their expression (Chegini et al., 2002
, 2003; Rhoton-Vlasak et al., 2005). However, the level of IL-15 released by EEC and ESC into their conditioned media was low, a common characteristic observed in other cell types, with the exception of activated macrophages and epithelial cells in response to inflammatory and immune reactions, and due to regulation at the level of transcription, translation and intracellular trafficking (Kurys et al., 2000; Fehniger and Caligiuri, 2001; Nishimura et al., 2005). IL-15 mediates its action through IL-15 receptor, consists of a ß-chain (shared with IL-2), common -chain and a unique -chain (IL-15). IL-15Rß complex signals through Janus kinases 1 and 3 and signal transducer and activator of transcription (STAT)-3 and -5, as well as through src-related tyrosine kinases and Ras/Raf/mitogen-activated protein kinase to fos/jun activation. A high-affinity interaction with soluble form of IL-15R may serve as a specific inhibitor of IL-15-mediated actions (McInnes and Gracie, 2004). Because a significant portion of IL-15 detected in EEC and ESC was cell-associated (non-secreted), cellular trafficking, at least in part, may account for regulation of IL-15 in the endometrial cells. The biological significance of cell-associated IL-15 is the ability to suppress the endogenous IL-15 production, a novel regulatory mechanism that may control the intracellular fate of IL-15 (Nishimura et al., 2005). Furthermore, cell-associated IL-15 has been found in cytoplasmic and nuclear compartments in association with IL-15-receptor, with nuclear translocation to serve as regulator of IL-15 action on target genes (Nishimura et al., 2005). Effective translation of IL-15 has also been considered an immediate cellular response to events, such as intracellular infection, inflammation and malignancies (Kurys et al., 2000; Fehniger and Caligiuri, 2001; Waldmann, 2002; McInnes and Gracie, 2004). In contrast, the expression of IL-13 is regulated through mRNA stabilization at transcriptional and posttranscriptional levels and influenced by several factors in other cell types (Chomarat and Bannchereau, 1998; Brombacher, 2000; Wynn, 2004). To our knowledge, our study is the first to demonstrate a regulatory function for ovarian steroids on IL-13 expression and cell-associated IL-15 production. We also found that in addition to ovarian steroids, TNF- and TGF-ß, cytokines with key inflammatory/immune properties, in a cell-specific manner differentially regulate the expression of IL-13 and IL-15 mRNA and protein in EEC and ESC.
With respect to endometrial IL-13 and IL-15 regulation, our observations are consistent with their expression during the menstrual cycle, indicating a differential regulatory function for ovarian (Chegini et al., 2002, 2003). Interestingly, treatment of EEC and ESC with E2 and MPA resulted in a rapid induction of IL-13 and IL-15 expression, contrasting in part with previous reports showing that prolonged exposure to P4 or P4 + E2, but not E2 was necessary to induce IL-15 expression in ESC (Okada et al., 2002, 2004). Because prolonged culturing of ESC has been used as an in vitro model of decidualization, we exposed ESC to MPA, E2 and MPA + E2 for 10 days and found no significant difference between IL-15 or IL-13 production as compared with short-term culturing (18–36 h). We consider culture condition rather than hormonal regulation as the most likely explanation for the difference between these studies. As the endometrial expression of IL-13 and IL-15 was also increased before and during the onset of menses, a period with declined sex steroid production, locally expressed non-steroidal factors must regulate their expression. As such, recent reports have identified PGE2 and IL-1ß as potential regulator of IL-15 expression in ESC (Dunn et al., 2002; Okada et al., 2004). We identified TNF- and TGF-ß, cytokines with key regulatory function on inflammatory and immune responses, which regulate the expression of IL-13 and IL-15 in EEC and ESC in a cell- and dose-dependent manner. Because the peak endometrial expression of TNF-, TGF-ß and their receptors during the menstrual cycle occurs during menses and the secretory phase, respectively (Chegini et al., 1994, 1999a,b; Tabibzadeh et al., 1999; von Wolff et al., 1999), their dose-dependent action on IL-13 and IL-15 expression in EEC and ESC reflects their potential autocrine/paracrine regulatory actions on these cytokines. Interestingly, TNF- increased IL-13 and IL-15 expression in ESC, with an increased expression of IL-15 in EEC, whereas TGF-ß was a potent inducer of IL-13 expression in EEC, but equally affected IL-15 expression in both EEC and ESC. Based on these observations, it could be concluded that interactions among ovarian steroids and cytokines such as IL-1, TNF- and TGF-ß may result in a compartmental balance of expression and extracellular/intracellular distributions of IL-13 and IL-15 influencing the outcome of their actions during the normal menstrual cycle and disorders affecting endometrium.
The biological significance of IL-13 and IL-15 in the endometrium is their elevated expression during menses, late secretory phase, dysfunctional uterine bleeding and endometriosis which coincide with increased infiltration of inflammatory- and immune-related cells and enhanced tissue remodelling. Since IL-13 and IL-15 regulate the recruitment, maintenance and activation of T cells, neutrophils, macrophages and mast cells, and the expression of proteases and ECM (Chomarat and Bannchereau, 1998; Brombacher, 2000; Fehniger and Caligiuri, 2001; Waldmann, 2002; McInnes and Gracie, 2004; Wynn, 2004), they could influence the outcome of endometrial response to injury during menses and dysfunctional uterine bleeding. However, IL-13 and IL-15 had limited mitogenic activities for EEC and ESC although these cells undergo an enhanced cell growth and differentiation during follicular and luteal phase, they may regulate the endometrial maintenance and activation of neutrophils, macrophages and mast cells (Fehniger and Caligiuri, 2001; Lodolce et al., 2002; Waldmann, 2002; McInnes and Gracie, 2004; Wynn, 2004). These inflammatory and immune-related cells are present in endometrium throughout the menstrual cycle and increase in number in several pathological conditions including dysfunctional uterine bleeding and endometriosis (Mori et al., 1997; Chegini and Williams, 2000; Salamonsen and Lathbury 2000; Crichley et al., 2001; Laird et al., 2003). Excess production of IL-13 has also been identified to influence the outcome of tissue fibrosis, either independently or through the induction of TGF-ß, which is a key profibrotic cytokine (Blackburn et al., 2003; Wynn, 2004). This is specifically important considering the need for endometrial tissue repair following menses, irregular bleeding or establishment of endometriosis-associated adhesion formation, where TGF–ß and interaction with IL-13 could play a key role (Chegini et al., 2002). In addition to IL-13, IL-15 is a key regulator of humoral aspects of mucosal immunity such as IgA production, and their expression and regulation by TNF- and TGF-ß in EEC may influence the outcome of the endometrial mucosal immunity.
We also found that IL-13 and IL-15 act as key regulators of TNFR1 expression and TNF soluble receptor production in EEC and ESC. Although the biological significance of endometrial TNFR1 expression and regulation by IL-13 and IL-15 is not clear from our study, changes in their expression can influence the endometrial inflammatory response mediated through TNF-. TNF- mediates its action through TNFR1 (p55) and TNFR2 (p75), in which TNFR1 acts as a receptor for a soluble ligand, and TNFR2 mediates signalling of the membrane-bound ligand (Aggarwal, 2003). In addition, TNF--induced apoptosis and mitogenic activity appears to be mediated through TNFR1 and TNFR2, respectively, although the two receptors may transduce their signals cooperatively (Aggarwal, 2003). Interestingly, the endometrium of women with endometriosis is reported to be deficient in TNFR2 expression, whereas their peritoneal fluids contained elevated levels of soluble TNFR1 and TNFR2 as compared with normal subjects (Koga et al., 2000; Kharfi et al., 2003). In addition, IL-13 and IL-15 through regulation of TNF-, TNFR1, TNFR2 and TNF soluble receptors expression as well as the induction IL-1, IFN- and other inflammatory cytokines, respectively, act as apoptotic and anti-apoptotic cytokines (Ruckert et al., 2000; Matthys et al., 2002; Pelletier et al., 2002; Berard et al., 2003; Hiromatsu et al., 2003; Figueras et al., 2004; McInnes and Gracie, 2004; Wynn, 2004). IL-15 also induces GM-CSF expression in NK cells, where along with production of IL-13, IFN- and TNF- regulates monocytic cells and humoral immune responses (Fehniger and Caligiuri, 2001; Loza et al., 2002). Because the endometrial expression of GM-CSF is regulated by ovarian steroid and TGF-ß (Chegini et al., 1999b), the interactions among these cytokines may influence the outcome of IL-13 and IL-15 action during normal menstrual cycle and in abnormalities affecting this tissue.
In conclusion, our results provided further evidence for the endometrial expression of IL-13 and IL-15 and showed that ovarian steroids, as well as TNF- and TGF-ß differentially regulate their expression in endometrial cells in a dose- and cell-dependent manner. IL-13 and IL-15 had limited mitogenic activities for endometrial cells; however, in a cell-dependent manner regulated the expression of TNFR1 and TNF soluble receptor, a mechanism that may influence the outcome of endometrial inflammatory/immune responses during the normal menstrual cycle, as well as in dysfunctional bleeding and endometriosis with altered cytokine expression.
|
Acknowledgements |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Dr Chegini is a recipient of the University of Florida Research Foundation Award, and we thank the Foundation for supporting part of the work presented in this study. Presented in part at 56th Annual Meeting of the American Society for Reproductive Medicine Orlando, Florida, October 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Aggarwal BB (2003) Signaling pathways of the TNF superfamily a double-edged sword. Nat Rev Immunol 3,745–756.[CrossRef][Web of Science][Medline]
Berard M, Brandt K, Paus SB and Tough DF (2003) IL-15 promotes the survival of naive and memory phenotype CD8 (+) T cells. J Immunol 170,5018–5026.
Blackburn MR, Lee CG, Young HW, Zhu Z, Chunn JL, Kang MJ, Banerjee SK and Elias JA (2003) Adenosine mediates IL-13-induced inflammation and remodeling in the lung and interacts in an IL-13-adenosine amplification pathway. J Clin Invest 112,332–344.[CrossRef][Web of Science][Medline]
Brombacher F (2000) The role of interleukin-13 in infectious diseases and allergy. Bioessays 22,646–656.[CrossRef][Medline]
Chegini N and Williams RS (2000) Cytokines and growth factor networks in human endometrium from menstruation to embryo implantation. In Hill JA (ed.), Cytokines in Human Reproduction. Wiley & Sons Publisher, New York, NY, pp. 93–132.
Chegini N, Rossi MJ and Masterson BJ (1992) Platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and EGF and PDGF beta-receptors in human endometrial tissue: localization and in vitro action. Endocrinology 130,2373–2385.
Chegini N, Zhao Y, Williams RS and Flanders KC (1994) Human uterine tissue throughout the menstrual cycle expresses transforming growth factor-beta 1 (TGF-ß1), TGF-ß2, TGF-ß3, and TGF-ß type II receptor messenger ribonucleic acid and protein and contains [125I]TGF-ß1-binding sites. Endocrinology 135,439–449.[Abstract]
Chegini N, Dou Q and Williams RS (1999a) An inverse relation between the expression of tumor necrosis factor alpha (TNF-) and TNF- receptor in human endometrium. Am J Reprod Immunol 42,297–302.
Chegini N, Tang XM and Dou Q (1999b) The expression, activity and regulation of granulocyte macrophage-colony stimulating factor in human endometrial epithelial and stromal cells. Mol Hum Reprod 5,459–466.
Chegini N, Ma C, Roberts M, Williams RS and Ripps BA (2002) Differential expression of interleukins (IL) IL-13 and IL-15 throughout the menstrual cycle in endometrium of normal fertile women and women with recurrent spontaneous abortion. J Reprod Immunol 56,93–110.[CrossRef][Web of Science][Medline]
Chegini N, Roberts M and Ripps B (2003) Differential expression of interleukins (IL)-13 and IL-15 in ectopic and eutopic endometrium of women with endometriosis and normal fertile women. Am J Reprod Immunol 49,75–83.
Chomarat P and Bannchereau J (1998) Interleukin-4 and interleukin-13: their similarities and discrepancies. Int Rev Immunol 17,1–52.[Medline]
Critchley HO, Kelly RW, Brenner RM and Baird DT (2001) The endocrinology of menstruation – a role for the immune system. Clin Endocrinol (Oxf) 55,701–710.[CrossRef][Medline]
Dunn CL, Critchley HO and Kelly RW (2002) IL-15 regulation in human endometrial stromal cells. J Clin Endocrinol Metab 87,1898–1901.
Fehniger TA and Caligiuri MA (2001) Interleukin 15: biology and relevance to human disease. Blood 97,14–32.
Figueras M, Busquets S, Carbo N, Barreiro E, Almendro V, Argiles JM and Lopez-Soriano FJ (2004) Interleukin-15 is able to suppress the increased DNA fragmentation associated with muscle wasting in tumour-bearing rats. FEBS Lett 569,201–206.[CrossRef][Web of Science][Medline]
Hershey GK (2003) IL-13 receptors and signaling pathways: an evolving web. J Allergy Clin Immunol 111,677–690.[Web of Science][Medline]
Hiromatsu T, Yajima T, Matsuguchi T, Nishimura H, Wajjwalku W, Arai T, Nimura Y and Yoshikai Y (2003) Overexpression of interleukin-15 protects against Escherichia coli-induced shock accompanied by inhibition of tumor necrosis factor-alpha-induced apoptosis. J Infect Dis 187,1442–1451.[CrossRef][Web of Science][Medline]
Kelly RW, King AE and Critchley HO (2001) Cytokine control in human endometrium. Reproduction 121,3–19.[Abstract]
Kharfi A, Labelle Y, Mailloux J and Akoum A (2003) Deficient expression of tumor necrosis factor receptor type 2 in the endometrium of women with endometriosis. Am J Reprod Immunol 50,33–40.
Kitaya K, Yasuda J, Yagi I, Tada Y, Fushiki S and Honjo H (2000) IL-15 expression at human endometrium and decidua. Biol Reprod 63,683–687.
Koga K, Osuga Y, Tsutsumi O, Okagaki R, Momoeda M, Yano T, Fujiwara T, Takai Y, Kugu K, Morita Y et al. (2000) Increased concentrations of soluble tumour necrosis factor receptor (sTNFR) I and II in peritoneal fluid from women with endometriosis. Mol Hum Reprod 6,929–933.
Kurys G, Tagaya Y, Bamford R, Hanover JA and Waldmannm TA (2000) The long signal peptide isoform and its alternative processing direct the intracellular trafficking of interleukin-15. J Biol Chem 275,30653–30659.
Laird SM, Tuckerman EM, Cork BA, Linjawi S, Blakemore AI and Li TC (2003) A review of immune cells and molecules in women with recurrent miscarriage. Hum Reprod Update 9,163–174.
Lodolce JP, Burkett PR, Koka RM, Boone DL and Ma A (2002) Regulation of lymphoid homeostasis by interleukin-15. Cytokine Growth Factor Rev 13,429–439.[CrossRef][Web of Science][Medline]
Loza MJ, Zamai L, Azzoni L, Rosati E and Perussia P (2002) Expression of type 1 (interferon gamma) and type 2 (interleukin-13, interleukin-5) cytokines at distinct stages of natural killer cell differentiation from progenitor cells. Blood 99,1273–1281.
Luo X, Ding L and Chegini N (2004) Gonadotropin-releasing hormone and TGF-beta activate MAP kinase and differentially regulate fibronectin expression in endometrial epithelial and stromal cells. Am J Physiol Endocrinol Metab 287,E991–E1001.
Matthys P, Dooms H, Rottiers P, Mitera T, Overgergh L, Leclercq G, Billiau A and Grooten J (2002) Induction of IL-15 by TCR/CD3 aggregation depends on IFN-gamma and protects against apoptosis of immature thymocytes in vivo. Clin Exp Immunol 130,379–385.[Medline]
McInnes IB and Gracie JA (2004) Interleukin-15: a new cytokine target for the treatment of inflammatory diseases. Curr Opin Pharmacol 4,392–397.[CrossRef][Web of Science][Medline]
Mori A, Zhai YL, Toki T, Nikaido T and Fujii S (1997) Distribution and heterogeneity of mast cells in the human uterus. Hum Reprod 12,368–372.
Nishimura H, Fujimoto A, Tamura N, Yajima T, Wajjwalku W and Yoshikai Y (2005) A novel autoregulatory mechanism for transcriptional activation of the IL-15 gene by a nonsecretable isoform of IL-15 generated by alternative splicing. FASEB J 19,19–28.
Okada S, Okada H, Sanezumi M, Nakajima T, Yasuda K and Kanzaki H (2000) Expression of interleukin-15 in human endometrium and decidua. Mol Hum Reprod 6,75–80.
Okada H, Nakajima T, Sanezumi M, Ikuta A, Yasuda K and Kanzaki H (2002) Progesterone enhances interleukin-15 production in human endometrial stromal cells in vitro. J Clin Endocrinol Metab 85,4765–4770.
Okada H, Nakajima T, Yasuda K and Kanzaki H (2004) Interleukin-1 inhibits interleukin-15 production by progesterone during in vitro decidualization in human. J Reprod Immunol 61,3–12.[CrossRef][Web of Science][Medline]
Pelletier M, Ratthe C and Girard D (2002) Mechanisms involved in interleukin-15-induced suppression of human neutrophil apoptosis: role of the anti-apoptotic Mcl-1 protein and several kinases including Janus kinase-2, 38 mitogen-activated protein kinase and extracellular signal-regulated kinases-1/2. FEBS Lett 532,164–170.[CrossRef][Web of Science][Medline]
Rhoton-Vlasak A, Chegini N, Hardt N and Williams RS (2005) Histological characteristics and altered expression of interleukins (IL) IL-13 and IL-15 in endometria of levonorgestrel users with different uterine bleeding patterns. Fertil Steril 83,659–665.[CrossRef][Web of Science][Medline]
Riesewijk A, Martin J, van Os R, Horcajadas JA, Polman J, Pellicer A, Mosselman S and Simon C (2003) Gene expression profiling of human endometrial receptivity on days LH+2 versus LH+7 by microarray technology. Mol Hum Reprod 9,253–264.
Ruckert R, Asadullah K and Seifert M (2000) Inhibition of keratinocyte apoptosis by IL-15: a new parameter in the pathogenesis of psoriasis? J Immunol 165,2240–2250.
Salamonsen LA and Lathbury LJ (2000) Endometrial leukocytes and menstruation. Hum Reprod Update 6,16–27.
Tabibzadeh S, Satyaswaroop PG, von Wolff M and Strowitzki T (1999) Regulation of TNF-alpha mRNA expression in endometrial cells by TNF- and by oestrogen withdrawal. Mol Hum Reprod 5,1141–1149.
Umekawa T, Chegini N and Khan SR (2002) Oxalate ions and calcium oxalate crystals stimulate MCP-1 expression by renal epithelial cells. Kidney Int 61,105–112.[CrossRef][Web of Science][Medline]
Verma S, Hiby SE, Loke YW and King A (2000) Human decidual natural killer cells express the receptor for and respond to the cytokine interleukin 15. Biol Reprod 62,959–968.
von Wolff M, Classen-Linke I, Heid D, Krusche CA, Beier-Hellwig K, Karl C and Beier HM (1999) Tumour necrosis factor-alpha (TNF-) in human endometrium and uterine secretion: an evaluation by immunohistochemistry, ELISA and semi-quantitative RT-PCR. Mol Hum Reprod 5,146–152.
Waldmann T (2002) The contrasting roles of IL-2 and IL-15 in the life and death of lymphocytes: implications for the immunotherapy of rheumatological diseases. Arthritis Res 4,S161–S167.
Wynes MW and Riches DW (2003) Induction of macrophage insulin-like growth factor-I expression by the Th2 cytokines IL-4 and IL-13. J Immunol 171,3550–3559.
Wynes MW, Frankel SK and Riches DW (2004) IL-4-induced macrophage-derived IGF-I protects myofibroblasts from apoptosis following growth factor withdrawal. J Leukoc Biol 76,1019–1027.
Wynn TA (2004) Fibrotic disease and the T (H) 1/T (H) 2 paradigm. Nat Rev Immunol 4,583–594.[CrossRef][Web of Science][Medline]
Submitted on May 9, 2005; accepted on September 21, 2005.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  A. Germeyer, A. M. Sharkey, M. Prasadajudio, R. Sherwin, A. Moffett, K. Bieback, S. Clausmeyer, L. Masters, R. M. Popovici, A. P. Hess, et al. Paracrine effects of uterine leucocytes on gene expression of human uterine stromal fibroblasts Mol. Hum. Reprod., January 1, 2009; 15(1): 39 - 48. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Franchi, J. Zaret, X. Zhang, S. Bocca, and S. Oehninger Expression of immunomodulatory genes, their protein products and specific ligands/receptors during the window of implantation in the human endometrium Mol. Hum. Reprod., July 1, 2008; 14(7): 413 - 421. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Li, X. Luo, Q. Pan, I. Zineh, D. F. Archer, R.S. Williams, and N. Chegini Doxycycline alters the expression of inflammatory and immune-related cytokines and chemokines in human endometrial cells: implication in irregular uterine bleeding Hum. Reprod., October 1, 2006; 21(10): 2555 - 2563. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||