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Mechanical Stretch Stimulates Interleukin-8 Production in Endometrial Stromal Cells: Possible Implications in Endometrium-Related Events

Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo, Tokyo 113-8655, Japan

Address all correspondence and requests for reprints to: Dr. Yutaka Osuga, Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail: yutakaos-tky{at}umin.ac.jp.

	Abstract

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Uterine movement is suggested to play roles in various events related to the uterus. In view of the current concept underscoring the biological implications of mechanical stretch, we speculated that the mechanical stretch exerted by uterine movement might stimulate the production of biochemical mediators in endometrial cells and contribute to inflammation-associating processes, such as menstruation and endometriosis. To address the possible effects of mechanical stretch in the endometrium, endometrial stromal cells (ESC) were cultured on flexible-bottomed culture plates, and cyclic stretch (25% elongation) was applied in serum-free conditions at a rate of two cycles per minute using a computer-operated cell tension system. IL-8 concentrations in the conditioned medium were measured using ELISA, and IL-8 mRNA expression in ESC was measured by RT-PCR. Cyclic stretch increased the secretion of IL-8 from ESC. The increase in IL-8 secretion was inhibited by PD98059, an inhibitor of extracellular signal-regulated kinase 1/2. The increase was also inhibited by progesterone. In addition, the conditioned medium of ESC cultured with cyclic stretch stimulated the mRNA expression of IL-8 in ESC cultured under stationary conditions. These findings imply that uterine movement has an impact on endometrium-related physiology and pathology by stimulating the production of a biochemical mediator(s) in the endometrium.

	Introduction

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IT IS WELL known that myometrium exhibits a spontaneous contractile activity even in nonpregnant women (1, 2). Normal uterine contractility may subserve certain physiological phenomena, such as menstruation, sperm transport, and implantation of the embryo. In contrast, abnormal uterine contractility is possibly related to several disorders, including dysmenorrhea and endometriosis. Because of its architectural characteristics, the endometrium is influenced by myometrial mechanical movement, as studied by intrauterine pressure recording and ultrasonography (2, 3).

Accumulating evidence underscores the biomedical significance of mechanical stress, which induces biochemical responses in cells and is suggested to be involved in the pathophysiology of several diseases (4). For example, cyclic stretch, which induces IL-8 expression in alveolar epithelial cells, is implicated in lung inflammation triggered by mechanical ventilation (5, 6). Notably, IL-8, a multifunctional chemokine, is well known to be involved in normal activities of the uterus as well as the pathogenesis of endometriosis (7).

In view of these findings, we hypothesized that myometrium-derived mechanical stress may induce certain cellular responses in the endometrium and may contribute to the physiology and pathology of the uterus. As a first step in addressing the thesis, we studied the effect of mechanical stress on IL-8 secretion from endometrial stromal cells (ESC), using an experimental device that stretched cultured cells.

	Subjects and Methods

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Patients and samples

Endometrial tissues were obtained from 43 women without endometriosis undergoing hysterectomy for uterine fibroids (n = 41) or adenomyosis (n = 2). The mean age of the women was 44.0 yr (SD, 4.4). All of the women had regular menstrual cycles, and none had received hormonal treatment for at least 6 months before surgery. At the time of surgery, 23 women were in the proliferative phase, and 20 were in the secretory phase. The tissues collected by curettage under sterile conditions were processed for primary cell cultures. The experimental procedures were approved by the institutional review board of University of Tokyo, and signed informed consent for use of the endometrium was obtained from each woman.

Isolation and culture of human ESC

The isolation and culture of human ESC were conducted as previously reported (8). Briefly, the tissues were minced into small pieces and incubated in DMEM/Ham’s F-12 containing type I collagenase (0.25%; Sigma-Aldrich Corp., St. Louis, MO) and deoxyribonuclease I (15 IU/ml; Takara Shuzo, Tokyo, Japan) for 60 min at 37 C with agitation. The resultant dispersed endometrial cells were separated by filtration through a 40-µm pore size nylon cell strainer (BD Biosciences, Franklin Lakes, NJ). Stromal cells in the filtrate were collected and plated in a 100-mm culture plate and kept at 37 C in a humidified 5% CO₂/95% air environment. At the first passage, the cells were seeded onto six-well, flexible-bottomed culture plates coated with collagen I (Flexcell International Co., Hillsborough, NC) at a plating density of 5 x 10⁵ cells/well. The purity of stromal cell preparations was more than 95%, as judged by positive cellular staining for vimentin. ESC from individual patients, not a mixture of cells from multiple patients, were used in each experiment. Before each treatment, the confluent cells were incubated overnight with serum-free medium. Before the treatment with ovarian hormones, the confluent cells were incubated overnight with medium containing 2.5% fetal bovine serum.

Application of stretch

Stretch was applied in a cyclic fashion at a rate of two cycles per minute (23-sec stretch and 7-sec release) in serum-free conditions using a Flexercell tension system FX-4000T (Flexcell International Co.), a computer-operated, vacuum-driven stretch device. The amplitude of stretch we used in all experiments was 25% elongation. These elongation levels represent the maxima, not the averages, within the radially nonuniform strain field produced by this device, as described previously (9). Control cells were cultured under identical conditions, but remained stationary.

Treatments of ESC

To evaluate the effect of PD98059 (Calbiochem, San Diego CA), an inhibitor of extracellular signal-regulated kinase 1/2 (ERK1/2), ESC were treated with PD98059 for 1 h before application of cyclic stretch for 24 h. Similarly, to study the effects of ovarian hormones, ESC were treated with 10 ng/ml (36.7 nM) estradiol, 100 ng/ml (318 nM) progesterone, or estradiol plus progesterone, for 1 h before the application of cyclic stretch for 24 h. The steroids were dissolved in 100% ethanol, and then were diluted with the medium to the final concentrations. The final concentration of ethanol in the medium was adjusted to 0.1%.

To examine whether autocrine/paracrine factor(s) induced IL-8 expression, conditioned medium was prepared by collecting the culture medium of ESC treated with and without cyclic stretch for 24 h. In an independent setting, ESC was plated onto ordinary plastic-bottomed, six-well plates at the first passage. Our preliminary study revealed that the IL-8 expression of ESC on the plastic-bottomed plate was virtually equivalent to that of ESC on the flexible-bottomed plate. After they had reached confluence, they were incubated with serum-free medium overnight. Subsequently, 40% of the volume of the medium was replaced with the prepared conditioned medium, and the incubation was continued for 4 h to examine the mRNA expression and for 24 h to examine the secretion of IL-8.

RT-PCR

Total RNA was extracted from ESC using an RNeasy minikit (Qiagen, Hilden, Germany). One microgram of total RNA was reverse transcribed in a 20-µl volume using Rever Tra Ace -- (TOYOBO, Tokyo, Japan) according to the manufacturer’s instructions. To assess IL-8 mRNA expression, 0.15 µg cDNA in 20-µl volume was applied to real-time quantitative PCR, and data analysis was performed using Light Cycler (Roche, Mannheim, Germany), according to the manufacturer’s instructions. Expression of IL-8 mRNA was normalized to RNA loading for each sample using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as an internal standard. IL-8 primers (sense, 5'-ACTTCCAAGCTGGCCGTGGCTCTCTTGGCA-3'; antisense, 5'-TGAATTCTCAGCCCTCTTCAAAAACTTCTC-3') were chosen to amplify a 295-bp fragment. GAPDH primers were purchased from TOYOBO. The PCR conditions for amplification of IL-8 were 30 cycles at 95 C for 15 sec, 66 C for 8 sec, and 72 C for 11 sec, followed by a melting curve analysis.

Measurement of IL-8

Conditioned culture media collected in the experiments were centrifuged and stored at –80 C until assay. Concentrations of IL-8 in the media were measured using a specific ELISA kit (TECHNE Co., Minneapolis, MN). The concentrations were normalized to cell counts on each well of the culture plates.

Counting cell numbers

Cell counting was performed using a Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions.

Western blot analysis

Cultured cells were homogenized in the lysis buffer containing 50 mM Tris-HCl (pH 6.8), 2% sodium dodecyl sulfate, 10% glycerol, 50 mM dithiothreitol, and 0.1% bromophenol blue. The lysates were further diluted with lysis buffer to give a final concentration of 1 mg total protein/ml. Samples of 20 µg protein/lane were resolved by 10% SDS-PAGE. Proteins were transferred onto a nitrocellulose membrane and incubated with rabbit antibody to total human ERK1/2 or rabbit antibody specific to phosphorylated (Thr²⁰²/Tyr²⁰⁴) ERK1/2 (1:1000; Cell Signaling Technology, Beverly, MA) as primary antibody, and antirabbit horseradish peroxidase antibody (1:1000; Amersham Biosciences, Little Chalfont, UK) as secondary antibody. Phosphorylation of Thr²⁰²/Tyr²⁰⁴ in ERK1/2 is known to be correlated with activation of the molecules. Immune complexes were visualized by the ECL Western blotting system (Amersham Biosciences).

Statistical analysis

Data were analyzed using ANOVA with post hoc analysis (Fisher’s protected least significance) for multiple comparisons. P < 0.05 was considered significant.

	Results

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Stretch-induced expression of IL-8 in ESC

We first conducted time-course experiments to determine the effect of stretch on the expression of IL-8 mRNA in ESC (Fig. 1A). Cyclic stretch significantly increased IL-8 mRNA levels in ESC. The maximum increase (12.5-fold over control) was observed at 4 h. In contrast, ESC kept stationary exhibited no change in IL-8 mRNA levels (data not shown). To examine IL-8 protein secretion, ESC were cultured for 24 h with or without stretch (Fig. 1B). Cyclic stretch significantly increased IL-8 secretion from ESC from 8 h compared with that from the control culture. IL-8 secretion increased over time to 24 h (4.4-fold over control at 24 h). No remarkable difference was observed in the secretion of IL-8 from ESC, whether ESC were subcultured from the endometrial tissues of proliferative phase or from those of secretory phase (data not shown).

View larger version (15K): [in this window] [in a new window]   FIG. 1. Effects of cyclic stretch on the expression of IL-8 mRNA in ESC (A) and on the secretion of IL-8 protein from ESC (B). A, Total RNA isolated from ESC stimulated by cyclic stretch (25% elongation) for the indicated hours was reverse transcribed and amplified by real-time PCR using primers of IL-8. The data were calculated by subtracting the signal threshold cycles (CT) of the internal standard (GAPDH) from the CT of IL-8. Values are the mean ± SEM of the combined data of four separate experiments using different ESC preparations. B, ESC were cultured with or without cyclic stretch (25% elongation) for the indicated hours. IL-8 concentrations in the conditioned media were measured by ELISA. Values are the mean ± SEM of quadruplicate cultures. *, P < 0.05; **, P < 0.005 (vs. without stretch).

As shown in Fig. 2A, phosphorylation of ERK1/2 was increased by stretch in ESC. The increase was most prominent at 5 min and gradually reduced.

View larger version (37K): [in this window] [in a new window]   FIG. 2. Involvement of ERK1/2 in cyclic stretch-induced IL-8 secretion from ESC. A, Phosphorylation of ERK1/2 by cyclic stretch in ESC. ESC were stimulated by cyclic stretch for the indicated times. Cell extracts were prepared and assayed for phosphorylated ERK1/2 (phospho-ERK1/2) and total-ERK1/2 by Western blotting. The data shown are representative of three separate experiments. B, Effect of PD98059 (25 µM), an ERK1/2 inhibitor, on cyclic stretch-induced IL-8 secretion from ESC. ESC were pretreated with or without PD98059 for 1 h. Then ESC were treated with or without cyclic stretch for 24 h. IL-8 concentrations in the conditioned media were measured by ELISA. The values represent relative ratios of IL-8 concentrations, compared with those without cyclic stretch and without PD98059 treatment. Values are the mean ± SEM of quadruplicate cultures. *, P < 0.005.

PD98059 significantly inhibited the stimulatory effect of stretch on IL-8 secretion, whereas PD98059 had no effect on basal IL-8 secretion in ESC (Fig. 2B).

Effect of conditioned medium of stretch-treated ESC on IL-8 expression in ESC

We addressed whether stretch-induced IL-8 expression in ESC is regulated by autocrine/paracrine factors. As shown in Fig. 3A, conditioned medium of ESC treated with stretch induced a significant increase (1.7-fold over control) in IL-8 expression in stationary cultured ESC compared with that in ESC without stretch. Conditioned medium of ESC treated with stretch also induced a significant increase in IL-8 secretion from stationary cultured ESC compared with that of ESC without stretch (Fig. 3B)

View larger version (17K): [in this window] [in a new window]   FIG. 3. Effects of conditioned medium of ESC cultured with cyclic stretch on the expression of IL-8 mRNA in ESC (A) and on the secretion of IL-8 protein from ESC (B). Conditioned medium had been prepared from the culture of ESC with or without cyclic stretch. Subsequently, ESC were cultured under a stationary condition with the medium containing the prepared conditioned medium (40%). A, After incubation for 4 h, total RNA isolated from ESC was reverse transcribed and amplified by real-time PCR using primers of IL-8. The data were calculated by subtracting the signal threshold cycles (CT) of the internal standard (GAPDH) from the CT of IL-8. Values are the mean ± SEM of the combined data of four separate experiments. *, P < 0.001. B, After incubation for 24 h, cell culture supernatants were collected, and IL-8 concentrations were measured by ELISA (final concentrations). IL-8 concentrations in the medium before the incubation were also measured (initial concentrations). The data shown are the net increases in the concentrations of IL-8 during the incubation period that was calculated by subtracting the initial concentrations from the final concentrations. Values are the mean ± SEM of quadruplicate cultures. *, P < 0.01.

As shown in Fig. 4, progesterone, regardless of the presence of estradiol, significantly suppressed the stretch-induced secretion of IL-8 from ESC, whereas estradiol alone did not have any significant effect. In contrast, neither hormonal status affected the secretion of IL-8 under stationary conditions.

View larger version (23K): [in this window] [in a new window]   FIG. 4. Effects of estradiol (E) and progesterone (P) on stretch-induced secretion of IL-8 in ESC. Treatment of ESC with E (10 ng/ml), P (100 ng/ml), E plus P, or vehicle (0.1% ethanol; C) was started 1 h before cyclic stretch was applied. After being stimulated by cyclic stretch or kept stationary for 24 h, the conditioned media were collected and assayed for IL-8 concentrations by ELISA. IL-8 concentrations were normalized to cell counts. The values represent relative ratios of IL-8 concentrations compared with the control without cyclic stretch. Values are the mean ± SEM of quadruplicate cultures. *, P < 0.001 vs. C with stretch.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

Uterine contraction during menstruation is similar to that during regular labor, with high intensity and high amplitude (2). The expression of IL-8 in the endometrium is most pronounced around menstruation, suggesting that IL-8 plays a role, such as chemoattraction of neutrophils, in the menstrual endometrium (10, 11). In light of the present findings, the vigorous uterine movement in the menstrual phase may regulate physiological events in the endometrium by up-regulating endometrial IL-8 secretion.

In addition to the menstrual phase, uterine movement is observed throughout the menstrual cycle, with changing properties under the effects of ovarian steroid hormones and other bioactive molecules. Uterine movement is suggested to play various roles, such as elimination of pathogens, transport of sperm, and excretion of shed endometrium. In addition to these mechanical effects, a novel implication of uterine movement in uterine physiology was found in the present study, demonstrating that the movement could exert an effect by biochemical mediators.

Eutopic endometrium has been shown to play a crucial role in the pathogenesis of endometriosis (12). According to the implantation theory (13), an increase in the amount of retrograde menstruation, which is observed in women with endometriosis, increases the chances for refluxed endometrial cells to implant in the peritoneum. The aberration in uterine contraction observed in women with endometriosis (14, 15, 16) may cause endometriosis by increasing retrograde menstruation. The present findings propose a distinct link between uterine contraction and the pathogenesis of endometriosis. Specifically, in view of the potential roles of IL-8 in the pathogenesis of endometriosis (7, 17, 18, 19, 20, 21), uterine contraction-induced IL-8 secretion from the endometrium may be involved in the process.

A remarkable finding in the present study is that progesterone inhibited the stretch-induced IL-8 secretion regardless of the presence of estradiol, whereas estradiol had no effect. An increase in IL-8 levels in the products of conception has been shown in abortion and premature delivery (22, 23). Thus, it is intriguing to speculate that progesterone suppresses uterine contraction-induced inflammatory responses in the endometrium to protect conceptus during pregnancy. Furthermore, the inhibitory effect of progesterone might explain in part the therapeutic effect of the hormone on endometriosis, given that stretch-induced IL-8 secretion plays a role in endometriosis. In this study progesterone treatment for 24 h did not affect IL-8 secretion from ESC in the stationary condition, whereas others demonstrated that long-term progestin treatment suppressed IL-8 secretion. The discrepancy might be due to the differences in experimental conditions (24).

In the present study conditioned medium of ESC that had been treated with cyclic stretch stimulated IL-8 mRNA expression and protein secretion in ESC. The finding suggests an autocrine/paracrine mechanism in stretch-induced IL-8 secretion. In contrast, phosphorylation of ERK, which was shown to be a mediator of IL-8 secretion, was observed as early as 5 min. Thus, stretch is suggested to directly activate an intercellular signal that induces IL-8 secretion. Autocrine/paracrine factors have been shown to be essential mediators for stretch-induced responses in certain cells (25, 26, 27, 28), whereas the effect of stretch unrelated to autocrine/paracrine factors has been observed in other cells (29). Our study suggested that stretch induces IL-8 secretion from ESC by direct and indirect (autocrine/paracrine) mechanisms.

To date, the precise mechanism by which mechanical stretch is converted to intracellular signals remains elusive. In the present study ERK phosphorylation was shown to be associated with stretch-induced IL-8 secretion. A similar mechanism has been suggested in human airway smooth muscle cells (30), whereas another signaling mechanism seems to be at work in alveolar epithelial cells (31).

In summary, the present study demonstrated that cyclic stretch stimulated IL-8 production in ESC. This finding suggests that mechanical uterine movement is transduced into biochemical signals in the endometrium, which play roles in uterus-related events such as menstruation and endometriosis.

	Acknowledgments

 
We thank Emi Nose for her technical assistance.

	Footnotes

 
First Published Online December 7, 2004

Abbreviations: ESC, Endometrial stromal cell(s); ERK, extracellular signal-regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Received June 9, 2004.

Accepted November 23, 2004.

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