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Thalidomide Inhibits Tumor Necrosis Factor--Induced Interleukin-8 Expression in Endometriotic Stromal Cells, Possibly through Suppression of Nuclear Factor-B Activation

NetForce Co. Ltd. (T.Y., H.M., K.W., K.I.), Nagoya, Aichi 453-0801, Japan; Department of Obstetrics and Gynecology (H.K., M.S., N.K., T.T.), Hamamatsu University School of Medicine, Hamamatsu, Shizuoka 431-3192, Japan; Computer Technology Integration Co. Ltd. (T.K.), Nagoya, Aichi 450-0003, Japan; and Department of Knowledge-Based Information Engineering (N.K., H.S.), Toyohashi University of Technology, Toyohashi Aichi 441-8580, Japan

Address all correspondence and requests for reprints to: Hiroshi Kobayashi, M.D., Ph.D., Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Handayama 1-20-1, Hamamatsu, Shizuoka 431-3192, Japan. E-mail: hirokoba{at}hama-med.ac.jp.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
Objective: Endometriosis, a common disease among women of reproductive age, is characterized by the presence of endometrial-like tissue outside the uterus. TNF- induces IL-8 production in endometriotic cells through nuclear factor-B (NF-B) activation. Thalidomide (Thal) inhibits inflammation by down-regulating the expression of proinflammatory cytokines in tumor cells and inflammatory cells. However, the mechanism of Thal action in human endometriotic stromal cells has not yet been elucidated.

Main Outcome Measures: We examined whether Thal abrogates TNF--induced up-regulation of IL-8 expression in endometriotic stromal cells.

Results: Here, we show 1) that treatment of endometriotic stromal cells with TNF- increased the expression of phosphorylated IB and degradation of total IB, which in turn activates NF-B; 2) Thal significantly inhibits the TNF--induced expression of phosphorylated IB and degradation of IB; 3) TNF- activation induced increased nuclear translocation of NF-B, which was inhibited by pretreatment with either Thal or N-tosyl-L-phenylalanine chloromethyl ketone, an NF-B inhibitor. Thal did not enhance the N-tosyl-L-phenylalanine chloromethyl ketone’s action; and 4) Pretreatment with Thal reduced TNF--induced IL-8 protein production as well as mRNA expression.

Conclusion: The current study showed for the first time that Thal treatment attenuated the expression of IL-8 by reducing TNF--induced NF-B activation.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
ENDOMETRIOSIS IS A DISORDER characterized by the presence and growth of endometrial tissue outside the uterus, primarily into the peritoneum. Integrated clinical and research teams are needed that combine expert medical, surgical, and holistic care with state-of-the-art research expertise in immunology, endocrinology, and genetics to discover new diagnostic methods and medical treatments for endometriosis (1). Today, a composite theory of retrograde menstruation with implantation of endometrial fragments in conjunction with peritoneal factors to stimulate cell growth is the most widely accepted explanation (2, 3, 4). Dynamic interplay among cytokines may contribute to realize a favorable microenvironment for the implantation of endometrial cells and the progression of the disease (5, 6). It has previously been showed that IL-6, IL-8, and TNF- are significantly elevated in the peritoneal fluid of women with endometriosis compared with that of women without endometriosis (7, 8), that peritoneal fluid levels of IL-8 significantly enhanced proliferation of stromal cells derived from ovarian endometriomas (8), and that TNF- promoted the proliferation of endometriotic stromal cells by inducing IL-8 expression (9). These results allow us to speculate that suppression of IL-8 expression may abrogate the development and progression of endometriosis.

Thalidomide (Thal) has several targets and mechanisms of action. Despite its known teratogenic effects, Thal has been used to treat a variety of diseases ranging from alleviation of autoimmune disorders to prevention of metastasis of cancers (10). The antiinflammatory effect of Thal is associated with suppression of cytokine expression and the antioncogenic effect with inhibition of angiogenesis (11). Thal has been used in several cutaneous inflammatory disorders (e.g. erythema nodosum leprosum in lepromatous leprosy, cutaneous lupus erythematosus, and severe aphthosis), cancers (e.g. relapsed/refractory multiple myeloma, malignant melanoma, and systemic signs in cancer), and inflammatory conditions (e.g. Crohn’s disease and rheumatoid arthritis) (11, 12, 13, 14, 15). To prevent the teratogenicity, however, use of Thal is strictly controlled with close adherence to a birth control program.

It has been reported that Thal blocks nuclear factor-B (NF-B) activation through a mechanism that involves the inhibition of activity of IB kinase (11). Consistent with the observed inhibition of NF-B, Thal blocked the cytokine-induced expression of NF-B-regulated genes (11, 16, 17). The pivotal role of cytokine in inflammation and the antiinflammatory activity of Thal raise the question of whether the induction of TNF--dependent IL-8 expression during inflammation serves as a target of antiinflammation by Thal. However, there is no report of whether Thal inhibits TNF--induced up-regulation of IL-8 expression in endometriotic cells. To our knowledge, this is the first report of inhibition of TNF--induced IL-8 production by Thal in human endometriotic stromal cells. Our findings provide new insights into the mechanism of protection against endometriosis by Thal.

	Patients and Methods

Top Abstract Introduction Patients and Methods Results Discussion References

 
Reagents

DMEM and fetal bovine serum were obtained from Invitrogen Japan K.K. (Tokyo, Japan). Anti-p65 NF-B (catalog no. sc-372; epitope, C terminus), anti-IB (product no. C21, catalog no. sc-371; epitope C terminus), and anti-phospho-IB (product no. B9, catalog no. sc-8404; epitope, p-Ser-32) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Thal (catalog no. 585970) was obtained from Calbiochem (manufactured by EMD Biosciences, Inc., San Diego, CA). It was resuspended in dimethyl sulfoxide (Me₂SO). N-tosyl-L-phenylalanine chloromethyl ketone (TPCK; an NF-B inhibitor) was from Sigma-Aldrich Japan (Tokyo, Japan). TNF- was from Genzyme (Minneapolis, MN).

Endometriotic stromal cell purification and cell culture

Eight women with moderate to severe stages of endometriosis [obtained in the proliferative phase (n = 6) and luteal phase (n = 2)] were enrolled in this study. All patients received laparoscopic examinations or laparotomy. Specimens were obtained from two different sites (ovarian endometriomas and peritoneal endometriotic spots). The capsules of ovarian endometrioma were removed from the normal ovarian cortex while the subjects were undergoing laparoscopic procedures for pathologic examinations and further culture preparations. Endometriosis was confirmed histologically by the presence of endometrial glands and stroma. Stromal cells were collected from endometriotic tissues essentially as described (18, 19). To confirm the majority of stromal cells obtained for this study were of endometriosis origin without contamination, stromal cells derived from peritoneal implant or ovarian endometrioma were induced using 8-bromo-cAMP (0.5 mm) (20). The production of prolactin, a marker of decidualization, was demonstrated in the culture media of stromal cells obtained from ovarian endometriomas. However, peritoneal implants produced prolactin to a lesser degree, possibly due to contamination of fibroblasts (data not shown). Furthermore, endometriotic stromal cells obtained from peritoneal implants were small in quantity for further cell culture. Therefore, in the present study, chocolate cyst linings of the ovaries in patients with endometriosis (n = 8) were a source of endometriotic tissue in the Hamamatsu University Hospital (Shizuoka, Japan) and its related hospitals. Informed consent was obtained before surgical removal of endometriotic tissue. The use of human tissues for this study was approved by Hamamatsu University’s Institutional Review Board and Committee for the Protection of Human Subjects. All tissues were acquired from patients who were not receiving medication for the treatment of endometriosis for at least the preceding 3 months. We used stromal cells in a monolayer culture after the first passage. This procedure resulted in populations of stromal cells of greater than 95% purity. Endometriotic stromal cells were plated in a 100-mm dish, and cultures were allowed to proliferate until 90% confluence.

Cell stimulation

Endometriotic stromal cells were plated in 24-well dishes at a concentration of 1 x 10⁵ cells/well. Monolayer cells pretreated with or without Thal (5 µM) or TPCK (10 µM) were stimulated with TNF- (0.1 ng/ml) and incubated at 37 C for 24 h. At the end of the incubation, supernatants were removed and assayed for cytokines.

In a parallel experiment, cells plated in culture dishes at a concentration of 1 x 10⁶ were preincubated with Thal (5 µM) or TPCK (10 µM) and then stimulated with TNF- (0.1 ng/ml) for 20 min (for Western blot analysis) or 6 h (for Northern blot analysis), respectively.

Determination of cytokines

The concentration of IL-8 in the culture supernatants was determined in duplicate using commercially available IL-8 ELISA kit (TFB, Tokyo, Japan). The assays were performed as described by the manufacturer. Culture supernatants were used at a dilution of 1:2 to 1:100 and measured twice.

3,-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay

To measure cell viability, the MTT assay was performed. The cultured cells (5 x 10⁵ /well) were incubated with MTT (420 µg/ml, Sigma), which is metabolized by living cells in 3 h. MTT crystals were solubilized with HCl-isopropanol (150 µl/well). MTT is cleaved by living cells to yield a dark-blue formazan product. Plates were analyzed in an ELISA plate reader at 570 nm with a reference wavelength of 690 nm.

Nuclear NF-B pull-down assay

Nuclear NF-B pull-down assay was performed as described (21). Endometriotic stromal cells were pelleted and resuspended in hypotonic lysis buffer. The nuclear pellet was extracted, and the supernatants were diluted and incubated with agarose beads conjugated to a consensus NF-B binding oligonucleotide (Santa Cruz Biotechnology). The result was analyzed by SDS-PAGE and Western blotting using an anti-p65 NF-B antibody.

Western blot analysis

The cells treated with or without various agents for indicated times were washed with PBS. Cells (1 x 10⁶) were lysed in 750 µl lysis buffer at 4 C for 15 min and scraped with a rubber policeman. The protein concentrations in the supernatants of cell extracts were measured by the Bio-Rad protein assay (Bio-Rad, Hercules, CA). All samples were stored at –70 C until use. In parallel, cells treated in the same condition in different dishes were harvested and counted using a hemocytometer. Centrifuged lysates (50 µg) were analyzed by SDS-PAGE and transferred to a polyvinylidene difluoride membrane by semidry transfer (Bio-Rad). Membranes were blocked for 1 h at room temperature in Tris-buffered saline containing 0.1% Tween 20 and 2% BSA. Blots were probed with the following primary antibodies overnight at 4 C: phospho-IB and IB were detected by specific primary antibodies and horseradish peroxidase-conjugated secondary antibodies. The immunoblots were visualized by chemiluminescence with the ECL kit from Amersham Biosciences (Tokyo, Japan). Filters were quantitated by scanning densitometry using a Bio-Rad model 620 video densitometer with one-dimensional Analyst software package for Macintosh.

Expression of IL-8 mRNA in endometriotic stromal cells

Total RNA was isolated using TriZol (Invitrogen, Tokyo, Japan) according to manufacturer’s protocol. Total RNA (5 µg/lane) was electrophoresed on a 1% denatured formaldehyde agarose gel, transferred to a nylon membrane (Bio-Rad), and UV cross-linked. The membranes were hybridized with a human radiolabeled IL-8 probe (289 bp), stripped, and subsequently hybridized with a human radiolabeled glyceraldehyde-3-phosphate dehydrogenase (G3PDH) probe (983 bp) (22). For preparation of the hybridization probes, total RNA isolated from endometriotic stromal cells was subjected to RT-PCR by using the Super-Script One-step kit from Invitrogen (Carlsbad, CA), and the corresponding PCR primers were: IL-8, forward, 5'-ATGACTTCCAAGCTGGCCGTGGCT-3' and reverse, 5'-TCTCAGCCCTCTTCAAAAACTTCTC-3'; and G3PDH, forward, 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3' and reverse, 5'-CATGTGGGCCATGAGGTCCACCAC-3'. Total RNA was converted to cDNA according to the kit’s instructions, and the PCR was conducted: denaturation at 94 C for 45 sec, annealing at 60 C for 45 sec, and primer extension at 72 C for 2 min, total of 30 cycles. The resulting PCR products were purified by QIAEX II gel extraction kit (QIAGEN, Valencia, CA). Membranes were prehybridized for 3 h at 55 C and subsequently hybridized overnight at 43 C with random primer [³²P]dCTP-labeled IL-8 cDNA or G3PDH probe (Prime It II random priming kit, Stratagene, La Jolla, CA). After autoradiography, the signals were quantified by densitometric analysis. Equal RNA loading efficiency was determined by visualization of G3PDH. Band intensities were quantitated using a Molecular Imager PhosphorImager (Bio-Rad).

Statistics

Data are expressed as mean ± SD. Statistical analysis was performed by one-way ANOVA followed by Fisher’s protected least significant difference test. P < 0.05 was considered statistically significant.

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
Thal Inhibits TNF--induced NF-B activation in endometriotic stromal cells

The activation of NF-B is usually associated with phosphorylation of IB, followed by its degradation by the proteasome and NF-B nuclear translocation and subsequent activation of target gene expression. Within 10 min of stimulation with TNF-, phosphorylated IB (p-IB) was clearly observed (18). We examined the effect of Thal on the activation of NF-B induced by TNF- in the endometriotic stromal cells.

As shown in Fig. 1A, TNF- treatment caused activation of NF-B as demonstrated by the measures of IB phosphorylation and degradation of IB. Therefore, the level of IB protein decreased as the phosphorylation level of IB protein increased (lane 1 vs. lane 2). Pretreatment of cells with Thal (5 µM) blocked TNF--induced IB phosphorylation by 60% and TNF--induced IB degradation by 65% (lane 4). Thal alone does not affect the expression levels of p-IB and IB (lane 3). Direct inhibition of NF-B with TPCK, an NF-B inhibitor (10 µM, 1 h) (lane 5), blocked TNF--induced IB phosphorylation by 70% and TNF--induced IB degradation by 70%, respectively. Thal did not enhance the TPCK’s action (lane 6). The band intensity values for p-IB were shown in Fig. 1B. This suggests that inhibition of TNF--induced activation by Thal is responsible for the inhibition of NF-B.

View larger version (28K): [in this window] [in a new window]   FIG. 1. Effects of Thal on the level of phosphorylation of IB induced by TNF- in endometriotic stromal cells. A, Confluent (90%) endometriotic stromal cells pretreated with test drugs (Thal, 5 µM, 2 h, lanes 3, 4, and 6; or TPCK, 10 µM, 1 h, lanes 5 and 6) were exposed to medium alone (lanes 1 and 3) or medium with TNF- (0.1 ng/ml, lanes 2 and 4–6) for 20 min. Expression levels of IB and phosphorylated IB (P-IB) were determined by Western blot analysis. B, Blots of P-IB in A were scanned, and the band intensities were quantitated. The band intensity values were used to determine the relative amount of P-IB. The mean and SD of four treatments are presented. *, P < 0.05 compared with lane 2.

TNF--induced nuclear translocation of NF-B was inhibited by pretreatment with Thal

Thal-dependent suppression of TNF--induced nuclear translocation of NF-B was directly examined in endometriotic stromal cells using nuclear NF-B pull-down assays. Nuclear extracts prepared from untreated and TNF--treated cells were incubated with agarose beads conjugated to consensus NF-B oligomers, and nuclear NF-B was assayed by Western blotting using anti-p65 NF-B antibody. As shown in Fig. 2, TNF- activation induced increased nuclear NF-B at a 20-min incubation (lane 3), which was inhibited by pretreatment with either Thal (5 µM, 2 h; lane 4) or TPCK (10 µM, 1 h; lane 5). Thal did not enhance the TPCK’s action (lane 6). Both Thal and TPCK do not cause cell death (data not shown). These results demonstrate that Thal prevents the degradation of the predominant inhibitory molecule, IB, and then inhibits the translocation of NF-B into the nucleus.

View larger version (25K): [in this window] [in a new window]   FIG. 2. Effects of Thal on TNF--induced NF-B nuclear translocation. A, Confluent (90%) cells pretreated with or without Thal (5 µM, 2 h; lanes 4 and 6) or TPCK (10 µM, 1 h; lanes 5 and 6) were exposed to medium alone or medium with TNF- (0.1 ng/ml) for 0 (lane 1), 10 (lane 2), or 20 (lanes 3–6) min. Expression levels of NF-B were determined by Western blot analysis using anti-p65 antibody. D, Blots of NF-B in A were scanned, and the band intensities were quantitated. The band intensity values were used to determine the relative amount of NF-B. The mean and SD of four treatments are presented. Those with unlike superscripts (a–d) are different (P < 0.05).

To further confirm the role of NF-B activation, the effect of TPCK and Thal on TNF--induced IL-8 protein and gene expression was assessed. It has been reported that TPCK significantly suppressed NF-B activation-mediated IL-8 expression (18). In the present study, we compared the levels of IL-8 protein production in endometriotic stromal cells pretreated with or without Thal (5 µM). As we expected, pretreatment with Thal reduced TNF--induced IL-8 protein production (Fig. 3A, lane 5 vs. lane 4) as well as mRNA expression (Fig. 3B, lane 5 vs. lane 4). Neither Thal alone (lane 2) nor TPCK alone (lane 3) did not reduce IL-8 production. Thal failed to further strengthen the TPCK’s action (lane 7 vs. lane 6).

View larger version (23K): [in this window] [in a new window]   FIG. 3. Effects of Thal on TNF--induced IL-8 protein production and IL-8 mRNA expression. A, Confluent (90%) endometriotic stromal cells were preincubated without or with Thal (5 µM, 2 h; lanes 2, 5, and 7) or TPCK (10 µM, 1 h; lanes 3, 6, and 7), and then TNF- (0.1 ng/ml; lanes 4–7) was added and incubated for 24 h. Concentrations of IL-8 in the supernatants were measured by ELISA. The minimum detectable level of IL-8 was 10 pg/ml. B, Cells were preincubated without or with Thal (5 µM, 2 h; lanes 2, 5, and 7) or TPCK (10 µM, 1 h; lanes 3, 6, and 7) and incubated in the presence of TNF- (0.1 ng/ml; lanes 4–7) for 6 h. Total RNA was isolated from the cells and analyzed by Northern blot analysis. Total RNA (5 µg) was size-fractionated on 1% formaldehyde-agarose gels, transferred to a nylon membrane, and then hybridized to an IL-8 probe. The visualization of G3PDH was used for normalization. The mean and SD of four treatments are presented. Bars represent SD. *, P < 0.05 vs. the addition of TNF- (lane 4).

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

TNF- was first identified as a cytokine secreted by endotoxin-activated macrophages (23). Sakamoto et al. (18) also demonstrated that TNF- promoted the production of IL-8 in endometriotic tissue and that NF-B activation has been involved in the induction of IL-8 in endometriotic tissues. They reported (18) that IL-8 is produced in the human endometrial stromal cells and further demonstrated that IL-8 exerts its growth-promoting actions in normal endometrium as well as in endometriotic cells (8, 9). Thus, TNF- may play a central role as a key cytokine that agitates many other cytokines in the peritoneal cavity of endometriosis patients. We confirmed the previous report (18) demonstrating that TNF- induced the expression of phosphorylated IB and degradation of IB, that phosphorylation of IB and its degradation are essential for liberation of NF-B from binding with IB, and that adding TNF- induced IL-8 gene and protein expression in endometriotic stromal cells, through nuclear translocation of NF-B.

In terms of treatment for endometriosis, GnRHa agonist is effective, reducing endometrial implants in 50–90% of patients and improving pain and physical findings in 75% of cases (24). This, in turn, produces a fall in the estrogen concentration, with resultant amenorrhea. Inconsistent with patients who received GnRHa agonist treatment, the addition of Thal may not exert a significant effect on endocrine system, including estrogen production. Our in vitro experiments suggest a direct action of Thal on endometriotic stromal cells. Although there are no data about changes in endocrine system after Thal treatment, we speculate that Thal treatment may not have any effects on secretion of estrogen and progesterone. A more recent paper (25) reported that Thal may induce hypergonadotropic amenorrhea. Although this effect appears to be reversible, the long-term effect of Thal on ovarian reserve is unclear.

Endometriosis is considered to be an inflammatory-like phenomenon (1). In the present study, we show that Thal inhibited TNF--induced up-regulation of IL-8 expression, possibly through suppression of NF-B activation in endometriotic stromal cells. To our knowledge, this is the first report that IL-8 expression was directly attenuated in endometriotic cells after treatment with Thal.

We could not test whether ectopic endometrial tissue from within the pelvis (peritoneal endometriosis) is the same as endometriotic tissue from within the ovary. We must distinguish endometriosis cells obtained from ovarian endometriomas vs. peritoneal implants. The latter is perhaps more common and not used in this study. With the retrograde menstruation theory of endometriosis, the endometriotic tissue surely implants in the pelvis and on the surface of the ovary. Therefore, the source we used may be representative of endometriosis at all sites in the pelvis. It is unclear, however, whether the results are able to be extrapolated as widely as we suggest.

Notwithstanding these limitations, the data provide a new therapeutic option that Thal treatment may reduce the ability of cytokine production in endometriotic cells and thus lead to suppression of its progression. This drug is a known, predictable teratogen (10) and should therefore not be used in women of reproductive age or used only with adequate contraception.

	Footnotes

 
This work was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan (to H.K.), by grants from the Fuji Foundation for Protein Research (to H.K.), by the Kanzawa Medical Foundation (to H.K.), by the Sagawa Cancer Research Foundation (to H.K.), and by the Aichi Cancer Research foundation (to H.K).

First Published Online February 1, 2005

Abbreviations: G3PDH, Glyceraldehyde-3-phosphate dehydrogenase; MTT, 3,-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; NF-B, nuclear factor-B; p-IB, phosphorylated inhibitor B; Thal, thalidomide; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone.

Received October 2, 2004.

Accepted January 25, 2005.

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