Yuri Takemura

Recruitment of CCR6-Expressing Th17 Cells by CCL 20 Secreted from IL-1β-, TNF-α-, and IL-17A-Stimulated Endometriotic Stromal Cells

In a novel paradigm of T cell differentiation, type 17 T helper (Th17) cells may play a significant role in endometriosis, a chronic inflammatory disease. However, the mechanism regulating the accumulation of Th17 cells in endometriotic tissues remains unknown. We hypothesized that Th17 cells migrate to endometriotic tissues through an interaction of the chemokine CC chemokine ligand (CCL)20 and its receptor CCR6. Using endometriotic tissues from women with endometriosis, we demonstrated, by flow cytometry, that Th17 cells in endometriotic tissues express CC chemokine receptor (CCR)6. Immunohistochemistry also revealed that CCL20 was expressed in the epithelial cells and stromal cells beneath the epithelium of endometriotic tissues. CCR6+ cells were small and round and scattered in the stroma in which abundant CCL20+ cells were detected. CCL20 caused selective migration of Th17 cells in the peripheral blood in a migration assay. IL-1β, TNF-α, and IL-17A increased the secretion of CCL20 in cultured endometriotic stromal cells. Inhibitors of p38- and p42/44-MAPKs, and stress-activated protein kinase/c-Jun kinase suppressed the secretion of CCL20 increased by IL-1β, TNF-α, and IL-17A. This suggests that the CCL20/CCR6 system is involved in the migration of Th17 cells to endometriotic tissues and that proinflammatory cytokines contribute to the development of endometriosis via up-regulation of CCL20 secretion from endometriotic stromal cells.

Interleukin-4 stimulates proliferation of endometriotic stromal cells.

Several lines of evidence indicate that the Th2 immune response is associated with endometriosis. Although an increased concentration of interleukin (IL)-4, a typical Th2 cytokine, has been reported in endometriotic tissues, the implication of this for endometriosis has not been determined. To investigate a possible role of IL-4 in the development of endometriosis, we examined the presence of IL-4-producing cells in endometriotic tissues and the effect of IL-4 on proliferation of endometriotic stromal cells. Endometriotic stromal cells were isolated from endometriotic tissues obtained from women undergoing surgery for endometrioma. Immunohistochemistry of endometriotic tissues revealed that IL-4-positive cells were abundant in the stroma. The effect of IL-4 on proliferation of endometriotic stromal cells was studied using cell counting and BrdU incorporation assays. IL-4 (0.1 to 10 ng/ml) significantly increased cell number and BrdU incorporation in a dose-dependent manner, and the proliferative effect of IL-4 was inhibited by anti-IL-4 receptor antibody. IL-4-induced activation of mitogen-activated protein kinases in endometriotic stromal cells was examined by Western blotting. IL-4 induced phosphorylation of p38 mitogen-activated protein kinase, stress-activated protein kinase/c-Jun kinase, and p42/44 mitogen-activated protein kinase and inhibitors of these kinases suppressed IL-4-induced proliferation of endometriotic stromal cells. These findings suggest that proliferation of endometriotic stromal cells induced by locally produced IL-4 is involved in the development of endometriosis.

The Expression and Possible Roles of Chemokine CXCL11 and Its Receptor CXCR3 in the Human Endometrium

IFN-γ secreted by a human embryo and trophoblast cells during implantation is suggested to play an important role in implantation and pregnancy. In the present study, we explored expression and possible functions of CXCL11, a CXC chemokine strongly induced by IFN-γ, and its receptor CXCR3 in the human endometrium. Secreted CXCL11 protein was not detected in cultured endometrial stromal cells (ESC) but was detected in cultured endometrial epithelial cells (EEC). IFN-γ stimulated the protein levels of CXCL11 in a dose-dependent manner in EEC and ESC. CXCL11 secreted from EEC with 100 ng/ml IFN-γ was 220-fold of the control, and 100-fold as compared with that secreted from ESC with the same dose of IFN-γ. CXCR3 was expressed in EEC, ESC, and trophoblast cells. Addition of IFN-γ to EEC increased the chemotactic activity of its culture medium to trophoblast cells and T cells, and the effect was suppressed by immunoneutralization with Abs of three CXCR3 ligands, including anti-CXCL11 Ab. CXCL11 significantly increased BrdU incorporation of ESC, which was inhibited by a p42/44 MAPK pathway inhibitor PD98059. In contrast, CXCL11 significantly decreased BrdU incorporation and increased the release of lactate dehydrogenase and the positive staining of annexin V in EEC. These findings suggest that IFN-γ promotes implantation by stimulating EEC to produce CXCL11, which induces migration of trophoblast cells and T cells, proliferation of ESC, and apoptosis of EEC.

Interleukin (IL)-1β Stimulates Migration and Survival of First-Trimester Villous Cytotrophoblast Cells through Endometrial Epithelial Cell-Derived IL-8

IL-1, secreted by human embryos and trophoblast cells, is important for successful implantation and pregnancy. We previously reported that IL-1beta induced IL-8 production in human endometrial stromal cells (ESCs) and that induction was regulated by substances implicated in implantation. In the present study using human primary cells in culture, we measured IL-1beta-induced production of IL-8 from endometrial epithelial cells (EECs) and ESCs and examined effects of the endometrium-derived IL-8 on migration and number of first-trimester villous cytotrophoblast cells (vCTs). Both basal and IL-1beta-induced IL-8 levels of cell supernatants were much higher in EECs than ESCs. Addition of IL-1beta to EECs increased the chemotactic activity of the supernatants to vCTs, and this effect was suppressed by immunoneutralization with anti-IL-8 antibody. Supernatants of IL-1beta-stimulated EECs yielded significantly higher number of vCTs compared with those of untreated EECs, and the effect was inhibited by IL-8 antibody. These findings suggest that IL-1 promotes implantation by stimulating EECs to produce IL-8, which subsequently induces migration of vCTs and contributes to survival of vCTs.

Increased risk of placenta previa is associated with endometriosis and tubal factor infertility in assisted reproductive technology pregnancy

Although assisted reproductive technology (ART) is suspected to increase the risk of placenta previa, a life-threatening complication of pregnancy, the reason is poorly understood. We recruited consecutive 318 pregnancies conceived by ART in our clinic and examined relation of ten variables, i.e. maternal age, gravidity, parity, male or female fetus, previous abortion, previous cesarean delivery, endometriosis, ovulatory disorder, tubal disease, and male infertility, to placenta previa, by logistic regression analysis. As a result, we found that endometriosis (odds ratio = 15.1; 95% CI = 7.6–500.0) and tubal disease (odds ratio = 4.4; 95% CI = 1.1–26.3) were significantly associated with placenta previa. It would be preferable to take the increased risk of placenta previa into account in treating ART pregnancy with endometriosis and tubal disease.

High mobility group box 1 (HMGB1) levels in the placenta and in serum in preeclampsia.

Citation 
Wang B, Koga K, Osuga Y, Hirata T, Saito A, Yoshino O, Hirota Y, Harada M, Takemura Y, Fujii T, Taketani Y. High mobility group Box 1 (HMGB1) levels in the placenta and in serum in preeclampsia. Am J Reprod Immunol 2011; 66: 143–148

Dienogest, a new conservative strategy for extragenital endometriosis: a pilot study

Extragenital endometriosis severely impairs the quality of life for affected women but its standard management has not yet been well established because of its relatively low incidence. As extragenital organs, intestine, followed by urinary tract, is the most common place affected by endometriosis, for which surgical treatment is sometimes difficult and accompanied by severe complications. Recently, dienogest, a novel progestin, has emerged as a new alternative for endometriosis, especially for endometriosis-associated pain. In this report, we presented four cases with rectosigmoidal and one with bladder endometriosis, treated with oral 2 mg/day dienogest for over 6 months. For all cases, the measurable extragenital lesions exhibited the reduction in their size after 10 to 11 months of use, accompanied with immediate relief of subjective symptoms related with extragenital lesions. This report suggests that dienogest can be a novel conservative alternative for extragenital endometriosis.

Concentration of Adiponectin in Peritoneal Fluid is Decreased in Women with Endometriosis

Adiponectin is a novel adipocytokine of extreme importance for the metabolism. Recent studies have indicated that adiponectin suppresses inflammation, angiogenesis, and fibrosis, which are central pathogenic factors for endometriosis. We addressed the possibility that adiponectin is implicated in endometriosis.

TGF-β1 induces proteinase-activated receptor 2 (PAR2) expression in endometriotic stromal cells and stimulates PAR2 activation-induced secretion of IL-6

BACKGROUND Proteinase-activated receptor 2 (PAR2) is a G-protein-coupled receptor that is activated by several serine proteases. PAR2 activation in endometriotic stromal cells (ESCs) has been implicated in the development of endometriosis but the regulatory mechanism of PAR2 expression in ESC is unknown. Our objective was to study the mechanism by which PAR2 expression may be regulated in endometriotic lesions. METHODS Primary cultures of ESCs were treated with transforming growth factor-β (TGF-β) 1, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), and the expression of PAR2 was examined by real-time quantitative PCR. ESCs pretreated with or without TGF-β1 were treated with PAR2 agonist peptide (PAR2AP) and the secretion of the pro-endometriotic cytokine, IL-6, was measured using a specific enzyme-linked immunosorbent assay. Effects of TGF-β type 1 inhibitor, SB431542, and PAR2 small interfering RNA (siRNA) on the TGF-β1 stimulation of PAR2 gene expression and PAR2AP-induced IL-6 secretion were also evaluated. To study intracellular signaling, effects of inhibitors of mitogen-activated protein kinases (MAPKs) and phosphoinositide 3-kinase (PI3K) and of Smad4 siRNA on the TGF-β1-induced PAR2 gene expression were studied. RESULTS Only TGF-β1, but neither TNF-α nor IL-1β, increased gene expression of PAR2. Activation of PAR2 with PAR2AP increased the secretion of IL-6 from ESCs. As expected, TGF-β1 pretreatment dose-dependently enhanced the PAR2AP-induced increase in IL-6 secretion from ESCs. Treatment of ESCs with the TGF-β type 1 inhibitor, SB431542, inhibited both TGF-β1-stimulation of PAR2 gene expression and PAR2AP-induced IL-6 secretion. Transfection of ESCs with PAR2 siRNA produced a similar inhibition of IL-6 secretion. The TGF-β1-induced increase in PAR2 gene expression was repressed by inhibition of p38 MAPK, p42/44 MAPK or PI3K, but not by knockdown of Smad4 expression. CONCLUSIONS In view of significant roles of PAR2 and IL-6 in endometriosis, the TGF-β1-induced increase in PAR2 expression may be an elaborate mechanism that augments the progression of the disease.

Progesterone decreases bone morphogenetic protein (BMP) 7 expression and BMP7 inhibits decidualization and proliferation in endometrial stromal cells

BACKGROUND Regulation of decidualization is decisive for proper implantation and the establishment of pregnancy. Recent studies have suggested that several bone morphogenetic proteins (BMPs) play physiological roles in reproduction. In the present study, we examined the expression of BMP7 in the endometrium and the effect of BMP7 on decidualization and proliferation of endometrial stromal cells (ESC). METHODS The gene expression of BMP7 in endometrial tissues collected from women with regular menstrual cycles was determined and the effect of ovarian steroid hormones on BMP7 gene expression was investigated in cultured ESC. The effect of BMP7 on the decidualization of ESC was determined by measuring the gene expression and protein secretion of insulin-like growth factor binding protein 1 (IGFBP1), a marker of decidualization. The effect of BMP7 on the proliferation of ESC was examined by the bromodeoxyuridine (BrdU) incorporation assay. RESULTS The gene expression of BMP7 in endometrial tissues was low at and after the mid-secretory phase of the menstrual cycle. Progesterone suppressed the gene expression of BMP7 in cultured ESC. Treatment with progesterone and estradiol for 12 days achieved decidualization of ESC, increasing the gene expression and protein secretion of IGFBP1. Addition of BMP7 protein to the culture almost completely inhibited these increases. BMP7 suppressed BrdU incorporation in ESC, which indicated an antiproliferative effect of BMP7 on ESC. CONCLUSIONS Progesterone-induced suppression of BMP7 and BMP7-induced inhibition of decidualization and proliferation of ESC suggest an elaborate regulatory mechanism for decidualization through BMP7 in the endometrium.