Expression of epithelial neutrophil-activating peptide 78 in cultured human endometrial stromal cells

It has been demonstrated that human endometrial stromal cells (ESC) produce a variety of chemokines in vivo and in vitro. To evaluate the expression of epithelial neutrophil-activating peptide 78 (ENA-78) in the endometrium, concentrations of ENA-78 in cyclic endometrial tissues were measured using enzyme-linked immunosorbent assay. The expression of ENA-78 was also detected in cyclic endometrium by immunohistochemistry. Endometrial tissues in the secretory phase contained higher amounts of ENA-78 protein than did those in the proliferative phase. Immunofluorescence staining revealed that ENA-78 protein was localized mainly in the stroma of endometrium. In addition, to evaluate the involvement of inflammatory mediators and ovarian steroid hormones in the production of ENA-78 by ESC was evaluated by in-vitro studies. Unstimulated ESC constitutively secreted ENA-78. Progesterone, lipopolysaccharide, tumour necrosis factor-alpha, and interleukin-1beta significantly stimulated the expression of ENA-78 by ESC. It is suggested that the production of ENA-78 by ESC is regulated by progesterone as well as by the inflammatory mediators. The modulation of ENA-78 concentration in the local environment by these mediators may contribute to the normal and pathological processes of human reproduction through regulation of leukocyte trafficking into the endometrium.     

Expression of interferon-gamma-inducible protein-10 in human endometrial stromal cells

Human endometrial stromal cells (ESC) can produce a variety of chemokines, especially after inflammatory stimulation. Interferon-gamma-inducible protein-10 (IP-10) is a potent chemoattractant for lymphocytes, and belongs to the family of non-ELR CXC chemokines. The expression of IP-10 in ESC after stimulation with interferon-gamma (IFN-gamma), interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha), or lipopolysaccharide (LPS) was evaluated using an enzyme-linked immunosorbent assay and Northern blot analysis. A small amount of IP-10 protein was detected in the culture media of unstimulated ESC. The expression of IP-10 mRNA was detected in ESC. IFN-gamma, IL-1 beta, TNF-alpha and LPS significantly stimulated the expression of IP-10 mRNA and protein in ESC. These results suggest that the production of IP-10 by ESC is regulated by inflammatory mediators. The modulation of IP-10 concentrations in the local environment may contribute to the normal and pathological processes of human reproduction by regulating leukocyte trafficking in the endometrium.     

Caspase cascade of Fas-mediated apoptosis in human normal endometrium and endometrial carcinoma cells

Human endometrial epithelial cells undergo apoptosis immediately before the menstrual period. Apoptotic signalling was analysed using human endometrial tissue and a human endometrial carcinoma cell line (HHUA). Activity levels of caspase-3, -8, and -9 were elevated in human endometrium during the late secretory phase and in HHUA cells incubated with an anti-Fas monoclonal antibody (mAb). Fas-mediated apoptosis of HHUA cells was blocked by prior exposure to inhibitors of caspase-9, -8 and -3. In HHUA cells treated with anti-Fas mAb, a release of cytochrome c was detected in the cytosolic fraction, in addition a full-length Bid was degraded. Full-length FLIP(L) (p55) was degraded during apoptosis, and p29 (regarded as the product of p55 cleavage) appeared instead of FLIP(L). In normal human endometrial tissue, Bid degradation was also observed in a cyclic manner with a peak during the early secretory phase of the menstrual cycle. Furthermore, the release of cytochrome c was seen in the early secretory phase. However, expression of FLIP(S) was only observed during the menstrual cycle in normal endometrial tissue. We concluded that the main apoptotic signalling in both normal human endometrial tissue and HHUA cells exposed to anti-Fas mAb is the mitochondrial pathway via Bid degradation. Although the function of FLIP is still unknown on normal endometrial tissue, it may be regulated by FLIP(L) expression on HHUA cells derived from human endometrial carcinoma.     

A quantitative evaluation of alpha1, alpha4, alphaV and beta3 endometrial integrins of fertile and unexplained infertile women during the menstrual cycle. A flow cytometric appraisal.

The expression of integrin molecules alpha1beta1, alpha4beta1 and alphaVbeta3 within endometrial tissue has been proposed as a marker of uterine receptivity during the implantation window. The present investigation examines by flow cytometric analysis the concentrations of alpha1, alpha4, alphaV and beta3 integrin subunits in endometrial stromal (ESC) and epithelial cells (EEC) in two groups of women throughout the menstrual cycle: normal fertile women (n = 27) and women with unexplained infertility (n = 26). Integrin concentrations in endometrial cells were calculated in relative fluorescence units against a negative cellular control. The assessment of integrin subunits detected the protein in ESC and EEC from the late proliferative to the late secretory phase. In both groups of women, the alpha1 was the highest integrin expressed in ESC and EEC throughout the menstrual cycle. All women exhibited low concentrations of alpha4-EEC at the time of the implantation window. Infertile women expressed lower concentrations of the alpha4-ESC during the proliferative and early secretory phase while lower concentrations of the alpha1-ESC were seen during the late secretory phase. Interestingly, the infertile women expressed lower concentrations of beta3-EEC in the early, mid-secretory and late secretory phases (P < 0.05). Infertile women also expressed lower concentrations of alpha1-EEC and alphaV-EEC during the late secretory phase (P < 0.05). It can be concluded that the quantitative determination of beta3-EEC by flow cytometry confirmed its potential feature as a marker of endometrial receptivity at the time of the implantation window. In addition, the defective expression of the alpha1-ESC found in the late secretory phase might be associated with the poor fertility outcome of women with unexplained infertility.     

Effects of interferon-gamma on cytokine production by endometrial stromal cells

To clarify the role of interferon-gamma (IFN-gamma) in reproduction, we have examined its effects on cytokine production by human endometrial stromal cells (ESC). Concentrations of interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1 (MCP-1), and macrophage colony- stimulating factor (M-CSF) in the culture media of normal ESC and an endometrial stromal sarcoma cell line, MaMi, were measured using an enzyme-linked immunosorbent assay. Both non-stimulated ESC and non- stimulated MaMi cells constitutively secrete IL-6, IL-8, MCP-1, and M- CSF. In a dose-dependent manner, IFN-gamma increased the concentrations of IL-6, MCP-1, and M-CSF and reduced the concentrations of IL-8 in ESC and MaMi cells. These results suggest that IFN-gamma produced by both decidual inflammatory cells and the developing embryo plays a role in the maintenance of early pregnancy by modulating the production of these cytokines by human ESC.      

17β-雌二醇和二噁英对子宫内膜间质细胞表达趋化因子IL-8及其受体CXCR1的调控作用

解析内外环境因素对子宫内膜间质细胞表达IL-8及其自分泌作用的调控。采用免疫组化法比较子宫内膜异位症患者异位灶和在位内膜CXCR1翻译水平表达;流式细胞术分析17β-雌二醇和二噁英单独或联合作用对子宫内膜间质细胞表面CXCR1表达的调控作用;ELISA法分析17β-雌二醇和二噁英单独或联合作用对子宫内膜间质细胞分泌IL-8的影响。结果显示CXCR1在子宫内膜异位症患者异位灶组织高表达。17β-雌二醇和二噁英单独作用均抑制子宫内膜间质细胞表面CX-CR1的表达以及IL-8的分泌。二者联合作用能够上调CXCR1的表达,上调幅度与雌二醇浓度呈正相关;但进一步抑制了IL-8的分泌。雌激素与二噁英对子宫内膜间质细胞复合作用抑制其IL-8的分泌及其自分泌作用;子宫内膜异位症患者腹腔液高水平IL-8并非由内外雌激素样物质直接作用于异位灶子宫内膜间质细胞所致。     

Leptin regulates the proliferation and apoptosis of human endometrial epithelial cells

The biological functions of leptin in the human endometrial epithelium were investigated using the human endometrial epithelial cell line, HHUA. Specifically, the effects of leptin on the proliferation and apoptosis of HHUA cells induced by treatment with anti-Fas IgM or anticancer drugs were examined. RT-PCR detected the expression of four leptin receptor isoform mRNAs in the cells and flow cytometric analysis revealed cell surface expression of the leptin receptor molecules. Leptin stimulated HHUA cell proliferation in a dose-dependent manner at concentrations below the normal serum leptin level. Leptin enhanced anti-Fas IgM-mediated growth inhibition and DNA fragmentation, but did not enhance the expression of either Fas antigen or Fas ligand. Moreover, leptin had no effect on anticancer drug-induced apoptosis. Based on these results, leptin at a physiological serum concentration, may regulate the remodeling of the human endometrial epithelium by stimulating cell proliferation and enhancing the Fas-specific intracellular apoptotic signaling pathway.     

Expression and function of interleukin-11 and its receptor alpha in the human endometrium

The interleukin-11 (IL-11) receptor alpha has an important function in decidualization of mouse endometrial stroma but the function of IL-11 and its receptor in the human endometrium remains unknown. The mRNA for IL-11 and its receptor alpha in human endometrial tissue samples were analysed by semi-quantitative RT-PCR and RNase protection assays respectively. The proteins were detected in frozen endometrial tissue samples by immunofluorescence. The effect of heparin-binding epidermal growth factor (HB-EGF) on secretion of IL-11 by cultured endometrial stromal cells was assessed by enzyme-linked immunosorbent assay. The proliferative potential of IL-11 in endometrial stromal cells was assessed by [(3)H]thymidine uptake. IL-11 and its receptor alpha mRNAs and proteins were detected in the endometrium throughout the cycle. Distinct patterns of localization of the ligand and receptor were observed. HB-EGF induced IL-11 secretion by cultured stromal cells, and IL-11 induced [(3)H]thymidine uptake by these cells. Our data suggest that IL-11-receptor interactions may perform different functions in the human endometrium at different stages of the cycle, and that secretion of IL-11 is modulated by local growth factors.     

Human chorionic gonadotropin and growth factors at the embryonic-endometrial interface control leukemia inhibitory factor (LIF) and interleukin 6 (IL-6) secretion by human endometrial epithelium

BACKGROUND: The elucidation of the molecular mechanisms by which the embryo contributes to its implantation is an area of extensive research. The main objective of this study was to investigate the pattern of leukemia inhibitory factor (LIF) and interleukin-6 (IL-6) secretion by human endometrial epithelium, and their regulation by human chorionic gonadotropin (hCG) and other growth factors present at the embryonic-endometrial interface. METHODS: Endometrial epithelial cells (EEC) were isolated from biopsies collected at both proliferative and secretory phases of fertile women. RESULTS: HCG (1-50 IU/ml) increased LIF secretion by EEC cultures derived from follicular phase (up to 285+/-75%) or from secretory phase (up to 212+/-16%). In contrast, hCG reduced IL-6 secretion by EEC in both phases. The hCG/LH receptor gene was transcribed by EEC as evidenced by RT-PCR. Insulin-like growth factors 1 and 2 increased LIF secretion by EEC. Transforming growth factor beta1 stimulated LIF and reduced IL-6 secretion. CONCLUSIONS: Through hCG, the blastocyst may be involved in the control of its implantation (via an increase of proimplantatory LIF) and tolerance (via an inhibition of proinflammatory IL-6). Other growth factors present at the embryonic-endometrial interface are also involved in the control of LIF and IL-6 endometrial secretion.     

Expression of aminopeptidase N and neutral endopeptidase on the endometrial stromal cells in endometriosis and adenomyosis.

Indirect immunofluorescence staining revealed that endometrial stromal cells (ESC) in the ectopic endometrium of patients with endometriosis or adenomyosis expressed aminopeptidase N/cluster of differentiation (CD) 13 antigen and neutral endopeptidase/CD10 antigen, both of which are expressed on ESC in the normal endometrium throughout the menstrual cycle. Thus, ESC in the ectopic endometrium resembled ESC in the normal endometrium not only morphologically but also antigenically. Both peptidase antigens may be useful markers for the histological diagnosis of endometriosis and adenomyosis.     

Differential regulation of interleukins IL-13 and IL-15 by ovarian steroids, TNF-alpha and TGF-beta in human endometrial epithelial and stromal cells

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 17beta 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-alpha (TNF-alpha; 10 and 25 ng/ml) and transforming growth factor-beta (TGF-beta; 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-alpha and TGF-beta 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.     

Activated platelets induce secretion of interleukin-1beta, monocyte chemotactic protein-1, and macrophage inflammatory protein-1alpha and surface expression of intercellular adhesion molecule-1 on cultured endothelial cells

Atherosclerosis is an inflammatory disease. Platelet-endothelium interaction plays an important role in the pathophysiology of atherogenesis. We investigated the role of activated platelets for secretion of interleukin (IL)-1beta, monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-1alpha and expression of intercellular adhesion molecule (ICAM)-1 on endothelial cells. Human umbilical vein endothelial cells (HUVEC) were incubated with non-stimulated or ADP-activated platelets for 6 hr. Secretion of interleukin (IL)-1beta, MCP-1 and MIP-1alpha and surface expression of ICAM-1 were measured by ELISA and flow cytometry. In the presence of activated platelets, the secretion of IL-1beta, MCP-1, and MIP-1alpha and surface expression of ICAM-1 were significantly increased compared with non-activated platelets. The present study shows that activated platelets may contribute to expression of various inflammatory mediators on endothelial cells.