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.     

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。     

The effect of inflammatory cytokines on secretion of macrophage colony-stimulating factor and monocyte chemoattractant protein-1 in human granulosa cells

PROBLEM: In order to investigate the role of macrophage colony-stimulating factor (M-CSF) and monocyte chemoattractant protein -1 (MCP-1) in human ovulation, we studied the regulation of M-CSF and MCP-1 in cultured human granulosa cells. METHOD OF STUDY: Immortalized granulosa cells (GC1a) were cultured in serum-free medium, and incubated with interleukin (IL)-1alpha, IL-1 receptor antagonist (ra) and tumor necrosis factor (TNF)-alpha. The supernatants were collected, and M-CSF and MCP-1 were measured by ELISA. RESULTS: The levels of M-CSF and MCP-1 were increased after treatment with IL-1alpha (1 nm) and TNF-alpha (1 nm) in a time-dependent manner. The levels of M-CSF and MCP-1 were significantly increased after treatment with IL-1alpha and TNF-alpha in a dose-dependent manner. However, the levels of M-CSF and MCP-1 were significantly decreased by treatment with IL-1alpha (1 nm) and/or increasing concentrations of IL-1 ra. CONCLUSIONS: Our data indicated that M-CSF and MCP-1 were regulated by IL-1alpha and TNF-alpha. It was suggested that M-CSF and MCP-1 may play an important role in human preovulatory processes.     

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CXCRI翻译水平表达;流式细胞术分析17β-雌二醇和二嗯英单独或联合作用对子宫内膜间质细胞表面CXCRI表达的调控作用;ELISA法分析17β-雌二醇和二嗯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CXCRI在子宫内膜异位症患者异位灶组织高表达。17β-雌二醇和二嗯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。雌激素与二嗯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。     

Hormonal and embryonic regulation of chemokines IL-8, MCP-1 and RANTES in the human endometrium during the window of implantation

Chemokines are a family of small polypeptides which specialize in the attraction of leukocytes. The presence of specific leukocyte subsets at the implantation site is an important element of the complex, and not completely understood, process of embryonic implantation. This report includes the investigation of the in-vivo immunolocalization and hormonal regulation of interleukin (IL)-8, monocyte chemotactic protein (MCP)-1 and RANTES (regulated upon activation normal T-cell expressed and secreted) in the human endometrium during hormone replacement therapy cycles for oocyte recipients in an IVF programme. In addition, we have analysed the embryonic regulation of these endometrial epithelial chemokines (IL-8 and MCP-1) using an in-vitro model for the apposition phase of human implantation by co-culturing single human embryos until the blastocyst stage with human endometrial epithelial cells (EEC). IL-8 and MCP-1 were immunolocalized in the human endometrium to the glandular and lumenal epithelium as well as to the endothelial cells. RANTES was mainly localized to the stromal compartment and endothelial cells. The immunoreactive levels of endometrial IL-8 and MCP-1 were up-regulated by the administration of progesterone during the receptive phase of the cycle. Furthermore, it was demonstrated that, in vitro, the human blastocyst does not produce measurable amounts of IL-8, MCP-1 or RANTES; however, it does up-regulate EEC IL-8 mRNA expression (P < 0.05) and protein production (P < 0.05), but not IL-8 secretion. The human embryo did not regulate EEC MCP-1 expression. These results provide evidence of hormonal and embryonic regulation of specific endometrial chemokines, suggesting two different but related mechanisms to induce the production of chemokines by the EEC, thus contributing to the attraction of specific leukocyte populations during the peri-implantation phase.     

Interleukin 11 advances progesterone-induced decidualization of human endometrial stromal cells

Differentiation of endometrial stromal cells into decidual cells is crucial for embryo implantation and placentation. Interleukin (IL)-11 signalling is essential for adequate decidualization in the mouse uterus. We examined the role of IL-11 during progesterone-induced decidualization of human endometrial stromal cells over a 10-12 day period, using prolactin (PRL) production as a decidual marker. These cells produced biologically active IL-11 and expressed IL-11, IL-11Ralpha and PRL mRNA during decidualization. Neutralization of endogenous IL-11 with an anti-human (hu)IL-11 antibody (AB) reduced production of PRL from day 8 and insulin-like growth factor binding protein (IGFBP)-1, another marker of decidualization, from day 10 of culture. Following AB washout, PRL and IGFBP-1 secretion increased. Addition of recombinant (r)huIL-11 (10 or 100 ng/ml) to endometrial stromal cells increased secretion of PRL from day 4 and IGFBP-1 from day 6 compared with progesterone alone. Morphological signs of differentiation accompanied biochemical differentiation in the progesterone-treated cells and were further induced by exogenous rhuIL-11. Our observations demonstrate that human endometrial stromal cells produce biologically active IL-11, which promotes progesterone-induced decidualization. These results suggest that IL-11 has both paracrine and autocrine actions on human endometrial stromal cells and plays an important role in preparing the human endometrium for implantation.     

Regulation of the cellular subpopulation ratios of normal human endometrial stromal cells by macrophage colony-stimulating factor

Normal human endometrial stromal cells (ESCs) stimulated with 8-Br-cAMP secrete significantly large amounts of PRL and granulocyte colony-stimulating factor (G-CSF), whereas unstimulated stromal cells secreted little. However, there is no relation between PRL and G-CSF levels secreted from the stimulated cells, which suggests that PRL-secreting stromal cells may not completely coincide with the G-CSF-producing stromal cells. Recently we found that macrophage colony-stimulating factor (M-CSF) inhibits 8-Br-cAMP-induced decidualization, differentiation to prolactin (PRL)-secreting cells, by suppressing viable decidualizing cells. Therefore, we investigated the effects of M-CSF on G-CSF secretion from normal human endometrial stromal cells using an in vitro decidualization activity assay. M-CSF did not show any significant effects on viable cell numbers of unstimulated ESCs, while M-CSF dose-dependently enhanced G-CSF release from the non-decidualized stromal cells. A high concentration of M-CSF strongly suppressed the viable cell numbers and PRL secretion of stromal cells co-stimulated with 8-Br-cAMP and M-CSF, although G-CSF release from the co-stimulated stromal cells was not affected by M-CSF. Moreover, M-CSF did not affect viable cell numbers, PRL secretion or G-CSF secretion of 8-Br-cAMP-stimulated cells. These results indicate that M-CSF enhances G-CSF secretion from 8-Br-cAMP-unstimulated human endometrial stromal cells but not from 8-Br-cAMP-stimulated stromal cells, thus suggesting that there exists a functional subpopulation of G-CSF-secreting stromal cells that are different from the predecidualized ESCs that differentiate into PRL-secreting ESCs under stimuli with 8-Br-cAMP. Hence, M-CSF may autoregulate functional cellular subpopulations of human endometrial stromal cells in an autocrine or a paracrine manner.     

The production and clinical evaluation of macrophage colony-stimulating factor and macrophage chemoattractant protein-1 in human follicular fluids

PROBLEM: In order to investigate the role of macrophage colony-stimulating factor (M-CSF) and macrophage chemoattractant protein-1 (MCP-1) in human ovulation, we measured the concentrations of M-CSF and MCP-1 in human follicular fluids (FFs) and correlated them with oocyte maturation. METHOD OF STUDY: The oocytes were obtained from the FFs of 46 women undergoing in vitro fertilization and embryo transfer (IVF ET). The concentrations of M-CSF and MCP-1 in the FFs were measured by enzyme-linked immunosorbent assay. In addition, granulosa cells obtained from the FFs of IVF patients were cultured and treated with forskolin and 12-O-tetradecanoylphorbol 13-acetate (TPA) for 24 48 hr. RESULTS: Concentrations of M-CSF and MCP-1 were significantly higher in the FFs than in the serum (P < 0.01). M-CSF concentrations tended to be higher, while MCP-1 concentrations were significantly higher in the FFs containing mature oocytes than in FFs containing immature oocytes (P < 0.05). The production of M-CSF was markedly increased over the basal level after treatment with forskolin (10 microM) for 24 (P < 0.02) and 48 hr (P < 0.01); however, the production of MCP-1 was unchanged. CONCLUSIONS: Our data suggest that M-CSF and MCP-1 may play an important role in human preovulatory processes and that M-CSF, in particular, may be regulated by cyclic adenosine monophosphate. M-CSF and MCP-1 may also be valuable biochemical markers in the evaluation of oocyte maturation.     

Multiplex analysis of cytokines, chemokines, growth factors, MMP-9 and TIMP-1 produced by human bone marrow, adipose tissue, and placental mesenchymal stromal cells

Comparative analysis of mesenchymal stromal cells isolated from human BM, adipose tissue, and placenta was carried out. The cells were compared by the levels of constitutive, spontaneous, and LPS-induced production of Th1/proinflammatory (IFN-γ, IL-2, IL-1β, TNF-α, IL-12, IL-17) and Th2/anti-inflammatory cytokines (IL-4, IL-5, IL-6, IL-10, IL-13), chemokines (IL-8, MCP-1, MIP-1β), growth factors (IL-7, granulocytic CSF, granulocytic macrophageal CSF, erythropoietin, VEGF, EGF, IGF-1, main FGF), matrix metalloproteinase-9, and tissue inhibitor of metalloproteinase-1. Mesenchymal stromal cells originating from different tissues were characterized by functional potential for hemopoiesis support (through production of granulocytic CSF, granulocytic macrophage CSF, erythropoietin), immunomodulation (through production of IFN-γ, IL-2, IL-6, IL-1β, TNF-α and chemokines IL-8, MCP-1, MIP-1β), and stimulation of reparative processes (through production of VEGF, FGF, IGF-1, IL-6 tissue inhibitor of metalloproteinase-1, and matrix metalloproteinase-9). By the type and levels of spontaneous (basal) production of cytokines, the adipose tissue mesenchymal stromal cells more distinctly demonstrated the proinflammatory (IL-1β TNF-α), immunoregulatory (IFN-γ, IL-2, IL-4, IL-6, IL-8, MCP-1, MIP-1β), and hemopoiesis-stimulating (granulocytic CSF, granulocytic macrophage CSF) phenotype and at the same time were characterized by lower sensitivity to lipopolysaccharide stimulation than BM and placental mesenchymal cells.     

Endometrial interleukin-6 in vitro is not regulated directly by female steroid hormones,but by pro-inflammatory cytokines and hypoxia

Endometrial interleukin-6 (IL-6) mRNA has been reported to be suppressed in the mid-secretory phase in patients with recurrent early spontaneous abortions. This prompted our study concerning the regulation of endometrial IL-6 in cell culture models of endometrial epithelial and stromal cells. Steroid-dependent secretion of IL-6 was analysed by 17beta-estradiol (10(-8) mol/l) or progesterone (10(-6) mol/l) treatment and withdrawal (n = 8). Regulation by pro-inflammatory cytokines was studied in co-cultures of endometrial cells with human blood peripheral mononuclear cells (PBMC; n = 5) and by stimulation with IL-1beta, IL-6 and tumour necrosis factor alpha (TNFalpha), secreted by PBMCs at high concentrations. Regulation by hypoxia was assessed by culture of endometrial cells in 2% oxygen for 6 and 24 h (n = 5). IL-6 mRNA and protein levels were analysed by RT-PCR and enzyme-linked immunosorbent assays respectively. Endometrial IL-6 was not directly affected by 17beta-estradiol and/or progesterone. Co-culturing endometrial cells with PBMCs led to an increase of stromal but not epithelial IL-6 mRNA levels. In stromal cells, IL-6 secretion increased 2-10-fold if stimulated with 10 ng/ml recombinant IL-1beta or TNFalpha (P < 0.05). Hypoxia stimulated IL-6 secretion in epithelial cells up to 2-fold and in stromal cells up to 48-fold (P < 0.05). In conclusion, IL-6 expression in stromal and epithelial cells in vitro is regulated differently by pro-inflammatory cytokines and hypoxia. These results suggest a tight and specific network of control for this important cytokine within different endometrial compartments.     

TSLP induced by estrogen stimulates secretion of MCP-1 and IL-8 and growth of human endometrial stromal cells through JNK and NF-κB signal pathways

It has reported that human endometrial stromal cells (ESCs) express thymic stromal lymphopoietin (TSLP), and TSLP concentrations in the serum and peritoneal fluid were higher in women with endometriosis. Endometriosis is an estrogen-dependent disease. The present study aimed to elucidate whether and how estrogen regulates the growth of ESCs through TSLP. The ESCs behaviors in vitro were verified by SRB assay and Ki67 level detection, respectively. In addition, the effects of estrogen on TSLP and TSLP on the correspondent functional molecules were investigated by ELISA and flow cytometry. Here we found that estrogen stimulated the secretion of TSLP in a dosage-dependent manner. Recombinant human TSLP stimulates the secretion of MCP-1 and IL-8, and markedly promotes the viability and proliferation relative gene Ki-67 expression of ESCs. These effects could be abolished by the inhibitor for JNK or NF-κB signal, respectively. Moreover, not only anti-TSLP neutralizing antibody, but also blocking JNK or NF-κB signal by inhibitor abrogated the stimulatory role in the production of MCP-1 and IL-8, and the growth of ESCs induced by estrogen. Our current study has demonstrated that TSLP is involved in the regulation of estrogen on the secretion of MCP-1 and IL-8, and the growth of ESCs through JNK and NF-κB signal pathways, which suggests that the abnormal high expression of TSLP induced by estrogen may play an important role in ESCs growth and finally contribute to the origin and development of endometriosis.     

Cooperation of interleukin-17 and interferon-gamma on chemokine secretion in human fetal intestinal epithelial cells

Interleukin (IL)-17 is a newly identified T cell-derived cytokine that can regulate the functions of a variety of cell types. In this study, we investigated the effects of IL-17 and interferon (IFN)-gamma on chemokine secretion in human fetal intestinal epithelial cells. IL-8 and monocyte chemoattractant protein (MCP)-1 secretion by the human fetal intestinal epithelial cell line, intestine-407, was evaluated by ELISA and Northern blot. The expression of IL-17 receptor (R) was analysed by a binding assay using [(125)I]-labelled IL-17. The activation of nuclear factor-kappa B (NF-kappa B), NF-IL6 and AP-1 was assessed by an electrophoretic gel mobility shift assay (EMSA). IL-17 induced a dose-dependent increase in IL-8 and MCP-1 secretion. The inducing effects of IL-17 on IL-8 and MCP-1 mRNA abundance reached a maximum as early as 3 h, and then gradually decreased. IL-17 and IFN-gamma synergistically increased IL-8 and MCP-1 secretion and mRNA abundance. IFN-gamma induced a weak increase in IL-17 R mRNA abundance, and incubation with IFN-gamma for 24 h enhanced [(125)I]-labelled IL-17-binding by 2.4-fold. IL-17 rapidly induced the phosphorylation and degradation of I kappa B alpha molecules, and the combination of IL-17 and IFN-gamma induced a marked increase in NF-kappa B DNA-binding activity as early as 1.5 h after the stimulation. Furthermore, this combination induced an increase in NF-IL-6 and AP-1 DNA-binding activity. In conclusion, it becomes clear that IL-17 is an inducer of IL-8 and MCP-1 secretion by human fetal intestinal epithelial cells. The combination of IL-17 with IFN-gamma synergistically enhanced chemokine secretion. These effects of IL-17 and IFN-gamma might play an important role in the inflammatory responses in the intestinal mucosa.     

腹膜间皮细胞在小鼠子宫内膜细胞刺激腹腔微环境中作用

目的：探讨腹腔微环境受子宫内膜细胞刺激改变中腹膜间皮细胞的作用及其对子宫内膜异位症发病机制的意义。方法： 注射小鼠子宫内膜上皮和间质细胞入小鼠腹腔，酶联免疫吸附法（ELISA）检测注射后4、24和72 h时点腹腔冲洗液细胞因子MCP-1/JE、IL-1α和IL-6蛋白表达，同步逆转录-聚合酶链式反应技术 (Real-Time RT-PCR) 检测腹膜组织 (主要含腹膜间皮细胞) 和腹腔巨噬细胞的细胞因子MCP-1/JE、IL-1 α和IL-6 mRNA表达。结果: 子宫内膜细胞刺激腹腔细胞因子蛋白含量迅速一过性升高，4 h时点为表达高峰，腹膜细胞因子基因表达与腹腔液蛋白表达同步，腹腔巨噬细胞基因表达高峰滞后于腹腔液蛋白表达。子宫内膜上皮细胞刺激腹腔炎症反应作用强于间质细胞。结论: 子宫内膜细胞刺激腹腔发生非特异性炎症反应，腹膜间皮细胞可能是其细胞因子效应的主要来源，提示腹膜在子宫内膜异位症中除作为病灶依附体外，还可能在腹腔微环境无菌性炎症反应中起重要作用。     

Cysteinyl leukotrienes induce monocyte chemoattractant protein 1 in human monocytes/macrophages

BACKGROUND: Monocytes/macrophages have a cysteinyl leukotriene 1 (CysLT1) receptor, but its function is poorly understood. Objective To elucidate the biological function of the CysLT1 receptor of human monocytes/macrophages. METHODS: We examined the production of TNF-alpha, IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, monocyte chemoattractant protein 1 (MCP-1), macrophage colony-stimulating factor (M-CSF), and eotaxin induced by CysLTs (leukotriene (LT)C4, -D4, and -E4) in THP-1 cells, a human monocytic leukaemia cell line, and peripheral blood CD14+ monocytes/macrophages. Moreover, we examined the effect of CysLTs on the expression of beta-chemokine receptor 2B (CCR2B) as the receptor of MCP-1 by Western blot analysis. RESULTS: ELISA revealed that CysLTs induced MCP-1 in THP-1 cells and peripheral blood CD14+ monocytes/macrophages, but not other cytokines. PCR demonstrated that CysLTs increased MCP-1 mRNA expression in THP-1 cells, and Western blotting showed that CysLTs increased the expression of CCR2B in THP-1 cells. Moreover, we demonstrated that pranlukast, a CysLT1 receptor antagonist, blocked MCP-1 production by CysLTs in THP-1 cells almost completely, and partially inhibited MCP-1 release by CysLTs in peripheral blood CD14+ monocytes/macrophages and CCR2B expression by CysLTs in THP-1 cells. CONCLUSION: CysLTs induce MCP-1 and increase CCR2B expression in human monocytes/macrophages.     

Effects of interferon-gamma on secretion of vascular endothelial growth factor by endometrial stromal cells

PROBLEM: The aim of this study was to clarify the physiological effects of interferon (IFN)-gamma on secretion of vascular endothelial growth factor (VEGF) by endometrial stromal (ES) cells. METHOD OF STUDY: ES cells were obtained from human uterine endometrium by enzymic digestion and filtration. The effects of IFN-gamma on production of VEGF by ES cells were examined by analyzing VEGF mRNA expression with Northern blotting analysis and by assaying VEGF protein. RESULTS: IFN-gamma inhibited VEGF mRNA and protein expression by ES cells in a dose-dependent manner. In ES cells treated with IFN-gamma, VEGF production was not significant until 6 hr of incubation and was significantly affected after 6 hr of incubation, but decreased significantly after 12 to 48 hr. IFN-gamma also suppressed VEGF mRNA expression by ES cells. CONCLUSIONS: ES cells produce VEGF, which may contribute to endometrial neovascularization and proliferation. IFN-gamma may play an important role in regulating VEGF production by ES cells.