Regulation of inflammatory and angiogenesis mediators in a functional model of decidualized endometrial stromal cells.

The mechanisms involving the expression of interleukin (IL) 1 family members in the process of preparing the endometrium to receive an embryo remain unclear. In this study, decidualization differentially skewed the balance of IL1 family receptor expression in a pattern that increases endometrial stromal cell receptivity to IL1, IL18 and IL33. Additionally, endometrial cells showed increased expression of homeobox HOXA10 and HOXA11 and LIFR, which are known to be involved in endometrial embryo receptivity. Further analyses of decidual endometrial cells revealed a significant increase in the release of potent proinflammatory, remodelling and angiogenic factors implicated in the embryo invasion process, such as VEGF (P = 0.0305), MMP9 (P = 0.0003), TIMP3 (P = 0.0001), RANTES (P = 0.0020), MCP1 (P = 0.0001) and MIF (P = 0.0068). No significant changes in endogenous IL1B secretion were observed. Decreased secretion of IL18 and decidualization increased secretion of IL33. These findings reveal a significant modulation of endometrial cell receptivity to IL1 family members during endometrial stromal cell decidualization, and suggest that the involvement of IL1 family members is important in physiological processes of endometrial receptivity, including adaptive immunology. This may be relevant to establishing a favourable uterine microenvironment for embryo implantation.

Selective modulation of the prostaglandin F2α pathway markedly impacts on endometriosis progression in a xenograft mouse model.

STUDY HYPOTHESIS: Selective activation or blockade of the prostaglandin (PG) F2α receptor (FP receptor) affects ectopic endometrial tissue growth and endometriosis development. STUDY FINDING: FP receptor antagonists might represent a promising approach for the treatment of peritoneal endometriosis. WHAT IS KNOW ALREADY: Eutopic and ectopic endometrium from women with endometriosis exhibit higher expression of key enzymes involved in the PGF2α biosynthetic pathway. It has also been shown that the PGF2α-FP receptor interaction induces angiogenesis in human endometrial adenocarcinoma. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: For this study, a mouse model of endometriosis was developed by inoculating human endometrial biopsies into the peritoneal cavity of nude mouse (n = 15). Mice were treated with AL8810 (FP receptor antagonist), Fluprostenol (FP receptor agonist) or PBS. Endometriosis-like lesions were collected and analysed for set of markers for angiogenesis, tissue remodelling, apoptosis, cell proliferation and capillary formation using qPCR and immunohistochemistry. MAIN RESULTS AND THE ROLE OF CHANCE: We found that selective inhibition of the FP receptor with a specific antagonist, AL8810, led to a significant decline in the number (P < 0.01) and size of endometriosis-like lesions (P < 0.001), down-regulated the expression of key mediators of tissue remodelling (MMP9, P < 0.05) and angiogenesis (VEGF, P < 0.01) and up-regulated the pro-apoptotic factor (Bax, P < 0.01) as compared with controls. Immunohistochemical analyses further showed a marked decrease in cell proliferation and capillary formation in endometrial implants from AL8810-treated mice, as determined by proliferating cell nuclear antigen (PCNA) and von Willebrand factor (vWF) immunostaining, respectively. Moreover, Fluprostenol, a selective FP receptor agonist, showed the opposite effects. LIMITATIONS, REASONS FOR CAUTION: We carried out this study in nude mice, which have low levels of endogenous estrogens which may affect the lesion growth. Caution is required when interpreting these results to women. WIDER IMPLICATIONS OF THE FINDINGS: This study extends the role of PG signalling in endometriosis pathogenesis and points towards the possible relevance of selective FP receptor antagonism as a targeted treatment for endometriosis. LARGE SCALE DATA: Not Applicable. STUDY FUNDING AND COMPETING INTERESTS: This work was supported by grant MOP-123259 to the late Dr Ali Akoum from the Canadian Institutes for Health Research. The authors have no conflict of interest.

Blood soluble interleukin 1 receptor accessory protein levels are consistently low throughout the menstrual cycle of women with endometriosis.

BACKGROUND: A deficiency in the counter-regulatory mechanisms of interleukin 1 (IL1) may play a significant role in endometriosis pathogenesis and associated chronic inflammation. The aim of this study was to investigate peripheral blood levels of soluble IL1 receptor accessory protein (sIL1RAP), a potent natural inhibitor of IL1, in women with and without endometriosis. METHODS: Peripheral blood samples were collected from women with endometriosis (n = 47) consulting for infertility, pelvic pain or tubal ligation, in whom the disease was diagnosed at laparoscopy. Control healthy women (n = 27) were requesting tubal ligation or reanastomosis and had no visible evidence of endometriosis at laparoscopy. sIL1RAP levels were determined by ELISA, whereas estradiol (E2) and progesterone (P4) levels were determined by competitive immunoassays. RESULTS: sIL1RAP levels were significantly decreased in women with early endometriosis stages compared to controls (p < 0.05) and markedly during the proliferative phase of the menstrual cycle (p < 0.001). Actually, while sIL1RAP were significantly increased in the proliferative compared to the secretory phase in normal women (p < 0.0001) and peaked at the end of this phase, sIL1RAP remained consistently low and showed non-significant variations throughout the menstrual cycle in women with endometriosis. CONCLUSIONS: Lower circulating levels of sIL1RAP points to a significant impairment in the counter-regulatory mechanisms of IL1, which in view of the cytokine's potent inflammatory and growth-promoting properties may play a significant role in the pathophysiology of endometriosis.

[Embryo implantation: role of interleukin 1 family members]. / L'implantation embryonnaire - importance de la famille de l'interleukine 1.

Endometrial receptivity to embryo implantation is one of the fundamental features of reproduction. Success of natural or assisted embryo implantation is low (20-25%). Implantation remains the result of a successful collaboration, tightly regulated and closely coordinated, between maternal and embryonic tissues located at the crossroads of endocrinology and immunology. In scientific terms, this collaboration is a mystery of human reproduction. The implanted blastocyst within the endometrium is dependent on a fine-tuned synchronization. Therefore, an accurate dialogue between the mother and the embryo is timely required to orchestrate mutual and well-synchronized changes in the developing embryo and maternal responsiveness in order to achieve a successful implantation. Maternal-derived mediators, such as steroid hormones, matrix-degrading enzymes, integrins, cytokines, chemokines, and many embryonic growth factors could be involved in the feto-maternal dialogue. Therefore, what is the maternal molecular signature compatible with embryo implantation?

Macrophage migration inhibitory factor is involved in a positive feedback loop increasing aromatase expression in endometriosis.

Immune-endocrine interplay may play a major role in the pathogenesis of endometriosis. In the present study, we have investigated the interaction between macrophage migration inhibitory factor (MIF), a major pro-inflammatory and growth-promoting factor markedly expressed in active endometriotic lesions, and estradiol (E(2)) in ectopic endometrial cells. Our data showed a significant increase of MIF protein secretion and mRNA expression in endometriotic cells in response to E(2). MIF production was blocked by Fulvestrant, an estrogen receptor (ER) antagonist, and induced by ERα and ERß selective agonists propyl-pyrazole-triol (PPT) and diarylpropionrile (DPN), respectively, thus demonstrating a specific receptor-mediated effect. Cell transfection with MIF promoter construct showed that E(2) significantly stimulates MIF promoter activity. Interestingly, our data further revealed that MIF reciprocally stimulates aromatase protein and mRNA expression via a posttranscriptional mRNA stabilization mechanism, that E(2) itself can upregulate aromatase expression, and that inhibition of endogenous MIF, using MIF specific siRNA, significantly inhibits E(2)-induced aromatase. Thus, the present study revealed the existence of a local positive feedback loop by which estrogen acts directly on ectopic endometrial cells to upregulate the expression of MIF, which, in turn, displays the capability of inducing the expression of aromatase, the key and rate-limiting enzyme for estrogen synthesis. Such interplay may have a considerable impact on the development of endometriosis.

Soluble human IL-1 receptor type 2 inhibits ectopic endometrial tissue implantation and growth: identification of a novel potential target for endometriosis treatment.

Endometriosis is often associated with a chronic pelvic immuno-inflammatory process, which is closely related to disease pathogenesis and major symptoms. Our studies led to the detection of a marked imbalance between IL-1 and its natural inhibitor IL-1 receptor type 2 (IL1R2) in women with endometriosis. This points to a deficiency in the local control of IL-1 that, in view of the cytokine's elevated levels and potent proinflammatory, angiogenic, and growth-promoting effects, may contribute to endometriosis development. Using an in vivo model in which human endometrial tissue was inoculated into nude mice and left to establish before any further treatment, our data showed that sIL1R2 interferes with the capability of endometrial tissue to invade, grow, disseminate, and stimulate angiogenesis into the host tissue. sIL1R2 significantly down-regulated the expression of major cell adhesion receptors (αv and ß3 integrins), matrix metalloproteinases (MMP-2 and -9), and vascular endothelial cell growth factor. Interestingly, treatment with sILR2 (5 µg/kg) led to a concomitant upregulation of matrix metalloproteinases natural inhibitors (TIMP1 and TIMP2) and down-regulation of BclII, a potent anti-apoptotic protein. This creates an imbalance between pro- and anti-proteolytic and apoptotic factors and may further contribute to IL1R2 growth-inhibitory effects. This study provides evidence that sIL1R2 alters ectopic endometrial tissue growth, remodeling, and survival in vivo and may represent an interesting potential therapeutic tool.

Human chorionic gonadotropin triggers angiogenesis via the modulation of endometrial stromal cell responsiveness to interleukin 1: a new possible mechanism underlying embryo implantation.

Deep functional changes occurring within the endometrium during implantation are orchestrated by embryonic and maternal signals. Human chorionic gonadotropin (hCG), a major embryonic signal, plays a critical role in the initiation and maintenance of pregnancy. Interleukin (IL) 1, one of the earliest embryonic signals, appears to exert a direct impact on the receptive endometrium and to induce major molecular changes that are essential for embryo implantation. Herein we investigate whether hCG can modulate endometrial stromal cell (ESC) receptivity to IL1 during the implantation window and assess the impact on angiogenesis in vitro. Primary cultures of ESCs from normal fertile women during the implantation window were treated for 24 h with different concentrations of hCG (0-100 ng/ml) and stimulated for 24 h with IL1B (0-0.1 ng/ml). IL1 receptors (IL1Rs), IL1R antagonist (IL1RA), and monocyte chemotactic protein (MCP) 1 were analyzed by real-time PCR, ELISA, and Western blotting. The angiogenic activity in vitro was studied using human microvascular endothelial cell line, scratch wound assay, and cell proliferation via BrdU incorporation into DNA. Human CG induced a dose-dependent imbalance in ESC receptivity to IL1 by significantly upregulating the functional signaling IL1R1 and concomitantly downregulating the decoy inhibitory IL1R2 and IL1RA upon subsequent exposure to IL1B. Prior exposure to hCG amplified MCP1 secretion by ESCs in response to IL1B and triggered the release of angiogenic activity in vitro in which MCP1 appeared to play a significant role. Overexpression of IL1R2 using cell transfection inhibited IL1 and hCG/IL1B-mediated MCP1 secretion. These findings suggest that hCG coordinates embryonic signal interaction with the maternal endometrium, and point to a new possible pathway by which it may promote embryonic growth.

Macrophage migration inhibitory factor up-regulates alpha(v)beta(3) integrin and vascular endothelial growth factor expression in endometrial adenocarcinoma cell line Ishikawa.

Human endometrium undergoes a series of dynamic physiological changes during the menstrual cycle of reproductive age women. Many factors, including hormones, cytokines, growth factors, matrix metalloproteinases and integrins, are essential for the success of embryonic implantation into endometrial tissue. Herein, we used a well-differentiated endometrial adenocarcinoma cell line, Ishikawa, to investigate in vitro the role played by macrophage migration inhibitory factor (MIF) in the regulation of endometrial receptivity markers. Quantitative real-time polymerase chain reaction (qRT-PCR) showed that MIF induced a slight increase in alpha(v) (alphav) mRNA integrin subunit expression during the first 12h, but reached a significant difference after 24h MIF treatment compared to control, whereas beta(3) (beta3) integrin subunit displayed significant increase in mRNA 2h following treatment. Immunocytofluorescence showed strong alphav and beta3 immunostaining at 25 ng/ml MIF, and Western blotting clearly indicated increased alphav and beta3 protein expression. MIF treatment significantly stimulated vascular endothelial growth factor (VEGF) mRNA expression in a dose- and time-dependent manner after 24 h treatment. Moreover, immunocytofluorescence revealed positive VEGF immunostaining compared to control, and analysis by ELISA of VEGF release in culture supernatants demonstrated that MIF (25 ng/ml) significantly induced VEGF secretion at 12 and 24 h. In conclusion, this study provides evidence that MIF directly up-regulates alphavbeta3 integrin and VEGF expression in human endometrial Ishikawa cells and may advance our understanding of factors involved in the establishment of endometrial receptivity and successful implantation.

Abnormal interleukin 1 receptor types I and II gene expression in eutopic and ectopic endometrial tissues of women with endometriosis.

Interleukin-1 (IL1) is believed to play a central role in the immuno-inflammatory process associated with endometriosis. IL1 triggers cell activation via its receptor type I (IL1R1), but its receptor type II (IL1R2) is known instead as a scavenger that buffers the cytokine's effects. Our previous studies have shown increased expression of IL1R1 in active endometriotic implants compared to normal and endometriosis women-derived endometrial tissues, and a simultaneous decrease in IL1R2 expression at the protein level. In the present study, in situ hybridization demonstrated a noticeable decrease in IL1R2 mRNA hybridization score in eutopic and matched ectopic endometrial tissues of women with endometriosis compared to normal women in the stroma (P<0.001 and P<0.001, respectively) and the epithelium (P<0.01 and P<0.05, respectively), whereas IL1R1 mRNA hybridization score was higher only in the ectopic implants, with a statistically significant difference in the stroma (P<0.05). This was corroborated by RT-PCR analysis of IL1R1 and IL1R2 mRNAs in ectopic (P<0.05 and P<0.05, respectively) and matched eutopic (P=0.22 and P<0.05, respectively) endometrial tissues from women with endometriosis compared to endometrial tissues from normal women. The decrease in IL1R2 mRNA levels in eutopic endometrial tissue of endometriosis women, and the concomitant increase in IL1R1 mRNA levels in ectopic implants, reveal a profound defect in IL1R 1 and IL1R2 gene expression which may accentuate the capability of this tissue to respond to IL1 and favor its ectopic growth.

Chorionic gonadotropin down-regulates the expression of the decoy inhibitory interleukin 1 receptor type II in human endometrial epithelial cells.

Secretion of embryonic IL-1beta seems to be the first response of the blastocyst to receptive endometrium, inducing molecular changes that are essential for attachment of the blastocyst. Here, we report that human chorionic gonadotropin (hCG), a glycoprotein hormone that plays a critical role in the initiation and maintenance of pregnancy, markedly down-regulates the expression of the decoy inhibitory IL-1 receptor type II (IL-1R2) in human endometrial epithelial cells. Treatment with hCG resulted in a dose-dependent decrease in IL-1R2 soluble and membrane bound protein forms and mRNA steady-state levels, whereas no significant effect on the expression of the activating IL-1 receptor type I (IL-1R1) was seen. Cell infection with the wild-type human LH/chorionic gonadotropin receptor corroborated the aforementioned data, whereas cell infection with the constitutively activated LH chorionic gonadotropin receptor led to similar effects on IL-1R2 and IL-1R1 expression without hCG treatment. Cloning of human IL-1R2 gene promoter in the pGL3 luciferase reporter vector and transient transfection experiments further showed a significant dose-dependent diminution of IL-1R2 promoter activity in response to hCG. These data suggest that hCG, by down-regulating the expression of IL-1R2, a potent and specific inhibitor of IL-1, without affecting the expression of the functional activating IL-1R1, diminishes the ability of endometrial epithelial cells to counterbalance the local effects of IL-1, making these cells probably more responsive to the cytokine. In view of IL-1's role as an embryonic signal, these data reveal a new mechanism by which hCG sustains human pregnancy and promotes embryonic growth.

Requirement of the extracellular cysteine at position six for CD40/CD40 dimer formation and CD40-induced IL-8 expression.

We recently showed that oligomerization of CD40 molecules on cell surface leads to disulfide-linked CD40/CD40 dimer formation, an event that is necessary for CD40-induced B7-2 expression in human B cells. Here, we demonstrate that CD40/CD40 dimers formation also occurs in different cell types such as T24 bladder cancer cells and CD40-transfected HEK 293 cells. Disulfide bonds mediate the formation of CD40/CD40 homodimers in CD40-activated cells. To determine the potential residue(s) involved in disulfide bonds formation and subsequent CD40-induced IL-8 expression, we generated a CD40 mutant in which the extracellular cysteine 6 was replaced by a glutamine (CD40-C6Q). CD40-induced IL-8 mRNA expression and protein synthesis were studied in stably transfected HEK 293 cells that were sorted out along with similar levels of expression of wild type (CD40-WT) and CD40-C6Q molecules. In contrast to cells expressing CD40-WT protein, disulfide-linked CD40/CD40 dimer formation was completely abolished in HEK 293 cells expressing CD40-C6Q proteins. Abolishment of disulfide-linked CD40/CD40 dimers in these transfected cells was sufficient to inhibit CD40-induced mRNA expression and secretion of IL-8. This study identifies the extracellular cysteine 6 of CD40 molecules as a potential molecular target to disrupt the expression of CD40-induced pro-inflammatory cytokines by epithelial cells.

Promotion of angiogenesis and proliferation cytokines patterns in peritoneal fluid from women with endometriosis.

Studies have long sought specific cytokines that could characterize endometriosis. Either due to variations between study designs regarding the assessment criteria for the cytokine or to low power resulting from small sample size, no factor proved to be sufficiently specific to endometriosis. In other clinical fields, a combination of several markers proved to be more powerful than a single-molecule approach. As well, in the context of endometriosis, simultaneous assessment of several cytokines present in the peritoneal fluid might help in unveiling patho-physiological processes, thus contributing to a better understanding of the condition. Therefore, the objective of this study was to investigate peritoneal fluid cytokines-derived of endometriotic women. For this retrospective case-control study, peritoneal fluid samples were obtained at laparoscopy and assessed by multiplex. Our data showed distinct patterns of peritoneal fluid cytokine concentrations in endometriotic women most notably a marked increase in EGF, FGF-2, IL-1α, MIP-1ß, TGFα, PDGF-AA, PDGF-BB, MCP-3, sCD40L, Gro Pan, IL-17α, MDC and Rantes. The overall effect of fertility status revealed a significant difference for only one cytokine, namely MDC. Furthermore, FLT-3L and IP-10 levels were decreased in endometriosis patients, the former in both menstrual cycle phases and the latter in the secretory phase. A significant inverse Pearson correlation (p<0.05) was noted between pro-angiogenic cytokines EGF and FGF and the anti-angiogenic cytokine IP-10 in endometriosis patients at stages III-IV and in the secretory phase. These changes may exacerbate the local peritoneal angiogenic and proliferative reaction observed in women with endometriosis, and contributes to its pathophysiology.

Augmented Angiogenic Factors Expression via FP Signaling Pathways in Peritoneal Endometriosis.

CONTEXT: Angiogenesis is required for ectopic endometrial tissue growth. Our previous studies showed that prostaglandin F2α (PGF2α) biosynthetic enzymes and receptor were markedly elevated in endometriotic lesions and that PGF2α is a potent angiogenic factor in endothelial cells. OBJECTIVE: We sought to determine whether or not the F-prostanoid receptor modulates angiogenesis in ectopic stromal cells. DESIGN: Release of angiogenic factors by ectopic endometrial stromal cell primary cultures stimulated with PGF2αand exposed to agents that target PGF2α signaling was assessed. SETTING: The study was conducted in an immunology laboratory at the Centre Hospitalier Universitaire (Québec City) medical research center. PATIENTS: Women found to have peritoneal endometriosis during laparoscopy were included in this study. MAIN OUTCOME MEASURE(S): Prostaglandin E2, PGF2α, vascular endothelial cell growth factor, and CXC chemokine ligand 8 mRNA and protein; FP prostanoid receptor expression. RESULTS: PGF2α markedly up-regulated prostaglandin E2, CXC chemokine ligand 8 and vascular endothelial cell growth factor secretion in endometriotic cells. This effect was suppressed in the presence of a specific F-prostanoid antagonist (AL8810) and its signaling pathway was dependent on F-prostanoid receptor variant. PGF2α can exert its proliferative and angiogenic activities either directly by stimulating endothelial cell proliferation, migration and angiogenesis through F-prostanoid receptor, or indirectly, by stimulating endometriotic stromal cells to produce potent angiogenic factors through either receptor variant. CONCLUSION: These results show for the first time that PGF2α exerts an angiogenic effect on ectopic stromal cells, inducing the secretion of major angiogenic factors via different F-prostanoid signaling pathways. This study suggests a new interpretation of the mechanism underlying endometriosis development involving PGF2α in endometriosis-associated angio-inflammatory changes.

Marked increase in macrophage migration inhibitory factor synthesis and secretion in human endometrial cells in response to human chorionic gonadotropin hormone.

Originally identified for its capacity to inhibit the random migration of macrophages in vitro, macrophage migration inhibitory factor (MIF) is now recognized as a multifunctional cytokine that modulates the immune response and acts as a growth and angiogenic factor. Recent studies showed that MIF is expressed in the human endometrium across the menstrual cycle as well as in chorionic villi from first-trimester human placenta, which suggests an involvement of MIF in reproduction. Herein, we report that human chorionic gonadotropin (hCG), a glycoprotein hormone that plays a critical role in the initiation and maintenance of pregnancy, markedly stimulates MIF expression in endometrial stromal cells. Cell treatment with hCG resulted in a dose-dependent increase in MIF protein secretion and mRNA steady-state levels, as shown by immunocytochemistry, ELISA, and RT-PCR. Assessment of MIF mRNA half-life showed that hCG treatment had no significant effect on MIF mRNA stability (P = 0.08). However, nuclear transcription assays (run-on) revealed that hCG acts predominantly by up-regulating MIF gene transcription. These data clearly indicate that MIF can mediate hCG effects on the human endometrium and, in view of the immunomodulatory and angiogenic properties of MIF, reveal a new mechanism by which hCG sustains human pregnancy and promotes embryonic growth.

An indirect quantitative method for the assessment of angiogenesis in human tissues.

We set up an ELISA that measures the concentration of von Willebrand factor (vWF) in human endometrial tissue and found a significant correlation with the mean vessel counts in vWF- and CD31-immunostained tissue sections. This ELISA allows an objective and quantitative evaluation of the vascular state in the endometrium and could be used as a method to evaluate the angiogenic state in other tissues.

Abnormal Expression of Prostaglandins E2 and F2α Receptors and Transporters in Patients with Endometriosis.

OBJECTIVE: To investigate the level of expression of prostaglandin receptivity and uptake factors in eutopic and ectopic endometrium of women with endometriosis. DESIGN: Prospective study. SETTING: Human reproduction research laboratory. PATIENTS: Seventy-eight patients with endometriosis and thirty healthy control subjects. INTERVENTION(S): Endometrial and endometriotic tissue samples were obtained during laparoscopic surgery. MAIN OUTCOME MEASURE(S): Real-time polymerase chain reaction assay of mRNA encoding prostaglandin E2 receptors (EP1, EP2, EP3, and EP4), prostaglandin F2α receptor (FP), prostaglandin transporter (PGT), and multidrug resistance-associated protein 4 (MRP4); immunohistochemical localization of expressed proteins. RESULTS: Marked increases in receptors EP3, EP4, and FP and transporters PGT and MRP4 in ectopic endometrial tissue were noted, without noticeable change associated with disease stage. An increase in EP3 expression and decreases in FP and PGT were observed in the eutopic endometrium of endometriosis patients in conjunction with the phases of the menstrual cycle. CONCLUSION(S): This study is the first to demonstrate a possible relationship between endometriosis and enhanced prostaglandin activity. In view of the wide range of prostaglandin functions, increasing cell receptivity and facilitating uptake in endometrial tissue could contribute to the initial steps of overgrowth and have an important role to play in the pathogenesis and symptoms of this disease.

Estradiol and interleukin-1beta exert a synergistic stimulatory effect on the expression of the chemokine regulated upon activation, normal T cell expressed, and secreted in endometriotic cells.

Endometriosis, commonly associated with intraperitoneal inflammation, is estrogen dependent. Possible links between the immunoinflammatory and endocrine changes observed in endometriotic women have been poorly understood. In this study, we report that estradiol (E(2)) and IL-1beta exert a synergistic stimulatory action on RANTES (regulated upon activation, normal T cell expressed, and secreted) expression by endometriotic cells. Treatment of endometriotic cells with IL-1beta had a dose-dependent effect on RANTES protein secretion and mRNA steady state levels, whereas cell treatment with E(2) or progesterone had no detectable effect. Interestingly, treatment of endometriotic cells with E(2) before stimulation with IL-1beta resulted in a further increase in RANTES protein secretion and mRNA steady state levels, compared with IL-1beta alone, whereas treatment with progesterone did not significantly affect cell responsiveness to IL-1beta. Assessment of RANTES mRNA half-life revealed that cell pretreatment with E(2) enhanced RANTES mRNA stability and increased gene transcription as shown by run-on analysis. Immunohistochemical analysis of RANTES in endometriotic tissue showed immunostaining to be predominant in the stroma with no noticeable differences in tissues from the proliferative and secretory phase of the menstrual cycle. This appears to be consistent with the cell culture data and indicates that RANTES expression in endometriotic tissue is not subject to cyclic variation. These findings reveal a new regulatory mechanism by which IL-1beta produced by activated macrophages can in synergy with ovarian and locally produced E(2) lead to enhanced macrophage and T-lymphocyte recruitment, thereby exacerbating the local immunoinflammatory process. Furthermore, the findings provide a further evidence for a close relationship between the endocrine and immunological changes observed in endometriosis.

Macrophage migration inhibitory factor is markedly expressed in active and early-stage endometriotic lesions.

The establishment of a new vascular supply is essential for the survival of endometrial tissue and its development in ectopic locations. We have previously shown that ectopic endometrial cells release an important mitogenic activity for human endothelial cells and identified macrophage migration inhibitory factor (MIF) as one of the principal bioactive molecules involved in endothelial cell proliferation. In the present study, immunohistochemical and dual immunofluorescence analyses showed that MIF is effectively expressed by endometriotic tissue, particularly in the glands, and identified endothelial cells, macrophages, and T lymphocytes as cells markedly expressing MIF in the stroma. Western blot analysis showed a single 12.5-kDa band corresponding to the known mol wt of the molecule. The highest concentrations of MIF protein in endometriotic tissue, as measured by ELISA, were found in flame-like red endometriotic lesions, compared with typical black-bluish (P < 0.01) or with white lesions (P < 0.01). Interestingly, MIF displayed a marked expression in lesions from the initial stage of endometriosis (stage I). Semiquantitative RT-PCR analysis of MIF mRNA levels in the same endometriotic tissues showed a pattern of expression comparable with that of the protein. In view of its potent proinflammatory and angiogenic properties, local production of MIF within endometrial implants, particularly in those that are highly vascularized and representing the earliest and most active forms of the disease, make plausible the involvement of this factor in the local immunoinflammatory process observed in endometriosis and the initial steps of endometriotic tissue growth and development.

Soluble interleukin-1 receptor type II blocks monocyte chemotactic protein-1 secretion by U937 cells in response to peripheral blood serum of women with endometriosis.

OBJECTIVE: To assess the ability of peripheral blood serum from women with endometriosis to induce monocyte chemotactic protein-1 (MCP-1) secretion by monocytes and the putative role of the interleukin-1 (IL-1) system in endometriosis-associated monocyte activation. DESIGN: Cultures of U937 monocytic cells exposed to serum from normal women (control group) or women with endometriosis. SETTING: Gynecology clinic and human reproduction research laboratory. PATIENT(S): Seventy-nine women with endometriosis and 38 control women with no evidence of endometriosis at laparoscopy. INTERVENTION(S): Peripheral blood obtained a few days before laparoscopy. MAIN OUTCOME MEASURE(S): MCP-1 secretion in the culture medium and serum concentrations of soluble IL-1 receptor type II (sIL-1RII), IL-1beta, and IL-1alpha by ELISA or by enzyme immunometric assay. RESULT(S): Serum concentrations of sIL-1RII were significantly lower in women with stage I-II endometriosis than in control women, whereas serum concentrations of IL-1beta and IL-1alpha were comparable between the two groups. The serum of women with endometriosis induced higher secretion of MCP-1 by U937 cells than that of control women, particularly in the initial stages of endometriosis (stages I-II), and recombinant IL-1RII (rIL-1RII) significantly blocked that secretion. CONCLUSION(S): These findings point toward a deficiency in the mechanisms involved in the down-regulation of IL-1 actions at the systemic level and reveal sIL-1RII as a key factor involved in that process.