Ali Akoum

Elevated concentration and biologic activity of monocyte chemotactic protein-1 in the peritoneal fluid of patients with endometriosis * † ‡

OBJECTIVE To estimate the concentration and the biologic activity of monocyte chemotactic protein-1 (MCP-1) in the peritoneal fluid (PF) of women with and without endometriosis. DESIGN A case control study was conducted. SETTING Gynecology clinic and Laboratories of endocrinology of reproduction and immunology. PATIENTS Women presenting for infertility, pelvic pain, or tubal ligation in which endometriosis was diagnosed at laparoscopy (n = 36) and normal fertile controls presenting for tubal ligation (n = 21). INTERVENTIONS Collection of PF via laparoscopy. MAIN OUTCOME MEASURES Determination of PF concentrations of MCP-1 by an ELISA and evaluation of its monocyte chemotactic activity using a human hystiocytic cell line (U937). RESULTS. The concentration of MCP-1 (median, range of values) was increased in the PF of endometriosis patients (283, 0 to 1,930 pg/mL; conversion factor to SI unit, 0.155) compared with the control group (140, 0 to 435 pg/mL). The most significant elevation of MCP-1 levels was found in the stage II of the disease (371, 200 to 1,930 pg/mL). An increased chemotactic activity for monocytes (mean number of migrating cells/mm2 +/- SD) also was found in stages I (1,460 +/- 312) and II (1,541 +/- 336) of the disease when compared with fertile controls (393 +/- 56). Forty percent to 53% of this activity was inhibited in the presence of an antibody specific to MCP-1. CONCLUSIONS These observations are consistent with previous data indicating increased leukocyte chemotaxis in the PF of patients with endometriosis and suggest that MCP-1 may play a relevant role in the peritoneal inflammatory reaction associated with the disease.

Secretion of monocyte chemotactic protein-1 by cytokine-stimulated endometrial cells of women with endometriosis *

OBJECTIVE To evaluate in vitro the production of monocyte chemotactic protein-1 (MCP-1) by endometrial cells of patients with and without endometriosis. DESIGN Primary cultures of stromal and epithelial cells isolated from human endometrium were exposed during 24 hours to different cytokines. Monocyte chemotactic protein-1 secretion was analyzed in the culture medium. SETTING Gynecology clinic and laboratories of endocrinology of reproduction and immunology. PATIENTS Women presenting for infertility or pelvic pain in which endometriosis was diagnosed at laparoscopy (n = 6) and women presenting for tubal ligation without laparoscopic evidence of the disease (n = 6). INTERVENTIONS None. MAIN OUTCOME MEASURES De novo secretion of MCP-1 in the culture supernatant by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis after metabolic labeling with 35S-cysteine. RESULTS The incubation of endometrial epithelial cells of endometriosis women with either interleukin-1 beta or tumor necrosis factor-alpha resulted in the appearance of at least two and sometimes three bands having approximately 15, 13, and 9 kd molecular weights. These bands were identified as three distinct species of MCP-1 as their immunoprecipitation was prevented effectively in presence of an excess of cold MCP-1. In contrast, the endometrial epithelial cells of only one of six normal women produce significant levels of MCP-1 under the same stimulation conditions. The stromal cells of both groups of subjects do not secrete appreciable amounts of MCP-1 or only small quantities in two cases of endometriosis. CONCLUSIONS Monocyte chemotactic protein-1 secretion is upregulated in cytokine-stimulated endometrial epithelial cells of women having endometriosis as compared with normal women without evidence of the disease. Such a difference at the level of eutopic endometrial cell may have a significance in the physiopathology of endometriosis.

Cytokine-induced secretion of monocyte chemotactic protein-1 by human endometriotic cells in culture

OBJECTIVE Local secretion of chemotactic factors could contribute to the attraction of macrophages into the peritoneal cavity of women with endometriosis. The purpose of this study was to investigate the ability of endometriotic cells to produce monocyte chemotactic and activating protein-1 in response to interleukin-1 beta and tumor necrosis factor-alpha, which are found in elevated levels in the peritoneal fluid of patients with endometriosis. STUDY DESIGN Cultures of fibroblast-like and epithelial cells isolated from endometriotic tissue were incubated with different concentrations of cytokines for varying periods of time. The de novo secretion of monocyte chemotactic protein-1 in the culture supernatants was analyzed by immunoprecipitation and electrophoresis after metabolic labeling with sulfur 35-labeled cysteine. RESULTS The incubation of endometriotic fibroblast-like cells with interleukin-1 beta and tumor necrosis factor-alpha resulted in a time- and dose-dependent release of monocyte chemotactic protein-1 into the culture supernatant. Coincubation of the cells with tumor necrosis factor-alpha and interferon gamma resulted in a synergistic and dose-dependent increase of the monocyte chemotactic protein-1 secretion, whereas interferon gamma alone had no significant effect. Preliminary results indicate that monocyte chemotactic protein-1 is also produced by endometriotic epithelial cells in response to the same cytokines. CONCLUSIONS Cytokine-stimulated endometriotic cells synthesize and secrete monocyte chemotactic protein-1 in culture, and they may play a relevant role in the recruitment of macrophages to the peritoneal cavity of patients by the local production of chemotactic factors.

Stimulation of Macrophage Migration Inhibitory Factor Expression in Endometrial Stromal Cells by Interleukin 1, beta Involving the Nuclear Transcription Factor NFκB

Abstract Endometriosis, the ectopic development of endometrial tissue, is, particularly in peritoneal endometriosis, believed to result from tubal reflux of menstrual tissue. The release of cytokines and growth factors by refluxed endometrial cells in response to peritoneal inflammatory stimuli may enhance the capability of endometrial cells to implant and grow into the peritoneal host tissue. Herein we report that interleukin 1 (IL1), a major proinflammatory cytokine that is overproduced by endometriosis women-derived peritoneal macrophages and found in elevated concentrations in the peritoneal fluid of patients with endometriosis, stimulates the synthesis and the secretion of macrophage migration inhibitory factor (MIF) by human endometrial stromal cells. IL1B (0.1–100 ng/ml) exerted dose- and time-dependent effects of MIF protein secretion and mRNA synthesis, as shown by ELISA and reverse transcription-polymerase chain reaction, respectively. IL1B appeared to induce MIF gene transcription via the κB nuclear transcription factor (NFκB), as shown by electrophoretic mobility shift assay and Western blot analysis of IκB phosphorylation. Curcumin (10−8 M), which is known for inhibiting NFκB activation, inhibited IL1B-induced MIF secretion as well as NFκB nuclear translocation and DNA binding. Taken together, these findings clearly show that IL1B up-regulates the expression of MIF in endometrial stromal cells in vitro and acts via NFκB. This may play an important role in the physiology of the human endometrium and the pathophysiology of endometriosis considering the immunomodulatory properties of MIF as well as its role in cell growth, angiogenesis and tissue remodeling.

Up-Regulation of Cyclooxygenase-2 Expression and Prostaglandin E2 Production in Human Endometriotic Cells by Macrophage Migration Inhibitory Factor: Involvement of Novel Kinase Signaling Pathways

Cyclooxygenase (COX) is the rate-limiting enzyme in the metabolic conversion of arachidonic acid to prostaglandins (PGs), including prostaglandin E(2) (PGE(2)), a major mediator of inflammation and angiogenesis. Herein, we report that macrophage migration inhibitory factor (MIF), a potent proinflammatory and growth-promoting factor found at elevated concentrations in the peritoneal fluid of women with endometriosis and active endometriosis lesions, acts directly on ectopic endometrial cells to stimulate the synthesis of COX-2, the inducible form of COX, and the release of PGE(2). MIF treatment strongly activated p38 and ERK MAPK, and specific inhibitors of both pathways completely blocked basal and MIF-induced PGE(2) synthesis. Whereas p38 inhibitors negatively affected the stimulated synthesis of COX-2 and that of PGE(2), ERK inhibitors only decreased the production of PGE(2). These findings show for the first time a direct role for MIF in the up-regulation of COX-2 synthesis and PGE(2) secretion in ectopic endometrial cells. They further indicate that whereas p38 and ERK MAPK signaling pathways both play a significant role in the regulation of basal and MIF-induced synthesis of PGE(2) by ectopic endometrial cells, only p38 kinase is involved in the regulation of COX-2 expression in these cells. This suggests that MIF acts at more than one level to stimulate the synthesis of PGE(2) and triggers the coordinate activation of multiple enzymes in the biosynthesis pathway. Our data provide evidence for a novel mechanism by which MIF can induce a proinflammatory phenotype in ectopic endometrial cells, and favor the establishment of endometriosis and its related clinical symptoms.

Marked elevation of macrophage migration inhibitory factor in the peritoneal fluid of women with endometriosis

OBJECTIVE To evaluate the presence of macrophage migration inhibitory factor (MIF) in the peritoneal fluid of normal fertile women and patients with endometriosis and its growth-promoting activity toward human endothelial cells. DESIGN Retrospective study using ELISA to measure peritoneal fluid MIF, and [3H]-thymidine incorporation into the DNA of human endothelial cells to assess its mitogenic activity. SETTING Gynecology clinic and human reproduction research laboratory. PATIENT(S) Thirty-six healthy women and 57 women with endometriosis. INTERVENTION(S) Peritoneal fluid samples were obtained at laparoscopy. MAIN OUTCOME MEASURE(S) Macrophage migration inhibitory factor concentrations in the peritoneal fluid samples and [3H]-thymidine incorporation into the DNA of human microvascular endothelial cells to assess proliferation. RESULT(S) This study demonstrated the presence of MIF in the peritoneal fluid and a 238% increase of MIF levels in women with endometriosis as compared with healthy women. Both fertile and infertile women with endometriosis had significantly higher MIF concentrations than did fertile women with normal gynecological status, but the difference was more significant in infertile endometriosis patients. Anti-MIF antibody significantly inhibited proliferation of human microvascular endothelial cells in response to peritoneal fluids from healthy women and women with endometriosis stages I-II and III-IV, as assessed by [3H]-thymidine incorporation. CONCLUSION(S) This study revealed the presence of MIF in the peritoneal fluid and its increased levels in endometriosis and suggests that MIF may be involved in endometriosis-associated infertility and angiogenesis.

Spontaneous and stimulated secretion of monocyte chemotactic protein-1 and macrophage migration inhibitory factor by peritoneal macrophages in women with and without endometriosis

OBJECTIVE To assess spontaneous and stimulated secretion of monocyte chemotactic protein-1 (MCP-1) and macrophage migration inhibitory factor (MIF) by peritoneal macrophages in women with and without endometriosis. DESIGN Macrophages were isolated from the peritoneal fluid and cultured for different periods of time (6, 20, and 44 hours) without any stimulation to determine spontaneous secretion of MCP-1 and MIF. Macrophages were also exposed to 1 microg/mL lipopolysaccharide for 6 hours to evaluate the stimulated secretion of these cytokines. SETTING Gynecology clinic and human reproduction research laboratory. PATIENT(S) Twelve fertile women and 11 women with endometriosis. INTERVENTION(S) Peritoneal fluid obtained at laparoscopy. MAIN OUTCOME MEASURE(S) Monocyte chemotactic protein-1 and MIF concentrations in the culture medium using ELISA. RESULT(S) Peritoneal macrophages of women with endometriosis demonstrated an increased capacity to secrete MCP-1 either spontaneously or after stimulation with lipopolysaccharide. They also showed a marked tendency for an increased secretion of MIF, but no statistically significant difference was found. CONCLUSION(S) Monocyte chemotactic protein-1 and MIF production by peritoneal macrophages may contribute to paracrine and autocrine activation and to macrophage accumulation in the peritoneal cavity of women with endometriosis. These mechanisms may exacerbate peritoneal inflammation and favor the growth of endometrial implants.

Decreased Expression of the Decoy Interleukin-1 Receptor Type II in Human Endometriosis

Many of the biological changes occurring in the endometrium during the menstrual cycle bear a striking resemblance to those associated with inflammatory and reparative processes. Hence, it would not be surprising to find that cytokines known for their pro-inflammatory properties, such as interleukin-1 (IL-1), could play a key role in the physiology of this tissue and that their action would be tightly controlled by local mechanisms. In the present study, immunohistochemical and Western blot analyses show that in normal women (n = 39), the endometrial tissue expresses, in a cycle-dependent manner, the IL-1 receptor type II (IL-1RII), a molecule of which the only biological property known to date is that of capturing IL-1, inhibiting thereby its binding to the functional type I IL-1 receptor. IL-RII immunostaining was particularly intense within the lumen of the glands and at the apical side of surface epithelium. Interestingly, the intensity of staining was markedly less pronounced in the endometrium of women with endometriosis (n = 54), a disease believed to arise from the abnormal development of endometrial tissue outside the uterus, especially in the early stages of the disease (stages I and II). This study is the first to show the local expression in endometrial tissue of IL-1RII, a potent and specific down-regulator of IL-1 action and its decreased expression in women suffering from endometriosis.

Triphasic expression of interleukin-1 receptor type I in human endometrium throughout the menstrual cycle of fertile women and women with unexplained infertility.

OBJECTIVE To evaluate expression of interleukin-1 receptor type I in the endometrium of fertile women throughout the menstrual cycle and to investigate whether unexplained infertility may be associated with abnormal expression of IL-1 receptor type I. DESIGN Retrospective study using immunohistochemical technique, Western blot assay, and reverse transcription polymerase chain reaction. SETTING Gynecology clinic and human reproduction research laboratory. PATIENT(S) 39 fertile women and 25 women with unexplained infertility. INTERVENTION(S) Endometrial biopsy of samples obtained at laparoscopy. MAIN OUTCOME MEASURE(S) Immunostaining intensity, molecular weight, and messenger RNA levels of IL-1 receptor type I. RESULT(S) Immunostaining showed that two threshold days (13 and 22) separate the menstrual cycle into three distinct periods of IL-1 receptor type I expression, both in epithelial and stromal cells. Results of Western blot assay and reverse transcription polymerase chain reaction confirmed the immunohistochemical data. Statistical analyses showed that the pattern of IL-1 receptor type I expression was similar in women with unexplained infertility and fertile women. CONCLUSION(S) IL-1 receptor type I exhibits three distinct levels of expression throughout the menstrual cycle in the endometrium of fertile women, suggesting different physiologic roles of the receptor within the cycle. However, IL-1 receptor type I does not seem to be involved in unexplained infertility.

Abnormal Interleukin-1 Receptor Type II Gene Expression in the Endometrium of Women with Endometriosis

Abstract Interleukin 1 (IL-1) is a major proinflammatory cytokine that is believed to play a central role in the pathophysiology of endometriosis. The IL-1 receptor type II (IL-1RII) is known to bind to IL-1 and to inhibit its biological effects. In our previous studies, we showed that human endometrium expresses IL-1RII, and we observed reduced expression of the protein in women with endometriosis. The aim of this study was to investigate IL-1RII mRNA in the endometrial tissue of normal women (n = 26) and of patients with various degrees of endometriosis (n = 53). In situ hybridization showed that IL-1RII mRNA expression was significantly decreased in endometriosis, particularly during the early stages of the disease (stages I and II). This was quite obvious in both glandular and stromal cells, and it was corroborated by reverse transcription-polymerase chain reaction analysis of IL-1RII mRNA in the endometrial tissue of women with (n = 10) and without (n = 8) endometriosis. The reduced levels of IL-1RII mRNA in the endometrium of women suffering from endometriosis reveals a profound defect in IL-1RII gene expression and, consequently, a reduced capability of endometrial tissue to down-regulate IL-1 activity. Defective IL-1RII gene expression during the early stages of endometriosis (stages I and II) may contribute to the etiology of the disease.