{"paper_id":"467135bf-55a1-4c2e-9669-045222bc2e06","body_text":"The high level of RANTES in the ectopic milieu recruits\nmacrophages and induces their tolerance in progression\nof endometriosis\nXiao-Qiu Wang1*, Jing Yu 1*, Xue-Zhen Luo 1, Ying-Li Shi 1, Yun Wang 1, Ling Wang 1\nand Da-Jin Li1,2\n1Laboratory for Reproductive Immunology, Hospital and Institute of Obstetrics and Gynecology, IBS, Fudan University Shanghai Medical College, Sha nghai 200011,\nPeople’s Republic of China\n2Department of Obstetrics and Gynecology, Hainan Medical College Affiliated Hospital, Haikou 570102, People’s Republic of China\n(Correspondence should be addressed to D-J Li at Laboratory for Reproductive Immunology, Hospital and Institute of Obstetrics and Gynecology, IBS,\nFudan University Shanghai Medical College; Email: djli@shmu.edu.cn)\n*(X-Q Wang and J Yu contributed equally to this work)\nAbstract\nRANTES (C–C chemokine, regulated on activation, normal T cell expressed and secreted) is involved in progression of\nendometriosis, but the precise mechanism is understood inadequately. This study is to elucidate the roles of RANTES in\nmacrophage recruitment and tolerance in the endometriotic milieu. The expression of RANTES was analyzed by\nimmunohistochemistry. The cell co-cultures were applied to simulate the endometriotic milieu to investigate the\nregulation of RANTES secretion and its receptor CCR1 expression. Transwell migration assay was used for chemotaxis\nof U937 cells (macrophage line) to endometrial stromal cells (ESCs) and/or human pelvic mesothelial cells. The\nexpression of CCR1 was analyzed by RT-PCR and qPCR in transcription and by western blot in translation respectively.\nConcentrations of RANTES, IL10, and IL12p70 were determined by ELISA. The phenotype of U937 cells and apoptosis\nof ESCs were analyzed by flow cytometry. We have found that the expression of RANTES is significantly higher in the\nendometriotic tissue and eutopic endometrium than that of the normal endometrium without endometriosis. The\ncombination of 17b-estradiol and dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin increases significantly RANTES secretion in\nthe endometriosis-associated cell co-culture which can recruit more macrophages, upregulate CCR1 expression, and\ninduce tolerant phenotype, which inhibits the apoptosis of ESC in the milieu. In conclusion, the higher levels of RANTES\nin the ectopic milieu facilitate the onset and progression of endometriosis by macrophage recruitment and tolerance that\nin turn inhibits apoptosis and enhances growth of ESC.\nJournal of Molecular Endocrinology (2010) 45, 291–299\nIntroduction\nEndometriosis is a common gynecological disorder in\nfertile women that is characterized by the presence and\ngrowth of functional endometrial tissues outside the\nuterine cavity. Although it has been generally accepted\nthat implantation of endometrial cells and fragments\nrefluxed during the menstrual period is the precondi-\ntion for the onset of endometriosis, we are still\nperplexed why the endometrium cannot be efficiently\ncleared in the ectopic milieu. The number of\nleukocytes, particularly macrophages, increases in the\nperitoneal fluid of patients with endometriosis (Hill et al.\n1988). These macrophages seem to have phenotypic\nand functional alterations leading to poor phagocytotic\ncapacity, which is closely related to the severity of\nendometriosis ( Raiter-Tenenbaum et al . 1998 ).\nHowever, the mechanisms by which the ectopic milieu\nmodulates macrophage functions, especially tolerance,\nhave not been fully elucidated. Our previous work has\nindicated that macrophages are involved in ectopic\nadhesion, implantation, and growth of the endome-\ntriotic tissue instead of clearing ( Shi et al . 2006 , 2007,\nYu et al. 2008). Therefore, the peritoneal macrophages\nmay contribute to the development of endometriosis.\nAn understanding of the regulatory mechanism that\ncontrols macrophage functions is valuable to prophy-\nlaxis and therapeutics for endometriosis.\nRANTES, one of the members of C–C chemokine\nfamily, is found to be elevated in peritoneal fluid of\nwomen with endometriosis, and commensurates with\nthe stage of the disease which appears to mediate\nchemotactic activity of the monocytes in peritoneal\ncavity (Hornung et al. 2001a,b), suggesting that it might\ncontribute to the progression of this disease. However,\nthe precise mechanism underlying roles of RANTES in\nendometriosis, has not been well elucidated. It has been\nknown that RANTES mRNA and protein expression can\nbe induced by the pro-inflammatory cytokines IL1 b,\ntumor necrosis factor- a (TNF-a), and interferon g\n291\nJournal of Molecular Endocrinology (2010) 45, 291–299 DOI: 10.1677/JME-09-0177\n0952–5041/10/045–291 q 2010 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org\nDownloaded from Bioscientifica.com at 06/12/2026 03:54:40PM\nvia free access\n\n\n(IFN-g) in the endometrial stroma ( Hornung et al .\n1997, Lebovic et al . 2001 ). Treatment of the endo-\nmetrial cells with estradiol before stimulation with IL1 b\nresults in an increase in RANTES transcription and\nsecretion ( Akoum et al. 2002 ).\nEnvironmental contaminant is also involved in the\npathogenesis of endometriosis. Exposure to 2,3,7,8-\ntetrachlorodibenzo-p-dioxin (TCDD), a highly toxic\nenvironmental contaminant, is associated with an\nincreased prevalence and severity of endometriosis\n(Rier & Foster 2002 ). TCDD appears to be antagonistic\nand analogous to estrogen, so effects of TCDD on the\naction of estrogen and inflammation may be mainly\nmechanic in the pathogenesis of endometriosis. Our\nprevious work has shown that the combination of\n17b-estradiol (E\n2) and TCDD upregulates CXCR1 and\nCCR8 expressions in endometrial stromal cells (ESCs),\nand promotes the secretion of their respective ligands,\nIL8 and I-309, in co-culture of the endometriotic focus-\nassociated cells ( Shi et al . 2006 , 2007). We have also\nfound that the combination of E\n2 and TCDD increases\nsecretion of RANTES in the co-culture that increases\nthe invasion of ESCs ( Yu et al . 2008 ).\nMacrophages have been classified along what is\nviewed as a linear scale: M1 designation is reserved for\nclassically activated macrophages, and the M2 for\ntolerant macrophages ( Martinez et al . 2008 ). M1\nmacrophages that are induced by the combination of\nIFN-g and TNF can produce high levels of pro-\ninflammatory cytokines and mediators as important\ncomponents of host defense ( O’Shea & Murray 2008 ).\nIn addition to immune complexes, other factors can\nprovide a signal for the differentiation of M2\nmacrophages, including apoptotic cells ( Erwig &\nHenson 2007 ), adenosine ( Hasko et al . 2007 ), and\nhyaluronan of moderate weight ( Kuang et al . 2007 ).\nM2 macrophages that produce high levels of the\nimmunosuppressive cytokine IL10 may dampen the\nimmune response and limit inflammation ( Mosser\n2003). Some tumor-associated macrophages may share\ncharacteristics with M2 macrophages ( Biswas et al .\n2006). In addition to IL10 production, M2 macro-\nphages also downregulate IL12 production, and then\nthe ratio of IL10 to IL12 can be used to define M2\nmacrophage ( Martinez et al . 2008 ). Because IL10 can\ninhibit the production and activity of many kinds of\npro-inflammatory cytokines, M2 macrophages are\npotent inhibitors of inflammation, despite the fact\nthat they retain the ability to produce many pro-\ninflammatory cytokines. M1 macrophages have low\nexpression of CD14 and high expression of HLA-DR\nand CD86 on their surface, while M2 macrophages\nhave increased expression of CD14 and decreased\nexpression of HLA-DR and CD86, so the altered\nphenotype can be also applied for identification of\nM2 macrophages ( Kuang et al . 2007 ).\nIn this study, we first observed the effects of E\n2 and\nTCDD on recruiting macrophages mediated by\nRANTES, and then analyzed the expression of CCR1\non macrophages. To better understand the role of\nRANTES in the progression of endometriosis, we\ninvestigated the secretion of IL10 and IL12p70 and\nthe expression of CD14, HLA-DR, and CD86 in\nmacrophages for the regulation of RANTES in the\ntolerance formation of macrophage. Lastly, we investi-\ngated the effect of the tolerant macrophages on\napoptosis of ESC.\nMaterials and methods\nTissue collection and cell culture\nAll the normal endometrium, eutopic endometrium,\nand endometriotic tissues were obtained from 20\npatients with or without endometriosis (mean age\n42\n.5 years; range 34–46), which had been confirmed\nby laparoscopy in the Hospital of Obstetrics and\nGynecology, Fudan University Shanghai Medical\nCollege. The endometriosis patients were classified\naccording to the revised American Fertility Society\n(AFS) classification: six in Stage 2 and nine in Stage 3.\nThe normal endometrium was from five patients\nundergoing surgery for CIN II–III. These patients had\nnot received any GnRH analog or other hormonal drug\nin the 6 months prior to surgery, and the informed\nconsent was obtained before surgery from each patient\nusing protocols approved by the Human Investigation\nCommittee in the Hospital of Obstetrics and Gynecol-\nogy, Fudan University Shanghai Medical College. All\nthe samples were obtained in the proliferative phase of\nthe menstrual cycle, which was confirmed histologically\naccording to the established criteria. The minced\neutopic endometrium was digested with collagenase\ntype I (0\n.1%; Sigma) for 30 min at 37 8C with constant\nagitation. The tissue pieces were filtered through a 200\nand 400 mm wire sieve to remove debris and epithelial\ncells. Following gentle centrifugation, the supernatant\nwas discarded, and the cells were resuspended in\nDMEM/F-12 (Gibco). The cell suspension was layered\nover Ficoll, and centrifuged at 800 g for 20 min to\nfurther remove leukocytes and erythrocytes, and the\nmiddle layer was collected and then washed with\nD-Hanks. The ESCs were cultured in a flask, and\nallowed to adhere for 20 min. The adherent stromal\ncells were cultured as monolayer with DMEM/F-12\ncontaining 10% FCS (Hyclone, Logan, UT, USA),\n20 mmol/l HEPES, 100 IU/ml penicillin, and\n100 mg/ml streptomycin and incubated in 5% CO\n2 at\n37 8C. The purity of ESCs is over 95%.\nHMrSV5 (HPMC, a human peritoneal mesothelial\ncell line provided by Prof. Jian Yao, the First People’s\nX-Q WANG , JY U and others . RANTES in macrophage tolerance to ectopic cells292\nJournal of Molecular Endocrinology (2010) 45, 291–299 www.endocrinology-journals.org\nDownloaded from Bioscientifica.com at 06/12/2026 03:54:40PM\nvia free access\n\n\nHospital, Shanghai, China) and human monocyte\nU937 cell line (purchased from Bank of Cell, Chinese\nAcademy of Sciences, Shanghai, China) were main-\ntained in DMEM (Gibco) with 10% FCS and RPMI 1640\nmedium (Life T echnologies) with 10% bovine calf\nserum respectively and containing 20 mmol/l HEPES,\n100 IU/ml penicillin, and 100 mg/ml streptomycin at\n37 8C in a humidified, 5% CO\n2 incubator. The medium\nwas changed every other day.\nImmunohistochemistry\nThe normal endometrium, eutopic endometrial, and\nendometriotic tissues were cut into serial sections of\n5 mm and were stained by Vectastain Elite ABC kit\n(Vector Laboratories, Burlingame, CA, USA). An\nimmunoperoxidase staining was performed by mono-\nclonal mouse antihuman RANTES antibody (15 mg/ml;\nR&D Systems, Wiesbaden, Germany).\nCell co-culture unit\nContact co-culture of two sorts of cells\nThe ESCs or HPMCs were cultured in 24-well plates at a\nconcentration of 1!105 cells/well until adhering to the\nflask wall. The medium was removed, and then ESCs or\nU937 cells were placed respectively over HPMC or ESC\nrespectively at the same concentration. The cells were\ncultured in a final volume of 1 ml fresh DMEM\nwith 2\n.5% FCS for 48 h. HPMCs, ESCs, and U937 cells\nin 1 !105 cells/well were cultured alone as controls.\nEach experiment was carried out in triplicate and\nrepeated three times.\nContact co-culture of U937–ESC–HPMC\nThe three sorts of cells were plated in the proportion of\n1:1:1. HPMCs were cultured in 24-well plates at a\nconcentration of 1!10\n5 cells/well until adhering to the\nplastic bottom. The medium was removed, and then\nESCs were applied over HPMCs at the same concen-\ntration. After ESCs adhered to the plastic bottom and\nHPMCs, the medium was removed again. U937 cells\nwere applied over ESCs and HPMCs at the same\nconcentration. The cells were cultured in a final volume\nof 1 ml fresh DMEM with 2\n.5% FCS for 48 h. Each\nexperiment was carried out in triplicate and repeated\nthree times.\nNoncontact transwell co-culture of two sorts of cells\nThe two sorts of cells were also plated in the proportion\nof 1:1. The ESCs, HPMCs, and U937 cells at 1 !10\n5\ncells/well were plated respectively at lower or upper\ncompartment of Costar transwell cell culture chamber\ninserts (0 .4 mm, 12 mm diameter). Therefore, three\ndifferent transwell co-cultures of two cells were\nestablished: E-H (ESC–HPMC), E-U (ESC–U937), and\nH-U (HPMC–U937) co-cultures. Each experiment was\ncarried out in triplicate and repeated three times.\nTreatment in vitro with estrogen and/or TCDD\nAfter serum starvation for 12 h, U937 cells were treated\nwith E 2 at a concentration ranging from 10 K10 to\n10K7 mol/l (Sigma), TCDD at a concentration ranging\nfrom 0.01 to 10 nmol/l (Sigma), or the combination of\nE2 (10K8 mol/l) and TCDD (1 nmol/l) for 48 h. Every\nco-culture unit was treated with E 2 (10K8 mol/l) or\nTCDD (1 nmol/l) or with the combination of both\nfor 48 h, with vehicle (DMSO) as controls. Each\nexperiment was carried out in triplicate and repeated\nthree times.\nTranswell migration assay\nAccording to the different cells in the lower chamber,\nthe following two groups were included: first group –\nESCs, HPMCs, or ESC–HPMC co-culture were put into\nthe lower chamber, and U937 cells (2 !105 cells/well)\nwere put into the upper chamber after incubation for\n48 h. Anti-RANTES-neutralizing antibody (R&D\nSystems) at 2\n.5 mg/ml was added to several lower\nchambers half an hour before chemotaxis assay. After\n3 h of incubation, the U937 cells in the lower chamber\nwere counted. Second group – ESCs at 1!105 cells/well\nin the lower chamber are treated with E 2 at 10K8 mol/l,\nTCDD at 1 nmol/l, or the combination of both, and\nother experimental procedure is same as above. U937\ncells were pretreated with anti-CCR1-neutralizing\nantibody (MBL, Nagoya, Japan) at 10 mg/ml or anti-\nCCR5-neutralizing antibody (BD, Franklin Lakes, NJ,\nUSA) at 5 mg/ml half an hour before chemotaxis assay\nfor role of the receptors in U937 recruitment.\nTotal RNA extraction and RT-PCR\nTotal RNA of U937 cells was isolated by using Trizol\nreagent (Gibco). The RT reaction was performed by a\nnonamer primer and 1 mg of RNA in a volume of 20 ml.\nPCR primers of CCR1 and the housekeeping gene\nglyceraldehyde-3-phosphate dehydrogenase ( GAPDH)\nwere as indicated elsewhere ( Shi et al . 2006 ). The PCR\ncycles and conditions for denaturation, annealing, and\nelongation were 35 cycles, 1 min at 94 8C, 30 s at 55 8C,\nand 30 s at 72 8C respectively. The PCR products were\nresolved in 1\n.5% agarose gel and visualized by ethidium\nbromide staining.\nRANTES in macrophage tolerance to ectopic cells . X-Q WANG , JY U and others 293\nwww.endocrinology-journals.org Journal of Molecular Endocrinology (2010) 45, 291–299\nDownloaded from Bioscientifica.com at 06/12/2026 03:54:40PM\nvia free access\n\n\nQuantitative real-time PCR\nTotal RNA of U937 cells was isolated by using Trizol\nreagent (Gibco). Triplicate samples containing cDNA\nwere prepared as mentioned above. SYBR Premix Ex\nTaq, ROX Reference Dye II (TaKaRa Biotechnology Co.\nLtd, Kyoto, Japan), and specific primers were mixed\nand analyzed on an ABI7000 thermal cycler (Applied\nBiosystems, Foster City, CA, USA). The primer\nsequences of CCR1 and housekeeping gene GAPDH\nare indicated in Table 1 and were synthesized by Sangon\nBiotech Co., Ltd (Shanghai, China). The cycling\nconditions consisted of a denaturation step at 95 8C\nfor 30 s, 40 cycles at 95 8C for 5 s, and a 34 s annealing\nstep at 60 8C. To determine the amount of gene product\npresent in the sample, cycle time ( C\nt) was determined.\nThe average Ct value was calculated from triplicate wells\nfor each sample with each primer set. Most duplicate\nsamples varied by !0\n.5 Ct. The relative gene expression\nwas determined by calculating DCt values ( DCt)b y\nsubtracting the Ct value for GAPDH primers from the Ct\nvalue for target gene prim ers. The relative fold\nexpression for each gene was determined by comparing\nwith controls in the experiment. The experiments were\ncarried out in triplicate.\nWestern blots\nWestern blot analysis was performed by antihuman\nCCR1 polyclonal antibody (Abcam, Cambridge, UK),\nand was resolved on SDS-PAGE and transferred to\nimmobilon PVDF membranes. After being soaked in\nblocking buffer, the membrane was incubated with\nprimary antibody overnight at 4 8C. The blots were\ndeveloped using the HRP-linked secondary antibody\nand a chemiluminescent detection system. The experi-\nments were repeated three times.\nELISA for determination of RANTES, IL10,\nand IL12p70\nIn ESC, HPMC, and U937, cultured alone and different\ntwo cells co-culture models, the culture supernatant was\ncollected, and the concentration of RANTES was\nquantified by the ELISA kits (R&D Systems). U937 cells\n(1!10\n6 cells/well in 96-well plates) were cultured with or\nwithout the presence of recombinant human RANTES\n(rhRANTES; Peprotech, Rocky Hill, NJ, USA) at a\nconcentration of 10 ng/ml for 0–96 h, and the concen-\ntration of IL12p70 and IL10 in the culture supernatant\nwas quantified by ELISA kits (R&D Systems) according to\nthe manufacturer’s instructions. The limit of detection\nwas !2\n.0 pg/ml. Each experiment was carried out in\ntriplicate, and repeated three times.\nFlow cytometry for phenotypes of macrophage\nThe U937 cells (5 !105 cells/well) were cultured in six-\nwell plates with or without the presence of rhRANTES\n(Peprotech) at a concentration of 10 ng/ml for 72 h,\nafter which, lipopolysaccharide (LPS; 10 ng/ml) was\nadded to several wells above for 24 h, and then the cells\nwere incubated for 30 min at room temperature with\n80 ml PBS containing 0\n.2% BSA (PBS–BSA) supple-\nmented with 20 ml antihuman CD14, CD86, and HLA-\nDR antibody (eBioscience, San Diego, CA, USA). After\nthat the cells were washed with PBS–BSA, and were\nanalyzed by a FACScan flow cytometer (Becton\nDickinson, Mountain View, CA, USA).\nApoptosis assay for ESC\nTo evaluate the early apoptosis, exposure of phospha-\ntidylserine on a cell surface was examined. The ESCs\nwere cultured in six-well plates with the concentration\nof 5!10\n5 cells/well until adhering to the flask wall. The\nmedium was removed, and then the U937 cells cultured\nalone or pretreated with 10 ng/ml rhRANTES for 72 h\nwere placed over ESCs at the same concentration. After\n48 h, the ESCs were detached, washed, and resus-\npended to 1 !10\n5 cells in 100 ml PBS containing 0 .2%\nBSA (PBS–BSA). The ESCs were then incubated for\n15 min at room temperature with 5mla n n e x i nV – F I T Ca n d\n10 ml propidium iodide (PI; Invitrogen). Here, the apop-\ntosis cells are annexin V–FITC positive and PI negative.\nStatistical analysis\nAll values are shown as mean GS.D., and are analyzed\nby variance (one-way ANOVA: post hoc analysis). P value\n!0.05 is considered statistically significant.\nTable 1 Primer sequences of CCR1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)\nSize (bp) Primer sequences\nGene names\nCCR1 123 Sense: 5 0-AAGGGCTTGGACTCAAGCAAGA-30\nAntisense: 50-TGGAGCCCACAGTCACCACTAC-30\nGAPDH 235 Sense: 5 0-GGGGAGCCAAAAGGGTCATCATCT-30\nAntisense: 50-GAGGGGCCATCCACAGTCTTCT-30\nX-Q WANG , JY U and others . RANTES in macrophage tolerance to ectopic cells294\nJournal of Molecular Endocrinology (2010) 45, 291–299 www.endocrinology-journals.org\nDownloaded from Bioscientifica.com at 06/12/2026 03:54:40PM\nvia free access\n\n\nResults\nImmunohistochemistry of RANTES in the normal\nendometrium, eutopic endometrium, and endome-\ntriotic tissues\nImmunohistochemistry was used to localize RANTES\nprotein in the frozen section. In the normal endome-\ntrium without endometriosis, stromal RANTES\nexpression was observed in seven of the ten prolife-\nrative samples, and was hardly observed in the\nepithelial cells. In all of the 13 eutopic endometrial\nsamples, we observed a significant increase in RANTES\nexpression in epithelial and stromal cells; here the\nstromal cells were stained more intensely than\nthe epithelial cells. The immunostain was localized in\nthe cytoplasm and the membrane of epithelial and\nstromal cells. In 9 of the 11 endometriotic tissues,\nRANTES immunoreactivity in the stromal compart-\nment was observed, but the epithelial cells were hardly\nobserved. The optical density of RANTES immuno-\nstaining in the stromal compartment of the eutopic\nendometrial samples and endometriotic tissues\nappears significantly more intense than that of\nthe normal endometrium without endometriosis\n(P!0\n.05; Fig. 1 ).\nEffect of the co-culture and estradiol or/and TCDD on\nthe secretion of RANTES\nThe cultured U937 cells secreted more RANTES than\nESC and HPMC did. The noncontact co-cultures by\ntranswell had no effect on RANTES secretion. In the\ncontact co-cultures of 24-well plates, neither U937 cells\nnor HPMCs co-cultured with ESCs affected RANTES\nsecretion, whereas the co-culture of HPMC with U937\ncells promoted significantly RANTES production and\nrelease (P!0\n.05; Fig. 2A ).\nEither estradiol or TCDD tended to stimulate\nRANTES secretion in the contact HPMC–U937 co-cul-\nture, but showed no significant difference ( PO0.05).\nThe combination of them appeared to have a\nsynergistic stimulatory effect on RANTES secretion in\nthis co-culture ( P!0\n.05; Fig. 2B ).\nEffect of the co-culture on U937 migration\nThe ESC–HPMC co-culture could significantly\nenhance the chemotaxis of U937 compared with the\ncontrol or ESCs or with HPMCs cultured alone, and\nanti-RANTES-neutralizing antibody partly inhibited\nthis effect but this was still higher than that of ESCs\nor HPMCs cultured alone ( P!0.01; Fig. 3A ).\nEffect of estradiol and/or TCDD on U937 migration\nin the co-culture\nE2 or TCDD alone had no effect on the migration of\nU937 cells to ESCs, but the combination of both could\nobviously promote the chemotaxis of U937 cells\n(P!0.01). Anti-CCR1, -CCR5, and -RANTES-neutralizing\nantibody inhibited partly the effect of the combination\n(P!0.01; Fig. 3B).\nEffect of estradiol or/and TCDD on CCR1 expression\nin U937 cells\nIt was shown in Fig. 4 that the mRNA transcription and\nprotein translation of CCR1 in U937 cells were\nincreased in a dose-dependent manner after treated\nwith E 2 or TCDD. The combination of both could\nfurther promote the expression of CCR1 ( P!0.01).\nFigure 1 Immunohistochemistry of RANTES in the normal\nendometrium, eutopic endometrium, and the ectopic tissues.\nRepresentative micrographs of immunohistochemical staining for\nRANTES in the normal endometrium, eutopic endometrium, and\nthe endometriotic tissues. Magnification, !400.\n4000 Non-contact\nA\nB\n*Contact\nESC HPMC U937 E-U E-H H-U\nH-U\nRANTES secretion (pg/ml)\n3000\n2000\n1000\nRANTES secretion (pg/ml)2000\nControl E 2 TCDD E 2+TCDD\n*\n2200\n2400\n2600\n2800\n0\nFigure 2 The H-U co-culture and the combination of 17b-estradiol\nand TCDD promote the secretion of RANTES. ESCs, HPMCs,\nand U937 cells alone and E-U, E-H, and H-U co-culture were\ncultured in the noncontact transwell or cell–cell contact 24-well\nplates for 48 h (A). The H-U co-culture in 24-well plates was\ntreated with 17b-estradiol (10\nK8 mol/l) or/and TCDD (1 nmol/l) for\n48 h (B). The levels of RANTES in the supernatants were\ndetermined by ELISA. Data are expressed as meanG\nS.E.M.\n*P!0.05 compared with others.\nRANTES in macrophage tolerance to ectopic cells . X-Q WANG , JY U and others 295\nwww.endocrinology-journals.org Journal of Molecular Endocrinology (2010) 45, 291–299\nDownloaded from Bioscientifica.com at 06/12/2026 03:54:40PM\nvia free access\n\n\nEffects of the co-culture and estradiol or/and TCDD on\nthe expression of CCR1 in U937 cells\nCo-culture could promote the expression of CCR1 in\nU937 cells compared to U937 cultured alone ( P!0.01;\nFig. 5 ), especially the contact U-H-E co-culture. TCDD\ncould promote the expression of CCR1 in U937 cells\ncompared to the control in U-H and U-H-E co-culture\n(P!0.01; Fig. 5), and the combination of E2 with TCDD\nfurther increased the protein translation of CCR1\n(P!0.01). In all the co-cultures, the combination of\nE2 with TCDD could upregulate the expression of CCR1\ncompared to the control ( P!0.01; Fig. 5 ).\nRANTES can induce macrophage tolerance\nTo investigate the effect of RANTES on polarization of\nmacrophages, rhRANTES (10 ng/ml) was used to treat\nmacrophages for 0–96 h. The results revealed that\nRANTES could change the balance between the release\nof IL10 and IL12p70 of macrophages with the\nupregulation of IL10 production and downregulation\nof IL12p70 production ( Fig. 6A ). rhRANTES could\ninduce macrophage tolerance with increased expre-\nssion of CD14, and decreased expression of HLA-DR\nand CD86 (P!0\n.05; Fig. 6B). The RANTES-treated cells\nwere refractory to stimulation with LPS ( Fig. 6B). These\nresults above indicate that RANTES can induce the\nformation of tolerant phenotype of macrophages.\nThe effect of tolerant macrophages on the apoptosis\nof ESCs\nESCs co-cultured with U937 cells showed significantly\nlower apoptosis rate than ESCs cultured alone, and\nthe apoptosis of ESCs did decrease further if\nco-cultured with the U937 cells pretreated with\nRANTES ( P!0\n.05; Fig. 7 ).\nDiscussion\nEndometriosis is a chronic inflammation disease. There\nare increased numbers of activated peritoneal macro-\nphages in patients with endometriosis ( Wu et al. 2002).\nThese macrophages cannot effectively clear the ectopic\nendometrial cells ( Raiter-Tenenbaum et al . 1998 ), and\ninversely secrete multiple kinds of inflammatory\nmediator to contribute to the progression of endo-\nmetriosis, for instance IL8 and I-309 ( Shi et al . 2006 ,\n2007). In view of this, macrophages play an important\nrole in the onset and development of this disease.\nHowever, our understanding of the macrophages’\nactions in the endometriotic milieu is still inadequate.\n6\n*\nMigration rate of U937 (%)\n5\n*\n++++ ++++Upper chamber  U937\nLower chamber\n–+–– –+––ESC\n––+– ––+–HPMC\n–––+ –––+ESC+HPMC\n–––– ++++Anti-RANTES\n*\n* *\n*\n#4\n3\n2\n1\n0\n6\nMigration rate\nof U937 (%)\n5\n*\n*\n#\n*\n#\n*\n#\n–+–++++E2\n––+++++TCDD\n––––+––a-CCR1\n–––––+–a-CCR5\n––––– –+a-RANTES\n4\n3\n2\n1\n0\nA\nB\nFigure 3 The co-culture and combination of 17 b-estradiol and\nTCDD increase chemotaxis of U937 through secreting RANTES.\nIn the transwell assay, ESCs, HPMCs, or ESC–HPMC co-culture\nwere put in the lower chamber, and U937 cells were put in the\nupper chamber. The ESC–HPMC co-culture significantly\nincreased the chemotaxis of U937, and anti-RANTES-neutralizing\nantibody partly inhibited the increased chemotaxis of U937 cells\n(A). The combination of 17b-estradiol and TCDD promoted\nmigration of U937 cells to ESCs, and anti-CCR1, -CCR5, or\n-RANTES-neutralizing antibody partly inhibited the effect of\n17b-estradiol and TCDD (B). *P!0\n.01 compared with control.\n#P!0.01 compared with U-E-H co-culture unit control (A) or ESC\ntreated with the combination of 17b-estradiol and TCDD (B).\n5·0 RT-PCR\nE2 TCDD\nCCR1\nGAPDH\nCCR1\nβ-Actin\nCombine\nE2 TCDD Combine\nReal-time qPCR\nTranscription Translation\n*\n*\n*\n*\n*\n*\n* *\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n*\n* *\n*\n*\nCCR1/GAPDH\n4·5\n4·0\n3·5\n3·0\n2·5\n2·0\n1·5\n1·0\n0·5\n0·0\nmRNA expression of CCR1\n(in fold)\n0\n5\n10\n15\n25\n20\n3·5\nE2 TCDD Combine\nCCR1/β-actin\n3·0\n2·5\n2·0\n1·5\n1·0\n0·5\n0·0\nFigure 4 17b-estradiol or/and TCDD promote the expression of\nCCR1 in U937. Treatment with 17b-estradiol (10K10,1 0K9,1 0K8,\nand 10K7 mol/l successively) or TCDD (0.01, 0.1, 1, and 10 nmol/l\nsuccessively) upregulated the mRNA transcription and protein\ntranslation of CCR1 in U937 cells; here, 17b-estradiol at\n10K8 mol/l and TCDD at 1 nmol/l showed a synergetic effect.\n*P!0.01 compared with control.\nX-Q WANG , JY U and others . RANTES in macrophage tolerance to ectopic cells296\nJournal of Molecular Endocrinology (2010) 45, 291–299 www.endocrinology-journals.org\nDownloaded from Bioscientifica.com at 06/12/2026 03:54:40PM\nvia free access\n\n\nIn this study, immunohistochemistry has confirmed\nthat RANTES is translated significantly higher in the\neutopic endometrium and ectopic tissues than in the\nnormal endometrium. The observations are consistent\nwith the findings of previous studies ( Hornung et al .\n2001a,b, Fang et al. 2009), which led us to propose that\nRANTES overexpression in the eutopic endometrium\nand endometriotic tissues may exert a potential role in\nthe pathogenesis of endometriosis.\nOur present data show that U937 cells spontaneously\nsecrete much more RANTES than ESCs and HPMCs do.\nHPMCs cultured alone in nature secrete few RANTES,\neven if stimulated by the combination of E 2 and TCDD\n(Wang XQ, unpublished observations). The direct cell\ncontact of HPMC and U937 significantly promotes the\nsecretion of RANTES, which means that the interaction\nof the endometriotic focus-associated cells does aggra-\nvate peritoneal inflammation by stimulating the release\nof RANTES. Further study showed that E\n2 or TCDD\nincreased RANTES secretion in the HPMC–U937\nco-culture unit, and the combination of them has a\nsynergistic effect, which implies that the increased\nmacrophages in peritoneal cavity of endometriosis\ninteract with HPMCs to stimulate RANTES production.\nThe chronic exposure to E\n2 and TCDD aggravates the\ninflammatory status by stimulating pro-inflammatory\ncytokine RANTES secretion, which leads to a persistent\nand serious inflammation, and finally results in the\nformation of an endometriotic focus.\nThe macrophages in peritoneal fluid are terminal\ncells without proliferation. Therefore, the monocytes’\nrecruitment from peripheral blood into peritoneal\nfluid is an important step for the onset of endome-\ntriosis. It has been shown that RANTES accounts for the\nmajority of the monocyte chemotactic activity in the\nectopic ESC-conditioned media ( Fang et al . 2009 ). In\nthis study, we have demonstrated that ESC–HPMC\nco-culture apparently promotes the chemotaxis of\nmacrophages more than ESC or HPMC cultured\nalone, and anti-RANTES-neutralizing antibody can\npartly inhibit the migration of macrophages in ESC–\nHPMC co-culture. Our previous work has shown that\nESC–HPMC co-culture promotes RANTES secretion of\nESCs ( Yu et al . 2008 ), which suggests that the retro-\ngraded ESCs in peritoneal fluid interact with HPMCs\nleading to the recruitment of macrophages through\nincreasing the secretion of RANTES. The combination\nof E\n2 and TCDD increases the migration of macro-\nphages by stimulating ESC to secret more RANTES and\nanti-RANTES-neutralizing antibody, and anti-CCR1 or\nanti-CCR5-neutralizing antibody can decrease but not\nblock completely the migration of macrophages, which\nsuggests that other cytokines and chemokines induced\nby E\n2 and TCDD may be involved in this process.\n0·6CCR1/β-actin\n0·5\n0·4\n0·3\n0·2\n0·1\n0\nCCR1\nβ-Actin\nCon\n*\n*\n*\nU937\nE\n2 TCDD E 2+TCDD\n1·2CCR1/β-actin\n1\n0·8\n0·6\n0·4\n0·2\n0\nCCR1\nβ-Actin\nCon\n* *\nU937-HPMC\nE\n2 TCDD E 2+TCDD\n0·7\n0·6CCR1/β-actin\n0·5\n0·4\n0·3\n0·2\n0·1\n0\nCCR1\nβ-Actin\nCon\n*\nU937-ESC\nE\n2 TCDD E 2+TCDD\n2·5CCR1/β-actin\n2\n1·5\n1\n0·5\n0\nCCR1\nβ-Actin\nCon\n*\n*\nU937-ESC-HPMC\nE2 TCDD E 2+TCDD\nFigure 5 The combination of 17b-estradiol and TCDD upregulates\nCCR1 protein translation in U937 and the co-cultures. U937 cells\nwere cultured alone or co-cultured with HPMCs, ESCs, or HPMCs\nand ESCs respectively, and were treated with 17b-estradiol at\n10\nK8 mol/l or/and TCDD at 1 nmol/l for 48 h. The CCR1\nexpression was detected by western blot. Data are expressed\nas meanGS.D.* P!0.01 compared with control.\n70\nIL10A\nB\npg/ml\n60\n50\n40\n30\n20\n10\n00 1 22 43 64 86 07 28 49 6  ( h )\n120\nIL12p70\npg/ml\n100\n80\n60\n40\n20\n00 1 22 43 64 86 07 28 49 6  ( h )\n40\nCD14 HLA-DR CD86 CD14 HLA-DR CD86\nPositive rate (%)\n35\n30\n25\n20\n15\n10\n5\n0\n60 Positive rate (%)\n50\n40\n30\n20\n10\n0\n50\nFI\n45\n40\n30\n35\n25\n20\n15\n10\n5\n0\nrhRANTES\n+rhRANTES\n–\nLPS\n–\n–\n+\n–\n–\n–\n+\n–\n–\n–\n+\n–\n–\n+\n+\n+\n–\n+\n+\n+\n–\n+\n+\n+\n70\n60 FI\n50\n40\n30\n20\n10\n0\n*\n*\n*\n* *\n* * *\n*\nFigure 6 RANTES induces the tolerance phenotype formation of\nmacrophage. U937 cells were cultured for 0–96 h with or without\nthe presence of 10 ng/ml rhRANTES, and then levels of IL12p70\nand IL10 in the supernatant were determined by ELISA (A). U937\ncells were treated with or without 10 ng/ml rhRANTES for 72 h,\nand were then stimulated with or without LPS (10 ng/ml) for 24 h.\nExpressions of CD14, HLA-DR, and CD86 in U937 cells were\ndetermined respectively by flow cytometry for positive rate and\nfluorescence intensity (FI) of the correspondent surface\nmolecules (B). Data are expressed as meanG\nS.E.M.* P!0.05.\nRANTES in macrophage tolerance to ectopic cells . X-Q WANG , JY U and others 297\nwww.endocrinology-journals.org Journal of Molecular Endocrinology (2010) 45, 291–299\nDownloaded from Bioscientifica.com at 06/12/2026 03:54:40PM\nvia free access\n\n\nCCR1 and CCR5 are the receptors of RANTES, and\nCCR5 is highly expressed in U937 cells, but is not\naffected by E 2 or TCDD (Wang XQ, unpublished\nobservations). In this study, we have found that\nalthough CCR1 is lowly expressed in U937 in nature,\nE\n2 or TCDD alone can upregulate CCR1 mRNA\ntranscription and protein translation, and the com-\nbination of both further enhances this effect. There is\na high level of RANTES in peritoneal fluid with\nendometriosis, which is related to the severity of the\ndisease ( Bersinger et al .2 0 0 6). Therefore, in the\npresence of E\n2 and TCDD, the high expression of\nCCR5 and CCR1 in macrophages may make them more\nsensitive to RANTES, thereby promoting their\nmigration to the peritoneal cavity.\nAfter monocytes are recruited into the peritoneal\ncavity, the abdominal milieu became more complex\ndue to the interaction of ESC, HPMC, and mono-\nmacrophages, which aggravates peritoneal inflam-\nmation. Hence, we have established different co-culture\nunits of these cells respectively to mimic various\nperitoneal local inflammation situations of the ectopic\nmilieu. It has been found that all the co-culture units\ncan promote the protein translation of CCR1 in\nmacrophages with comparison to the macrophages\ncultured alone. Furthermore, the combination of E\n2\nand TCDD can promote CCR1 expression in the\nmacrophages in all the co-culture with ESCs or/and\nHPMCs. Therefore, the combination of E\n2 and TCDD\ncan not only promote the secretion of RANTES, but can\nalso upregulate the expression of CCR1 in macro-\nphages. The dual regulation for the chemokine and its\nreceptor determines the pathogenic roles of RANTES\nin endometriosis.\nIn this study, we have found that rhRANTES can\ninduce the formation of tolerant macrophages with\nCD14\nhighHLA-DRlowCD86low phenotype and increased\nIL10/IL12 ratio. During their exposure to the ectopic\nmilieu, the newly recruited monocytes may be induced\ntolerant. Our results give a new insight into the formation\nof tolerant macrophages in endometriosis. RANTES\nderived from the endometriotic focus-associated cells\ncan change the polarization of macrophage, creating\nthe conditions that redound to endometriosis pro-\ngression. Therefore, the regulation of the tolerant\nmacrophage phenotype may shed light on developing\nnew therapeutic regimens for endometriosis.\nWe then address how the tolerant macrophages affect\nthe apoptosis of ESC. It has been found that the\napoptosis of ESCs appears significantly decreased when\nco-cultured with macrophages, which implies that\nmacrophages may inhibit the apoptosis of ESC by\nsecreting soluble cytokines. The macrophages pre-\ntreated with RANTES would be tolerant, and these\ntolerant macrophages show an increased capacity to\ninhibit the apoptosis of ESC in comparison with the\nmacrophages without RANTES treatment, which\nindicates that high levels of RANTES produced in the\nectopic milieu can induce the formation of tolerant\nmacrophages that in turn promote the ectopic ESC\ngrowth in the progression of endometriosis.\nIn conclusion, based on the results of this study as\nwell as others, a hypothetical model may be proposed to\nelucidate the onset and progression of endometriosis.\nThe retrograde endometrium activates inflammation,\nand the interaction of the endometriotic focus-associ-\nated cells increases the secretion of RANTES and other\ncytokines, which is further aggravated by combination\nof high levels of estrogen with TCDD. The increased\nRANTES recruits more macrophages into the ectopic\nmilieu. Meanwhile, the interaction of endometriosis-\nassociated cells and the combination of estrogen and\nTCDD upregulate CCR1 and CCR5 expression in\nmacrophages. Therefore, the macrophages recruited\ninto the ectopic milieu present increased sensitivity to\nRANTES. The high levels of RANTES not only recruit\nbut also induce macrophages tolerant. These tolerant\nmacrophages inhibit the apoptosis and promote growth\nof ESC in the endometriotic milieu, which shows\npotential value for prophylaxis and therapeutics of\nendometriosis.\nDeclaration of interest\nThe authors declare that there is no conflict of interest that could be\nperceived as prejudicing the impartiality of the research reported.\n14\n**\n*\nESC apoptosis (%)\n12\n10\n8\n6\n4\n2\n0\nESC + + +\nU937 – + –\nU937 pretreated\nwith rhRANTES\n104\n103\nPI102\n101\n100\n100 101\nAnnexin V-FITC\n12\n102 103 104\n104\n103\nPI102\n101\n100\n100 101\nAnnexin V-FITC\n4·71\n102 103 104\n104\n103\nPI102\n101\n100\n100 101\nAnnexin V-FITC\n0·42\n102 103 104\n–– +\nFigure 7 The RANTES-induced tolerant macrophages inhibit\napoptosis of ESC. ESCs were cultured alone or co-cultured with\nU937 cells which were pretreated with or without rhRANTES\n(10 ng/ml), and then ESCs were collected to evaluate their\napoptosis by flow cytometry. *P!0\n.05.\nX-Q WANG , JY U and others . RANTES in macrophage tolerance to ectopic cells298\nJournal of Molecular Endocrinology (2010) 45, 291–299 www.endocrinology-journals.org\nDownloaded from Bioscientifica.com at 06/12/2026 03:54:40PM\nvia free access\n\n\nFunding\nThis work is supported by National Basic Research Program of China\n2006CB944007 (to D-J L), National and Shanghai Leading Academic\nDiscipline Project 211XK22 (to D-J L), Program for Outstanding\nMedical Academic Leader of Shanghai (to D-J L), and Creative\nFoundation for Graduate Students of Fudan University EYF157015\n(to X-Q W).\nAuthor contribution statement\nX-Q W conducted all the experiments, prepared the figures, and wrote\nthe manuscript. J Y undertook migration, RT-PCR, and western blot\nanalysis, X-Z L completed immunohistochemistry, and Y-L S\ndetermined RANTES by ELISA. Y W and L W collected specimens\nand clinical data. 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Human Reproduction 23 1614–1626.\n(doi:10.1093/humrep/den125)\nReceived in final form 21 June 2010\nAccepted 23 August 2010\nMade available online as an Accepted Preprint 23 August 2010\nRANTES in macrophage tolerance to ectopic cells . X-Q WANG , JY U and others 299\nwww.endocrinology-journals.org Journal of Molecular Endocrinology (2010) 45, 291–299\nDownloaded from Bioscientifica.com at 06/12/2026 03:54:40PM\nvia free access","source_license":"CC0","license_restricted":false}