Produção Nacional
Revisado por pares
IL-1β Stimulates Brain-Derived Neurotrophic Factor Production in Eutopic Endometriosis Stromal Cell Cultures
2018; Elsevier BV; Volume: 188; Issue: 10 Linguagem: Inglês
10.1016/j.ajpath.2018.06.011
ISSN1525-2191
AutoresJie Yu, A.M.C. Francisco, Bansari G. Patel, J. Mark Cline, Eric Zou, Sarah L. Berga, Robert N. Taylor,
Tópico(s)Reproductive System and Pregnancy
ResumoEndometriosis implants are comprised of glandular and stromal elements, macrophages, nerves, and blood vessels and are commonly accompanied by pelvic pain. We propose that activated macrophages are recruited to and infiltrate nascent lesions, where they secrete proinflammatory cytokines, promoting the production of chemokines, neurotrophins, and angiogenic growth factors that sustain an inflammatory microenvironment. Immunohistochemical evaluation of endometriosis lesions reveals in situ colocalization of concentrated macrophages, brain-derived neurotrophic factor (BDNF), and nerve fibers. These observations were coupled with biochemical analyses of primary eutopic endometriosis stromal cell (EESC) cultures, which allowed defining potential pathways leading to the neuroangiogenic phenotype of these lesions. Our findings indicate that IL-1β potently (EC50 = 7 ± 2 ng/mL) stimulates production of EESC BDNF at the mRNA and protein levels in an IL-1 receptor–dependent fashion. Selective kinase inhibitors demonstrate that this IL-1β effect is mediated by c-Jun N-terminal kinase (JNK), NF-κB, and mechanistic target of rapamycin signal transduction pathways. IL-1β regulation of regulated on activation normal T cell expressed and secreted (RANTES), a prominent EESC chemokine, also relies on JNK and NF-κB. An important clinical implication of the study is that interference with BDNF and RANTES production, by selectively targeting the JNK and NF-κB cascades, may offer a tractable therapeutic strategy to mitigate the pain and inflammation associated with endometriosis. Endometriosis implants are comprised of glandular and stromal elements, macrophages, nerves, and blood vessels and are commonly accompanied by pelvic pain. We propose that activated macrophages are recruited to and infiltrate nascent lesions, where they secrete proinflammatory cytokines, promoting the production of chemokines, neurotrophins, and angiogenic growth factors that sustain an inflammatory microenvironment. Immunohistochemical evaluation of endometriosis lesions reveals in situ colocalization of concentrated macrophages, brain-derived neurotrophic factor (BDNF), and nerve fibers. These observations were coupled with biochemical analyses of primary eutopic endometriosis stromal cell (EESC) cultures, which allowed defining potential pathways leading to the neuroangiogenic phenotype of these lesions. Our findings indicate that IL-1β potently (EC50 = 7 ± 2 ng/mL) stimulates production of EESC BDNF at the mRNA and protein levels in an IL-1 receptor–dependent fashion. Selective kinase inhibitors demonstrate that this IL-1β effect is mediated by c-Jun N-terminal kinase (JNK), NF-κB, and mechanistic target of rapamycin signal transduction pathways. IL-1β regulation of regulated on activation normal T cell expressed and secreted (RANTES), a prominent EESC chemokine, also relies on JNK and NF-κB. An important clinical implication of the study is that interference with BDNF and RANTES production, by selectively targeting the JNK and NF-κB cascades, may offer a tractable therapeutic strategy to mitigate the pain and inflammation associated with endometriosis. Endometriosis is a debilitating gynecologic disorder characterized by the growth of endometrial tissue outside the uterine cavity, which commonly is accompanied by infertility and pelvic pain. A recent epidemiologic survey indicates that the overall prevalence of endometriosis among reproductive-age women is approximately 11%.1Buck Louis G.M. Hediger M.L. Peterson C.M. Croughan M. Sundaram R. Stanford J. Chen Z. Fujimoto V.Y. Varner M.W. Trumble A. Giudice L.C. ENDO Study Working GroupIncidence of endometriosis by study population and diagnostic method: the ENDO study.Fertil Steril. 2011; 96: 360-365Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar From data extrapolated from the World Bank, it is estimated that >176 million women are affected globally.2Adamson G.D. Kennedy S.H. Hummelshoj L. Creating solutions in endometriosis: global collaboration through the World Endometriosis Research Foundation.J Endometriosis. 2010; 2: 3-6Crossref Scopus (120) Google Scholar Careful estimates of annual health care expenses and loss of productivity secondary to endometriosis-associated pain averaged the equivalent of $11,300 in affected women from the United States and nine European nations.3Simoens S. Dunselman G. Dirksen C. Hummelshoj L. Bokor A. Brandes I. Brodszky V. Canis M. Colombo G.L. DeLeire T. Falcone T. Graham B. Halis G. Horne A. Kanj O. Kjer J.J. Kristensen J. Lebovic D. Mueller M. Vigano P. Wullschleger M. D'Hooghe T. The burden of endometriosis: costs and quality of life of women with endometriosis and treated in referral centres.Hum Reprod. 2012; 27: 1292-1299Crossref PubMed Scopus (569) Google Scholar Endometriotic implants are commonly found on the pelvic peritoneal surface and penetrating the ovarian cortex, but the most symptomatic variant is deep infiltrating endometriosis (DIE) in which lesions invade the cul-de-sac and rectovaginal septum.4Koninckx P.R. Ussia A. Adamyan L. Wattiez A. Donnez J. Deep endometriosis: definition, diagnosis, and treatment.Fertil Steril. 2012; 98: 564-571Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar The pathogenetic mechanisms that link endometriosis and pelvic pain remain unclear, although their association is clinically well established.5Morotti M. Vincent K. Becker C.M. Mechanisms of pain in endometriosis.Eur J Obstet Gynecol Reprod Biol. 2017; 209: 8-13Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 6Becker C.M. Gattrell W.T. Gude K. Singh S.S. Reevaluating response and failure of medical treatment of endometriosis: a systematic review.Fertil Steril. 2017; 108: 125-136Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar A prevailing immunocentric theory attributes inflammation through the recruitment of macrophages and other activated leukocytes from bone marrow to the endometriotic lesions by chemokines synthesized in situ.7Taylor R.N. Hummelshoj L. Stratton P. Vercellini P. Pain and endometriosis: etiology, impact, and therapeutics.Middle East Fertil Soc J. 2012; 17: 221-225Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 8Taylor R.N. Lebovic D.I. Endometriosis.in: Strauss J.F. Barbieri R.L. Yen and Jaffe's Reproductive Endocrinology. ed 7. Saunders Elsevier, Philadelphia2014: 565-585Crossref Scopus (9) Google Scholar, 9Wu J. Xie H. Yao S. Liang Y. Macrophage and nerve interaction in endometriosis.J Neuroinflammation. 2017; 14: 53Crossref PubMed Scopus (73) Google Scholar One of the postulated consequences of immune cell activation in the endometriosis microenvironment is the elaboration of cytokines, growth factors, and eicosanoids that simultaneously stimulate lesion innervation and neovascularization through a coordinated mechanism that was coined neuroangiogenesis.10Asante A. Taylor R.N. Endometriosis: the role of neuroangiogenesis.Annu Rev Physiol. 2011; 73: 163-182Crossref PubMed Scopus (127) Google Scholar Eutopic endometrial tissues from women with endometriosis were found to have significantly higher nerve density than those from unaffected control participants.11Tokushige N. Markham R. Russell P. Fraser I.S. Nerve fibres in peritoneal endometriosis.Hum Reprod. 2006; 21: 3001-3007Crossref PubMed Scopus (222) Google Scholar, 12Bokor A. Kyama C.M. Vercruysse L. Fassbender A. Gevaert O. Vodolazkaia A. De Moor B. Fulop V. D'Hooghe T. Density of small diameter sensory nerve fibres in endometrium: a semi-invasive diagnostic test for minimal to mild endometriosis.Hum Reprod. 2009; 24: 3025-3032Crossref PubMed Scopus (98) Google Scholar In an agnostic proteomic screen of factors that stimulate nerve growth, endometrial brain-derived neurotrophic factor (BDNF) expression was increased more than eightfold in women with endometriosis.13Browne A.S. Yu J. Huang R.P. Francisco A.M. Sidell N. Taylor R.N. Proteomic identification of neurotrophins in the eutopic endometrium of women with endometriosis.Fertil Steril. 2012; 98: 713-719Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar Circulating serum BDNF concentrations also are reported to be significantly (1.5-fold) higher in cases than in controls.14Wessels J.M. Kay V.R. Leyland N.A. Agarwal S.K. Foster W.G. Assessing brain-derived neurotrophic factor as a novel clinical marker of endometriosis.Fertil Steril. 2016; 105: 119-128Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar In keeping with the theory of neuroangiogenesis, BDNF is known to be trophic both for nerves and capillaries.15Reichardt L.F. Neurotrophin-regulated signalling pathways.Philos Trans R Soc Lond B Biol Sci. 2006; 361: 1545-1564Crossref PubMed Scopus (1625) Google Scholar, 16Matsuda S. Fujita T. Kajiya M. Takeda K. Shiba H. Kawaguchi H. Kurihara H. Brain-derived neurotrophic factor induces migration of endothelial cells through a TrkB-ERK-integrin alphaVbeta3-FAK cascade.J Cell Physiol. 2012; 227: 2123-2129Crossref PubMed Scopus (44) Google Scholar In the present studies we set out to test the hypothesis that macrophages in the endometriosis microenvironment may contribute to neuroangiogenesis by secretion of proinflammatory cytokines. Tissue macrophages, BDNF, and nerve fibers were colocalized in DIE lesions, and a primary cell culture model from subjects with endometriosis-associated pain was used to address the mechanisms by which macrophage-derived cytokines may induce BDNF mRNA and protein expression in eutopic endometriosis stromal cells (EESCs). The findings support the concept that cytokine cross talk among heterogeneous cell types in endometriosis lesions can lead to the recruitment of vessels and nerves, supporting lesion vascularization, survival, invasion, and nociception. Eight patients undergoing laparoscopy provided written informed consent under study protocols approved by the institutional review boards at Wake Forest School of Medicine, Winston-Salem, NC, and the Health Sciences School, Universidade do Vale do Sapucaí, Pouso Alegre, Minas Gerais, Brazil. All of the patients had regular menstrual cycles, had not received hormonal therapy for at least 3 months before surgery, and were undergoing laparoscopy for evaluation and treatment of pelvic pain. Six of the eight patients were nulligravid and all eight reported dysmenorrhea and dyspareunia. Immediately before laparoscopy, eutopic endometrial biopsies were collected by Pipelle aspiration under sterile conditions in five of the patients, and these were promptly transported to the laboratory on ice in phosphate-buffered saline. At laparoscopy, a thorough visual inspection of the pelvic cavity was performed by experienced gynecologic surgeons familiar with the appearance of typical and atypical endometriotic lesions. Intraoperative excisional biopsies from all eight patients (some of whom had multiple lesion types) confirmed the presence of histopathologic endometriosis (ie, endometrial glands, stroma, and hemosiderocytes). Five of the patients met criteria for DIE, and each had a single, dominant nodular lesion that involved the sigmoid colon, rectovaginal septum, or uterosacral ligament with invasion >5 mm. The clinical characteristics and intraoperative revised American Society for Reproductive Medicine endometriosis staging17Revised American Society for Reproductive Medicine classification of endometriosis: 1996.Fertil Steril. 1997; 67: 817-821Abstract Full Text PDF PubMed Scopus (2286) Google Scholar of the participants are provided in Table 1.Table 1Study ParticipantsAge, yearsGravity/parityPhase (d)rASRM stageSymptomsLesion locationsDIEStudy270/0Prolif (8)IVPain/infertilitySig noduleYesIHC311/1Prolif (1)IVPain/infertilityVag noduleYesIHC330/0Prolif (11)IVPain/infertilityVag noduleYesIHC310/0Prolif (9)IVPain/infertilitySig noduleYesIHC/CX312/1Prolif (6)IIIPainPerit/L ovNoCX360/0Prolif (10)IIPainL tube, ovNoCX350/0Prolif (8)IVPain/infertilityR ov, vag noduleYesCX230/0Prolif (5)IIPain/infertilityPerit/R ovNoCXCX, cell culture; d, cycle day; DIE, deep infiltrating endometriosis; IHC, immunohistochemistry; L, left; ov, ovary; perit, peritoneal implant; prolif, proliferative phase of menstrual cycle; R, right; rASRM, revised American Society for Reproductive Medicine endometriosis stage; sig, sigmoid; study, protocol; vag, vaginal. Open table in a new tab CX, cell culture; d, cycle day; DIE, deep infiltrating endometriosis; IHC, immunohistochemistry; L, left; ov, ovary; perit, peritoneal implant; prolif, proliferative phase of menstrual cycle; R, right; rASRM, revised American Society for Reproductive Medicine endometriosis stage; sig, sigmoid; study, protocol; vag, vaginal. Paraffin-embedded nodular DIE lesions from each of four participants were sectioned 4- to 5-μm thick and subjected to hematoxylin and eosin staining and immunohistochemistry (IHC) as described previously18Wood C.E. Borgerink H. Register T.C. Scott L. Cline J.M. Cervical and vaginal epithelial neoplasms in cynomolgus monkeys.Vet Pathol. 2004; 41: 108-115Crossref PubMed Scopus (41) Google Scholar, 19Yu J. Berga S.L. Johnston-MacAnanny E.B. Sidell N. Bagchi I.C. Bagchi M.K. Taylor R.N. Endometrial stromal decidualization responds reversibly to hormone stimulation and withdrawal.Endocrinology. 2016; 157: 2432-2446Crossref PubMed Scopus (42) Google Scholar with some modifications. After mounting, the slides were deparaffinized in xylene, rehydrated in graded concentrations of ethanol, and endogenous hydrogen peroxide was quenched in 3% hydrogen peroxide in methanol. Primary antibodies to CD68 (macrophages; catalog number M0876; Dako, Carpinteria, CA), BDNF (neurotrophic factor; catalog number 3160-1; Epitomics, Burlingame, CA), and PGP9.5 (nerve fibers; catalog number Z5116; Dako) were diluted to 1:50, 1:100, and 1:200, respectively, in Tris buffer, pH 7.4, containing 0.5% casein as a blocking reagent. Sections were incubated overnight at 4°C with the primary antibodies. For heat-induced epitope retrieval, slides were incubated in 10 mmol/L citrate buffer (pH 6.0) for 4 minutes in a pressure cooker. Biotinylated secondary Fc antibody was used as a linking reagent for all antibodies, and alkaline-phosphatase–conjugated streptavidin (BioGenex, San Ramon, CA) was applied for labeling. The chromogen was Vector Red Substrate (kit number 1; Vector Laboratories, Burlingame, CA), diluted in Tris (pH 8.2 to 8.5), and applied to slides for 5 to 10 minutes at 30°C to 35°C. Mild counterstaining with Mayer's hematoxylin was performed. Negative staining controls were performed by omitting the primary antibody and substituting nonimmune IgG (BioGenex). For positive controls, myenteric plexi from small intestine were evaluated by using the same reagents (data not shown). Eutopic endometrial biopsies for EESC cultures were performed in five of the patients (three without and two with DIE) (Table 1), all undergoing operation during the early–mid-proliferative phase (menstrual days 5 to 10) of the cycle to avoid effects of endogenous luteal progesterone. In our experience, the success of culture establishment and consistency of cell growth rates are menstrual cycle–specific and optimal when proliferative phase biopsies are obtained.20Ryan I.P. Schriock E.D. Taylor R.N. Isolation, characterization, and comparison of human endometrial and endometriosis cells in vitro.J Clin Endocrinol Metab. 1994; 78: 642-649Crossref PubMed Scopus (0) Google Scholar Tissue fragments were used to prepare EESC cultures, as described.20Ryan I.P. Schriock E.D. Taylor R.N. Isolation, characterization, and comparison of human endometrial and endometriosis cells in vitro.J Clin Endocrinol Metab. 1994; 78: 642-649Crossref PubMed Scopus (0) Google Scholar, 21Yu J. Boicea A. Barrett K.L. James C.O. Bagchi I.C. Bagchi M.K. Nezhat C. Sidell N. Taylor R.N. Reduced connexin 43 in eutopic endometrium and cultured endometrial stromal cells from subjects with endometriosis.Mol Hum Reprod. 2014; 20: 260-270Crossref PubMed Scopus (30) Google Scholar Briefly, after collagenase digestion, glandular epithelial cells and debris were separated from EESCs by filtration through 200-μm and 40-μm sieves. EESCs were subcultured at least twice to eliminate contamination by macrophages or other leukocytes and were used before the sixth passage to avoid dedifferentiation. Prior studies from our laboratory confirmed that the EESCs are >95% pure and retain functional estrogen, progesterone, and other nuclear receptors, as well as phenotypic endometrial markers, for at least five passages in vitro. The cells were grown to 60% to 80% confluence in 10-cm dishes with Dulbecco's modified Eagle's medium/Ham's F-12 50:50 Mix media (catalog number 10-092CV; CellGro, Manassas, VA) supplemented with 10% fetal calf serum. Some cells were incubated with up to 100 ng/mL IL-1β, which corresponds to the EC90 for IL-6 secretion by normal endometrial stromal cells.22Yu J. Berga S.L. Zou W. Yook D.G. Pan J.C. Andrade A.A. Zhao L. Sidell N. Bagchi I.C. Bagchi M.K. Taylor R.N. IL-1beta inhibits connexin 43 and disrupts decidualization of human endometrial stromal cells through ERK1/2 and p38 MAP kinase.Endocrinology. 2017; 158: 4270-4285Crossref PubMed Scopus (26) Google Scholar Replicate cultures from each patient were prepared. Representative examples are shown, and the number of experiments performed with EESCs from independent patients is indicated. EESC morphology was assessed by phase-contrast microscopy to determine cell shape, using a modified calculation of cell roundness23Malek A.M. Izumo S. Mechanism of endothelial cell shape change and cytoskeletal remodeling in response to fluid shear stress.J Cell Sci. 1996; 109: 713-726Crossref PubMed Google Scholar as described previously,19Yu J. Berga S.L. Johnston-MacAnanny E.B. Sidell N. Bagchi I.C. Bagchi M.K. Taylor R.N. Endometrial stromal decidualization responds reversibly to hormone stimulation and withdrawal.Endocrinology. 2016; 157: 2432-2446Crossref PubMed Scopus (42) Google Scholar whereby an index of 1.0 = round. To corroborate and localize the cellular distribution and expression of BDNF and c-Rel (a p65 component of the NF-κB complex), intact EESCs from three patients grown on Lab-Tek chamber slides before and after recombinant IL-1β exposure (Sigma-Aldrich, St. Louis, MO) were analyzed. The cells were fixed in 4% paraformaldehyde and cold acetone. Immunofluorescence cytochemistry (IFC) was performed on an EVOS cellular imaging microscope (Thermo-Fisher Scientific, Waltham, MA). Quantification of BDNF from IFC staining intensity was determined by using Image J software version 1.46r (NIH, Bethesda, MD; https://imagej.nih.gov/ij) to assess pixel counts per micrometer squared. Western blot analyses were performed on whole-cell extracts, obtained by using a vortex to mix EESCs in extraction buffer (catalog number FNN0011; Life Technologies, Grand Island, NY). Total proteins (20 to 60 μg per lane, determined with the Thermo Scientific-Pierce BCA Protein Assay kit; catalog number PI-23227; Thermo-Fisher Scientific) were run on NuPAGE Novex 4% to 12% Bis-Tris Protein Gels, transferred to polyvinylidene difluoride membranes, and blocked with 5% skim milk in phosphate-buffered saline. BDNF was detected by using mouse monoclonal anti-BDNF antibodies (dilution 1:250; catalog number ab10505; Abcam, Cambridge, MA). The BDNF bands were detected after incubation with secondary antibody linked to horseradish peroxidase and visualized by chemiluminescent detection and exposure to enhanced chemiluminescence hyperfilm (Amersham Pharmacia Biotech, Piscataway, NJ). Three dominant bands were observed, corresponding to an N-glycosylated and glycosulfated pro-BDNF precursor (32 kDa), a 28-kDa dimer, and the mature 14-kDa product of BDNF processing.24Mowla S.J. Farhadi H.F. Pareek S. Atwal J.K. Morris S.J. Seidah N.G. Murphy R.A. Biosynthesis and post-translational processing of the precursor to brain-derived neurotrophic factor.J Biol Chem. 2001; 276: 12660-12666Crossref PubMed Scopus (439) Google Scholar When anti-BDNF antibodies were immunoabsorbed with excess recombinant BDNF, the signals of all three isoforms were abrogated, validating antibody specificity. Blots were washed, reprobed with mouse monoclonal anti–β-actin antibodies (dilution 1:1000; catalog number 31430; Sigma-Aldrich), and developed in an identical manner to ensure even loading. Molecular weight standards were used to calibrate the migration of immunopositive bands. Western blot analyses of EESCs also were evaluated by using antibodies specific for proteins and phospho (p)-peptides corresponding to critical signaling pathways that have previously been characterized for IL-1β signaling in normal endometrial stromal cells.22Yu J. Berga S.L. Zou W. Yook D.G. Pan J.C. Andrade A.A. Zhao L. Sidell N. Bagchi I.C. Bagchi M.K. Taylor R.N. IL-1beta inhibits connexin 43 and disrupts decidualization of human endometrial stromal cells through ERK1/2 and p38 MAP kinase.Endocrinology. 2017; 158: 4270-4285Crossref PubMed Scopus (26) Google Scholar p–c-Jun N-terminal kinase (JNK) (catalog number 4668), p-SEK1 (catalog number 4514), p-ATF2 (catalog number 5112), p–c-Jun (catalog number 2361), Ras (catalog number 3339), p-mechanistic target of rapamycin (mTOR; Ser2448) (catalog number 5536), Raptor (catalog number 2280), and Rictor (catalog number 2114) antibodies (all diluted 1:1000; Cell Signaling Technology, Danvers, MA) were used to identify the predominant signaling mechanism(s) after IL-1β administration. Films were digitized on a flatbed scanner, and Image J was used to quantify the intensity of each band. Data are presented as the ratio of BDNF or p-peptide density/β-actin density of three cell preparations. IL-1ra, a natural inhibitor of type I and II IL-1 receptors,25Arend W.P. Interleukin-1 receptor antagonist.Adv Immunol. 1993; 54: 167-227Crossref PubMed Scopus (485) Google Scholar was purchased from R&D Systems (Minneapolis, MN) and used as described previously.22Yu J. Berga S.L. Zou W. Yook D.G. Pan J.C. Andrade A.A. Zhao L. Sidell N. Bagchi I.C. Bagchi M.K. Taylor R.N. IL-1beta inhibits connexin 43 and disrupts decidualization of human endometrial stromal cells through ERK1/2 and p38 MAP kinase.Endocrinology. 2017; 158: 4270-4285Crossref PubMed Scopus (26) Google Scholar NF-κB inhibitory peptides and selective kinase inhibitors were used to validate the signaling pathways activated by IL-1β treatment. SN50 (catalog number 481480; EMD-Millipore, Burlington, MA) blocks translocation of NF-κB. The inert (mutated) analogue SN50M was used as a negative control. PD98059 [extracellular signal-regulated kinase (ERK) inhibitor)], SB203580 [p38 mitogen-activated protein kinase (MAPK) inhibitor], and SP600125 (JNK inhibitor) were from Sigma-Aldrich (catalog numbers P215, S8307, and S5567, respectively); rapamycin (mTOR inhibitor) was obtained from Cell Signaling Technology (catalog number 9904). In all of the experiments that used various kinase and NF-κB inhibitors, the inhibitory peptides or compounds were added to the cultures 1 hour before the addition of IL-1β; the latter was allowed to incubate for up to 24 hours before termination of the studies. Three independent endometriosis-derived EESC preparations were evaluated by real-time quantitative PCR (qPCR). EESCs were incubated without or with IL-1β for 24 hours as described in the section above. Total RNA was isolated from the cells by using TRI-reagent (Life Technologies) according to the manufacturer's protocol and frozen at –80°C until analyzed. cDNA was synthesized from mRNA samples and subsequently used as template for qPCR assays. For qPCR, the SsoAdvanced Universal SYBR Green Supermix reagent (catalog number 1725271; Bio-Rad Laboratories, Hercules, CA) was used, the vendor guidelines were followed with some modifications, and BDNF primers were validated. A total reaction volume of 20 μL contained 10 μL of SYBR Green, primer mix 2 μL, 1 μL of 50 mmol/L MgCl2, 2 μL of H2O, and 5 μL of cDNA. The PCR was set for 40 cycles in a CFX real-time thermocycler (Bio-Rad Laboratories). These data were analyzed after normalization with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels, using the formula 2ΔΔct, where ct is the cycle threshold. Regulated on activation normal T cell expressed and secreted [RANTES; or chemokine (C-C motif) ligand 5], a prominent monocyte chemokine in endometriosis,26Reis F.M. Petraglia F. Taylor R.N. Endometriosis: hormone regulation and clinical consequences of chemotaxis and apoptosis.Hum Reprod Update. 2013; 19: 406-418Crossref PubMed Scopus (180) Google Scholar was measured with the use of a specific and sensitive enzyme-linked immunosorbent assay (R&D Systems) that was previously validated for EESC culture supernatants.27Hornung D. Ryan I.P. Chao V.A. Vigne J.L. Schriock E.D. Taylor R.N. Immunolocalization and regulation of the chemokine RANTES in human endometrial and endometriosis tissues and cells.J Clin Endocrinol Metab. 1997; 82: 1621-1628Crossref PubMed Scopus (232) Google Scholar, 28Yu J. Wu J. Bagchi I.C. Bagchi M.K. Sidell N. Taylor R.N. Disruption of gap junctions reduces biomarkers of decidualization and angiogenesis and increases inflammatory mediators in human endometrial stromal cell cultures.Mol Cell Endocrinol. 2011; 344: 25-34Crossref PubMed Scopus (48) Google Scholar The IHC, IFC, and Western blot images are from representative specimens and cell preparations, and each result was replicated in tissues or cells from a minimum of three independent patients. The sample size was estimated from previous empirical studies with the use of cell cultures derived from endometriosis patients.21Yu J. Boicea A. Barrett K.L. James C.O. Bagchi I.C. Bagchi M.K. Nezhat C. Sidell N. Taylor R.N. Reduced connexin 43 in eutopic endometrium and cultured endometrial stromal cells from subjects with endometriosis.Mol Hum Reprod. 2014; 20: 260-270Crossref PubMed Scopus (30) Google Scholar BDNF pixel counts were determined in five high-power fields from three independent cell cultures by IFC in EESCs grown on chamber slides. BDNF protein levels in EESC lysates on Western blot analyses were normalized to β-actin digitized band densities from three independent cell preparations. These data were normally distributed (Kolmogorov-Smirnov test), and results are presented as means ± SEM. Comparisons were determined with the use of paired t-tests. Dose responses to IL-1β of BDNF mRNA levels normalized to GAPDH mRNA were determined from 2ΔΔct calculations and were compared by analysis of variance. Statistical significance for all of the analyses was accepted when two-tailed tests yielded P < 0.05 between the treatment groups. To document the coordinated spatiotemporal coincidence of macrophages, neurotrophins (NTs), and nerve fibers, a series of IHC experiments were performed on nodular DIE lesions. A hematoxylin and eosin–stained section is provided for orientation (Figure 1) that revealed a focus of DIE invading striated muscle of the pelvic floor and rectovaginal septum. Tissue macrophages were identified by IHC for the low-density lipoprotein scavenger receptor CD68, whose antibodies revealed CD68+ macrophages scattered throughout the endometriosis stroma, being relatively spared in the endometriotic epithelial compartments and the investing rectovaginal muscularis (Figure 1). Specific BDNF immunostaining was identified in adjacent sections, predominantly localized in the glandular and stromal compartments of the DIE lesion, but not in the surrounding muscle tissue (Figure 1). PGP9.5+ nerve fibers were seen coursing through the stroma of the DIE lesion. Most of these fibers were viewed en face relative to the orientation of this DIE implant (Figure 1). Nonimmune IgG was used as a negative control (Figure 1). Similar distributions of CD68+, BDNF+, and PGP9.5+ cells were observed in DIE biopsies from all four independent patients evaluated by IHC. The histologic findings supported the concept that macrophage–stromal cell cross talk within endometriosis lesions could result in the elaboration of neurotrophic signals and recruitment of nociceptive nerves. A well-established, primary EESC culture model20Ryan I.P. Schriock E.D. Taylor R.N. Isolation, characterization, and comparison of human endometrial and endometriosis cells in vitro.J Clin Endocrinol Metab. 1994; 78: 642-649Crossref PubMed Scopus (0) Google Scholar, 21Yu J. Boicea A. Barrett K.L. James C.O. Bagchi I.C. Bagchi M.K. Nezhat C. Sidell N. Taylor R.N. Reduced connexin 43 in eutopic endometrium and cultured endometrial stromal cells from subjects with endometriosis.Mol Hum Reprod. 2014; 20: 260-270Crossref PubMed Scopus (30) Google Scholar, 27Hornung D. Ryan I.P. Chao V.A. Vigne J.L. Schriock E.D. Taylor R.N. Immunolocalization and regulation of the chemokine RANTES in human endometrial and endometriosis tissues and cells.J Clin Endocrinol Metab. 1997; 82: 1621-1628Crossref PubMed Scopus (232) Google Scholar was used to test this hypothetical mechanism. IL-1β is a potent proinflammatory cytokine synthesized by macrophages, whose concentration and activity were reported to be excessive in endometriosis.29Akoum A. Lawson C. Herrmann-Lavoie C. Maheux R. Imbalance in the expression of the activating type I and the inhibitory type II interleukin 1 receptors in endometriosis.Hum Reprod. 2007; 22: 1464-1473Crossref PubMed Scopus (38) Google Scholar, 30Lebovic D.I. Bentzien F. Chao V.A. Garrett E.N. Meng Y.G. Taylor R.N. Induction of an angiogenic phenotype in endometriotic stromal cell cultures by interleukin-1beta.Mol Hum Reprod. 2000; 6: 269-275Crossref PubMed Scopus (163) Google Scholar Tissue macrophages infiltrating the endometrial stroma22Yu J. Berga S.L. Zou W. Yook D.G. Pan J.C
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