ABSTRACT. Retrograde menstruation is postulated as the initiating event in the histogenesis of endometriosis; however, subsequent steps in the pathogenesis ...
0021-972X/97/$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1997 by The Endocrine Society
Vol. 82, No. 5 Printed in U.S.A.
Immunolocalization and Regulation of the Chemokine RANTES in Human Endometrial and Endometriosis Tissues and Cells* DANIELA HORNUNG, ISABELLE P. RYAN, VICTOR A. CHAO, JEAN-LOUIS VIGNE, ELDON D. SCHRIOCK, AND ROBERT N. TAYLOR Reproductive Endocrinology Center, Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco, California 94143-0556 ABSTRACT Retrograde menstruation is postulated as the initiating event in the histogenesis of endometriosis; however, subsequent steps in the pathogenesis of this common disorder remain poorly characterized. The ip accumulation of activated leukocytes and the infiltration of endometriosis lesions by macrophages and T cells are cytological markers of the inflammatory nature of this syndrome. The apparent recruitment of these leukocytes prompted us to search for chemokine expression by endometriosis cells. We reported previously that pelvic fluid RANTES (regulated upon activation, normal T cell expressed and secreted) concentrations correlated with the stage of endometriosis. In the current study, RANTES messenger ribonucleic acid (mRNA) was identified in normal endometrium and endometriosis lesions, and techniques were developed to localize RANTES protein
within these tissues. Using isolated endometrial and endometriosis cell cultures, we demonstrated that RANTES mRNA and protein can be induced by the proinflammatory cytokines tumor necrosis factor-a and interferon-g in endometrial stromal, but not in epithelial or adenocarcinoma cells. Immunocytochemical studies confirmed the biochemical findings. Metabolic labeling experiments verified that nascent RANTES secreted by cytokine-stimulated endometriosis stromal cells was the mature, 8-kDa protein predicted by the mRNA encoding this chemokine. The results indicate that RANTES is a normal constituent of the eutopic endometrium. We propose that secretion of RANTES by ectopic endometriosis implants provides a mechanism for peritoneal leukocyte recruitment. (J Clin Endocrinol Metab 82: 1621– 1628, 1997)
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implants are sites of local inflammation, and this reaction is responsible for the pain and infertility associated with the clinical disorder (5, 6). Increased numbers of peritoneal macrophages have been found in the pelvic fluid of infertile women with endometriosis (7–10). In addition, these cells display morphological evidence of activation in endometriosis patients (5, 11, 12). As the chemoattraction of circulating monocytes is regulated by the b-chemokine RANTES (regulated upon activation, normal T cell expressed and secreted) (13), we postulated that this protein might be secreted into the peritoneal cavity by endometriosis implants. We first tested this hypothesis in a case-control study with peritoneal fluid samples obtained from endometriosis patients and normal controls undergoing laparoscopy. Our findings demonstrated that the pelvic fluid concentrations of RANTES were elevated significantly in women with endometriosis and that these levels correlated with the stage of disease (14). Other monocyte chemoattractant activities (15, 16) also have been found to be increased in pelvic fluid of endometriosis patients, although the correlation with disease stage was variable. We have undertaken a series of experiments to verify the presence of RANTES messenger ribonucleic acid (mRNA) and localize the expression of RANTES protein in specimens of human endometrium and endometriosis implants and in primary cell cultures isolated from these tissues. Our findings indicate that RANTES is synthesized predominantly in the stromal compartment of normal endometrium and endometriosis tissue. That this chemokine is expressed in both eutopic and ectopic endometrium raises interesting ques-
NDOMETRIOSIS is a prevalent gynecological condition that affects between 5–15% of women of reproductive age (1). Although no single theory can fully account for the diverse clinical presentations of endometriosis, Sampson’s hypothesis (2) of transtubal regurgitation of viable endometrial cells is most consistent with the gravitationally dependent location of the implants on the peritoneal surfaces of the ovaries, broad ligaments, and cul-de-sac. Interestingly, the phenomenon of retrograde menstrual flow has been documented in more than 90% of menstruating women undergoing laparoscopy (3), and viable endometrial cells can frequently be recovered from the peritoneal cavity of cycling women (4). Thus, the expression of other cellular factors, such as integrins, cell adhesion molecules, angiogenic activities, or proinflammatory cytokines, may account for the susceptibility of implant formation in the subpopulation of affected women. The ip presence of ectopic endometrium per se may have little pathological impact. Recent studies suggest that these
Received November 13, 1996. Revision received January 21, 1997. Accepted January 31, 1997. Address all correspondence and requests for reprints to: Dr. Robert N. Taylor, Reproductive Endocrinology Center, Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco, California 94143-0556. * Presented in part at the 10th International Congress of Endocrinology, June 12, 1996, San Francisco, CA. This work was supported in part by a Deutsche Forschungsgemeinschaft Fellowship (to D.H.), an American College of Obstetricians and Gynecologists/Ortho Fellowship (to I.P.R.), and funds from the University of California-San Francisco Obstetrics and Gynecology Research and Education Foundation (to R.N.T.).
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tions about the roles of immunocyte recruitment in normal uterine physiology and in the inflammatory response observed in endometriosis. Materials and Methods Sources of tissues Tissue specimens were obtained from patients undergoing laparoscopy or laparotomy after providing written informed consent under a study protocol approved by the University of California-San Francisco committee on human research. Healthy ovulatory women, who had not received hormones or GnRH agonist therapy for at least 3 months before surgery, were recruited. Women with endometriosis (mean 6 sd age, 34 6 5 yr; n 5 11) were staged intraoperatively according to a modification of the revised American Fertility Society system (17). Control subjects were women with subserosal leiomyomata or without pelvic pathology requesting tubal ligation (age, 37 6 5 yr; n 5 11). Endometrial and endometrioma biopsies were collected under sterile conditions and transported to the laboratory on ice in aMEM with 10% FCS. Sufficient tissue for RNA, immunohistochemical, and cell culture analyses was available in about half the cases, so some biopsies were used only for single studies. All samples and cycle stages were estimated histologically (18). All normal endometrial biopsies were in phase and consistent with the patient’s menstrual dating. Biopsies from endometriosis lesions typically showed flattened epithelium and compact stroma.
Cell isolation, purification, and culture Primary endometrial and endometrioma cell cultures were prepared from biopsies as described previously (19). Biopsy specimens for cell isolation were obtained from women in the midproliferative phase of the menstrual cycle. Briefly, the tissue was dissected free from underlying myometrium or parenchyma, minced into small pieces, digested with collagenase (2 mg/mL) for 1 h at 37 C, and separated using serial filtration. Debris was removed by 100-mm aperture sieves, and epithelial glands were retained on 40-mm aperture sieves and backwashed onto tissue culture dishes. Stromal cells remaining in the filtrate were plated onto Primaria (Becton Dickinson, Lincoln Park, NJ) flasks and allowed to adhere for 30 min, after which blood cells were removed with phosphate-buffered saline rinses. The cells were cultured in aMEM reconstituted with 10% FCS, nucleosides, essential amino acids, penicillin G (100 U/mL), streptomycin (100 mg/mL), and fungizone (1 mg/mL). Exogenous steroids, unless indicated, were not added to the cultures. Stromal cultures were dissociated with 0.05% trypsin and 0.02% versene in saline, harvested by centrifugation, replated, and allowed to grow to confluence. Purification of the stromal cell population was confirmed by negative staining for the following antibodies: CD3 (T cells), CD11b (granulocytes), CD45 (monocytes and other leukocytes), and cytokeratin (epithelial cells). As a control, we used a well differentiated endometrial adenocarcinoma cell line (Ishikawa cells) obtained from the University of California-San Francisco Cell Culture Facility.
Cytokine treatment of cell cultures When the primary cell cultures or Ishikawa cells approached confluence, the complete medium was removed and replaced with fresh aMEM containing 2.5% FCS and antibiotics, and the cells were cultured for an additional 48 h. Some wells were treated for 48 h with interferon-g (IFNg; 100 ng/mL) and tumor necrosis factor-a (TNFa; 100 ng/mL; Sigma Chemical Co., St. Louis, MO), which have been shown to activate RANTES expression in human endothelial cells (20). Dose-response experiments indicated that this combination gave a maximal RANTES response in endometrial stromal cells (Ryan, I. P., and R. N. Taylor, unpublished results).
Extraction and purification of RNA from tissues and cells Total RNA was extracted from tissues (endometrium, endometrioma, myometrium, and ovary) and from cell cultures (endometrial and endometriosis stromal, epithelial, and Ishikawa cells) using the acid guanidinium isothiocyanate-phenol-chloroform extraction method of Chomczynski and Sacchi (21).
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Reverse transcription-PCR (RT-PCR) Polyadenylated mRNA was preferentially reverse transcribed using oligo(deoxythymidine) and random hexamer primers. Specific oligonucleotide primers were designed to amplify sequences from human RANTES mRNA (193 bp) and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 240 bp) as a positive control (22). The primers used for the RT-PCR experiments are shown in Table 1, and those used to generate complementary RNA (cRNA) probes for the ribonuclease (RNase) protection assays are shown in Table 2. PCR cycling was preceded by an initial denaturation at 94 C for 3 min. A thermal cycle program of 94/57/75 C for 34, 10, and 70 s was run for seven cycles, followed by a program of 94/66/75 C for 34, 15, and 70 s run for 25 cycles. A terminal extension was run at 75 C for 10 min. The PCR products were separated on 4% agarose gels (3% NuSieve GTG, 1% SeaKem GTG, FMC Bioproducts, Rockland, ME) and visualized by ethidium bromide staining (Life Technologies, Gaithersburg, MD).
RNase protection analyses For RNase protection analyses, we generated RANTES and GAPDH templates by PCR, having engineered T7 RNA polymerase-binding site sequences 59 to the antisense oligomers for the two gene products (see Table 2). RANTES and GAPDH cRNA were labeled with [a-32P]UTP (800 Ci/mmol; Easytides, DuPont, Boston, MA), synthesized by in vitro transcription using reagents from the Promega riboprobe system kit (Madison, WI), and gel-purified. The cRNA probes were hybridized with 10 mg total tissue or cellular RNA using the Hyb-Speed-RPA-Kit (Ambion, Austin, TX), and the products were separated on denaturing 8 mol/L urea-5% polyacrylamide gels as described previously (23). Human placental tissue served as a positive control for the RNase protection assays and immunostaining studies described below. Data were analyzed as ratios of the density of RANTES to GAPDH mRNA signals, determined by computer-assisted densitometry of the autoradiograms (Scan Analysis, Biosoft, Ferguson, MO).
Immunohistochemistry Endometrial and endometrioma tissues were fixed for 24 h in 2% paraformaldehyde and 0.5% glutaraldehyde, paraffin-embedded, cut in serial sections of 8 mm, and stained using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA). Immunoperoxidase staining was performed overnight at 4 C, using mouse monoclonal IgG antibodies against human cytokeratin 18 (1:2000 dilution; Sigma) and RANTES (0.8 mg/mL; VL2, provided by Drs. Peter Nelson and Alan Krensky, Stanford University, Palo Alto, CA) (24). Controls for the immunostaining specificity included sections stained with anti-RANTES antibodies immunoabsorbed with 10 mg/mL recombinant RANTES protein (R&D Systems, Minneapolis, MN) and sections stained with an identical concentration of an irrelevant mouse monoclonal IgG antibody against human synaptophysin (0.8 mg/mL; Boehringer Mannheim, Mannheim, Germany) that does not stain endometrium. Diaminobenzidine (Zymed, South San Francisco, CA) was used as the chromagen. All sections also were lightly counterstained with hematoxylin.
Immunocytochemistry Stromal and epithelial cells were plated onto Lab-Tek four-chamber slides, treated for 48 h in the absence or presence of IFNg (100 ng/mL) TABLE 1. Primer design for RT-PCR RANTES (193 bp) Sense primer: (148)-59-CAC TGC CCC GTG CCC ACA TCA A-39 Antisense primer: 59-GTA GGC TAA TAC GAC TCA CTA TAG GGT CCA TCT CCA TCC TAG CTC ATC TCC AAA-39-(287) GAPDH (240 bp) Sense primer: (825)-59-TGA TGA CAT CAA GAA GGT GGT GAA G-39 Antisense primer: 59-TCC TTG GAG GCC ATG TGG GCC AT-39-(1042)
RANTES IN HUMAN ENDOMETRIUM AND ENDOMETRIOSIS TABLE 2. Primer design for cRNA probe synthesis RANTES
PCR probe template: 202 bp Riboprobe: 178 bp Protected fragment: 163 bp
Sense primer: (148)-59-CAC TGC CCC GTG CCC ACA TCA A-39 Antisense primer: 59-cgt agg cta ata cga ctc act ata GGG AGG CTG TGA GAG TCT CCA TCC TAG CTC ATC TCC AAA-39-(287) Glyceraldehyde-3-phosphate dehydrogenase Probe template: 168 bp Riboprobe: 145 bp Protected fragment: 139 bp
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these tissues, an RNase protection assay was developed. Cycling endometrium from early, mid-, and late proliferative and midsecretory phases of normal controls (Fig. 2, upper panel, lanes 1– 4, respectively) and midproliferative phase endometrioma tissue (Fig. 2, upper panel, lane 6) contained high levels of RANTES mRNA, whereas myometrium contained scant levels of this transcript (Fig. 2, upper panel, lane 5). RNase protection analyses for GAPDH mRNA provided a positive control for RNA quantity and integrity (Fig. 2, lower panel, lanes 1– 6). These results were representative of seven independent normal endometrial and four endometrioma specimens.
Sense primer: (457)-59-AAC AGC CTC AAG ATC ATC AGC AA-39
Immunohistochemistry of endometrial and endometrioma tissues
Antisense primer: 59-acg cca taa tac gac tca cta taG GGA GGC AGT CTT CTG GGT GGC AGT GAT-39(574)
Immunohistochemistry was used to localize RANTES protein in fixed tissues. A section of normal proliferative endometrium stained with hematoxylin and eosin is shown in Fig. 3A. An adjacent section, stained with monoclonal antibodies against cytokeratin, specifically stained the glandular epithelium (Fig. 3B). Monoclonal IgG antibodies against RANTES showed the antigen to be localized primarily to the stromal compartment, with glandular epithelium appearing
Lower case letters represent T7 polymerase site sequence. Uppercase letters represent unprotected probe sequence. Bolded bases represent protected sequence. and TNFa (100 ng/mL), and fixed in 95% ethanol. Control cultures were stained with May-Gruenwald-Giemsa reagents. Primary monoclonal antibodies against human vimentin (Sigma), cytokeratin, RANTES, and synaptophysin were used as described above.
Immunoprecipitation of metabolically labeled RANTES protein Cytokine-stimulated endometrial stromal cells were labeled in vitro with [35S]methionine and [35S]cysteine for 48 h. The conditioned medium was collected by centrifugation and concentrated 10-fold by ultrafiltration using a YM3 Amicon membrane (Amicon, Beverly, MA) with a 3-kDa molecular mass cut-off. The concentrated media were precleared with protein A and divided into two parts: one part was treated with VL2 antibody, and the other part was treated with a commercially available monoclonal antibody against RANTES (catalog no. MAB278, R&D Systems). In both cases the same amount of IgG was added (4 mg). The incubation was conducted overnight at 4 C, 50 mL protein A bead slurry (Sigma) were added to the mixture, and the incubation was continued for another hour at 4 C. The protein A beads were collected by centrifugation, and the pellets were washed five times with 50 mmol/L Tris, pH 7.4. The pellets were boiled in 40 mL Laemmli buffer for 3 min and loaded onto a 13.5% polyacrylamide gel.
FIG. 1. RT-PCR analysis of gene transcripts. mRNA from human endometrium (lane 2), myometrium (lane 3), and endometrioma (lane 4) were reverse transcribed and amplified. Molecular mass markers are shown in lane 1. RT-PCR demonstrated a 193-bp band corresponding to the human RANTES mRNA in endometrium and endometrioma, but not in myometrium. GAPDH transcript products of 240 bp, a positive control for mRNA integrity, were detectable in all three samples. Negative controls, in which no complementary DNA (5) or no primer was added (6), revealed no amplification.
Results Identification of RANTES mRNA transcripts in endometrium and endometriosis
To verify the expression of RANTES mRNA in endometrial and endometrioma tissues, we used RT-PCR and RNase protection assays. RANTES mRNA transcripts were amplified in RNA isolated from normal endometrium and endometriosis tissues (Fig. 1, upper panel, lanes 2 and 4), but not in myometrium (lane 3) using RT-PCR. Normal ovarian tissue without evidence of endometriosis also showed no RANTES transcripts (data not shown). Intron-spanning primers used to amplify transcripts of a constitutive gene, GAPDH, indicated that the RNA preparations were of good quality and not contaminated by genomic DNA (Fig. 1, lower panel, lanes 2– 4). Negative controls for reverse transcription and PCR yielded no nonspecific bands (Fig. 1, lanes 5 and 6). To quantify the steady state levels of RANTES mRNA in
FIG. 2. RNase protection assays of RANTES and GAPDH transcripts. Increasing amounts of RANTES mRNA in human endometrium of early, mid-, and late proliferative and midsecretory phases (lanes 1– 4) of the menstrual cycle demonstrated by RNA protection analysis. Myometrium (lane 5) showed very low steady state levels of RANTES mRNA, whereas endometrioma (lane 6) had a high mRNA concentration. The amount of total RNA assayed was confirmed by concurrent hybridization with a GAPDH probe. The protected fragments for RANTES (163 bp) and GAPDH (139 bp) had the expected sizes (see Table 2).
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free of the antigen (Fig. 3C). Identical concentrations of an irrelevant monoclonal IgG antibody against synaptophysin served as a negative control (Fig. 3D). We observed a tendency for some endometrial epithelial cell staining in the luteal phase of the cycle, but the stroma remained immunopositive (not shown). Sections of endometrioma stained with cytokeratin (Fig. 3E) and RANTES (Fig. 3F) showed patterns very similar to those observed in normal endometrium. Note that portions of the ovarian stroma uninvolved with endometriosis showed very faint to absent RANTES immunostaining (Fig. 3F). Control experiments were performed on serial sections of endometrium stained with RANTES antibodies (Fig. 3G) or the same antibodies immunoabsorbed with excess RANTES protein (Fig. 3H). The latter treatment eliminated the staining pattern and verified the specificity of the immunolocalization. These results were representative of eight normal endometrial and four endometrioma specimens. RANTES mRNA expression in isolated endometrial and endometriosis cells
Using an established protocol we isolated and cultured epithelial and stromal cells from normal endometrium and endometriosis (19). RNase protection assays were used to quantify RANTES mRNA transcripts in the two cell types. In the absence of added cytokines, neither stromal nor epithelial cells isolated from endometriosis or normal endometrial biopsies were positive for RANTES mRNA (Fig. 4, lanes 4, 6, and 8). However, culturing the cells in the presence of TNFa (100 ng/mL) and IFNg (100 ng/mL) for 48 h dramatically induced RANTES mRNA expression in both the ectopic and eutopic endometrial stromal cells (Fig. 4, lanes 5 and 7), but not in endometrial epithelial cells (Fig. 4, lane 9). Ishikawa cells, a line derived from a well differentiated human endometrial epithelial carcinoma (25), also failed to express RANTES mRNA in the absence or presence of added cytokines (Fig. 4, lanes 10 and 11, respectively). GAPDH transcripts were detected in all cells studied and were unaffected by culture in the presence of cytokines. These results were representative of nine and eight normal endometrial and endometriosis stromal cell preparations, respectively, and six and four normal endometrial and endometriosis epithelial cell preparations, respectively. The Ishikawa cell findings were confirmed three times. Immunocytochemistry of purified endometrial cell populations
Endometrial stromal and epithelial cells were examined subsequently for RANTES protein expression by immunocytochemistry. Subconfluent monolayers of endometrial
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FIG. 4. RNase protection assay of human endometrial and endometriosis cell cultures. The full-length probes for GAPDH (145 bp) and RANTES (178 bp) are shown in lanes 1 and 2, respectively. Ten micrograms of RNA were simultaneously hybridized with excess RANTES and GAPDH riboprobes, and the reaction products were subjected to RNase digestion. Hybridization with transfer RNA revealed no specific protected fragments (lane 3). Unstimulated endometriosis (lane 4) and normal endometrial stromal cells (lane 6) showed no RANTES mRNA (163 bp), but were positive for GAPDH mRNA-protected fragments (139 bp). By contrast, cytokine-stimulated endometriosis (lane 5) and normal endometrial stromal cells (lane 7) were positive for both RANTES and GAPDH mRNA. Endometrial epithelial cells (lanes 8 and 9) and Ishikawa adenocarcinoma cells (lanes 10 and 11) failed to express RANTES mRNA under basal or stimulated conditions. However, GAPDH mRNA controls were positive in these epithelial and epitheloid cells.
stromal cells were stained with Giemsa (Fig. 5A), and their mesenchymal origin was confirmed by positive staining with vimentin antibodies (Fig. 5B). Stromal cells incubated with RANTES antibodies showed very faint staining under conditions in which the medium contained no added cytokines (Fig. 5C). After culturing the cells in the presence of TNFa (100 ng/mL) and IFNg (100 ng/mL) for 48 h (Fig. 5D), an increase in staining intensity was noted to be localized in nuclei and perinuclear cytoplasm. A 10-kDa intracellular RANTES precursor is predicted by its complementary DNA sequence (26). Immunoabsorption with excess RANTES antigen completely eliminated this staining (Fig. 5E). The addition of estradiol (0.1–10 nmol/L), medroxyprogesterone acetate (1–100 nmol/L), or a combination of the two steroids had no effect on the production of immunodetectable RANTES. These findings were representative of nine stromal cell preparations. Subconfluent monolayers of endometrial epithelial cells were stained with Giemsa (Fig. 5F), and their epithelial origin was confirmed by positive immunostaining with cytokeratin antibodies (Fig. 5G). None of four epithelial cell preparations revealed evidence of RANTES expression, even after cytokine stimulation (Fig. 5H), corroborating the absence of RANTES mRNA in cultured normal endometrial epithelial cells and human endometrial adenocarcinoma cells.
FIG. 3. Immunohistochemical localization of RANTES protein in human endometrium and endometriosis tissues. Sections of normal midproliferative endometrium were stained with eosin (A) or with antibodies to cytokeratin (B), RANTES (C), or synaptophysin (D). All sections also were counterstained with hematoxylin. As expected, cytokeratin antibodies localized to glandular epithelium, while RANTES antibodies stained endometrial stroma. Synaptophysin staining at the same antibody dilution served as a negative control. Immunostaining of an ovarian endometriosis implant revealed similar findings. Epithelial cells lining the endometriotic cyst were cytokeratin positive (E), whereas RANTES immunostaining was mainly confined to the endometriotic stroma, with sparing of the cyst epithelium (F). The adjacent ovarian stroma also showed no staining. The specificity of the immunostaining was confirmed by immunoabsorption of the primary RANTES antibodies. Stromal staining for RANTES (G) was eliminated after the antibodies were preabsorbed with pure RANTES protein at a concentration of 10 mg/mL (H). Magnification, 3200.
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FIG. 5. Immunocytochemistry of cultured human endometrial stroma and epithelial cells. May-Gruenwald-Giemsa (A) and vimentin (B) staining served as positive controls for isolated stromal cells. These cells showed low RANTES staining under basal conditions (C), but increased RANTES expression after cytokine stimulation (D). The specificity of the immunostaining was confirmed by immunoabsorption of the primary
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FIG. 6. Immunochemical verification of RANTES production and secretion by endometrial stromal cells. Pure recombinant human RANTES (15 ng) subjected to SDS-PAGE, transferred to a Trans-Blot membrane (Bio-Rad, Richmond, CA), and reacted with anti-RANTES antibodies (MAB278) demonstrated an immunopositive band at 8 kDa (A). Endometrial stromal cells were stimulated with cytokines and metabolically labeled with [35S]cysteine and methionine. The secreted products (B) were immunoprecipitated from conditioned medium with VL2 (lane 1) and MAB278 (lane 2) monoclonal antibodies and separated by SDS-PAGE. These demonstrated radiolabeled bands at 8 kDa, corresponding to secreted RANTES protein.
Immunoprecipitation of RANTES protein from endometriosis stromal cell supernatants
To verify antibody specificity, we performed immunoprecipitation analyses of metabolically labeled endometriosis stromal cell-conditioned medium. These cells were stimulated in vitro with TNFa and IFNg for 48 h and were labeled with [35S]methionine and [35S]cysteine during this time. The cell supernatants were concentrated and immunoprecipitated with two monoclonal antibodies (VL2 and the commercially available MAB278). The autoradiogram shown in Fig. 6 demonstrated that both antibodies immunoprecipitated an 8-kDa protein band, consistent with the known molecular mass of secreted human RANTES (26) and identical in size to recombinant human RANTES protein. Epithelial and unstimulated stromal cells contained no immunodetectable RANTES. Discussion
RANTES, a chemokine for monocytes and activated T cells, is actively synthesized by stromal cells derived from normal endometrium and endometriosis implants. The ability of stromal cells within endometriosis implants to produce this potent chemokine may be a factor in the recruitment of activated macrophages and T cells into the peritoneal cavity (14) and into the endometriosis implant itself (6). Peritoneal leukocytes also express RANTES mRNA and may themselves influence monocyte accumulation (Khorram, O., M. Yeung, D. Hornung, and R. N. Taylor, unpublished findings). Similar observations with related chemokines (16, 27) and other peritoneal chemotactic activities not yet fully characterized (15) suggest that convergent chemokine pathways contribute to a feedforward cascade that per-
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petuates the infiltration of inflammatory cells associated with endometriosis. Our findings are of further interest because they demonstrate that stromal cells of normal endometrium also express RANTES mRNA transcripts and protein. The significance of monocyte and T cell recruitment into the normal cycling human endometrium is unknown currently; however, the presence of lymphoid aggregates in the basalis region (28) and the leukocytic infiltration of luteal and early pregnancy decidua (29) have been described. The subtle increase in RANTES gene expression observed in normal endometrium during the ovulatory cycle was less than that noted for endometrial vascular endothelial growth factor mRNA (30). Nevertheless, it is likely that RANTES participates in the normal physiology of the endometrial immunological system. Some of the contrasting findings of our in vivo and in vitro studies provide insight into the complexity of RANTES regulation in the endometrium. RNase protection assays and immunohistochemical staining of RANTES protein in endometrial and endometriosis tissues indicate that the chemokine is expressed in the stroma throughout the ovulatory cycle in vivo. By contrast, isolated stromal cells cultured in the absence of added cytokines did not express RANTES mRNA transcripts, and immunocytochemical staining for RANTES protein was very faint in these cells. However, stimulation of the cultured stromal cells with IFNg and TNFa caused a substantial induction of RANTES mRNA and protein expression. In our in vitro model, added sex steroids had no apparent effect on RANTES expression. We documented previously that these stromal cell cultures were highly purified and were without T cell, macrophage, or epithelial cell contamination (19). We postulate that endometrial and endometriosis stromal cells in vivo are exposed to IFNg derived from nearby lymphoid aggregates (31) or resident leukocytes (6), TNFa derived from adjacent endometrial epithelial cells (32), or other endocrine or paracrine factors capable of inducing RANTES expression in endometrial and endometriosis tissues. In the current study only classical, blue-domed endometriosis lesions were examined. Studies are underway to collect biopsies of atypical endometriosis (e.g. flame-like and vesicular) lesions to determine whether these also express RANTES mRNA and protein. A complex network of proinflammatory cytokines and growth-promoting factors appears to be involved in the pathophysiology of endometriosis (14). Some of these proteins may play physiological roles in normal eutopic endometrium. Future clinical strategies aimed at neutralizing potentially pathological factors in the endometriosis syndrome must not lose sight of the possible importance of these molecules in normal endometrial physiology.
RANTES antibodies. Staining with RANTES antibodies was reduced when the antibodies were preabsorbed with pure RANTES protein at a concentration of 10 mg/mL (E). Isolated epithelial cells were stained with May-Gruenwald-Giemsa (F) and cytokeratin (G) as a positive control. These cells showed no RANTES staining under basal (not shown) or cytokine-stimulated conditions (H). Magnification, 3630.
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HORNUNG ET AL. Acknowledgments
The authors thank Evelyn Garrett for her assistance with histology, Charles Zaloudek for his help with the cytology methods, Jan Shifren for providing us with some of the RNA samples, and Cindy Voytek for imaging.
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