Neutrophils and Macrophages Promote Angiogenesis in the Early Stage of Endometriosis in a Mouse Model
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《内分泌学杂志》
Graduate Institute of Basic Medical Sciences (Y.-J.L., L.-Y.C.W.) and Departments of Immunology (H.-Y.L.) and Physiology (L.-Y.C.W.), Medical College, National Cheng Kung University, Tainan 70101, Taiwan
Department of Physical Therapy (M.-D.L.), Shu-Zen College of Medicine and Management, Kaohsiung 82144, Taiwan
Abstract
Substantial evidence suggests that inflammatory cytokines, immune cells, and angiogenesis are important for endometriosis. In this study, we investigated the role of the sequential events in the development of endometriosis in a mouse model. Uterine tissue was transplanted into the peritoneum of ovariectomized mice and then supplemented with estrogen or vehicle. On different days after transplantation, cell proliferation, angiogenesis, and infiltrated immune cells in ectopic tissue were examined using immunochemical staining. Many disintegrated blood vessels but no bromodeoxyuridine-positive cells in ectopic tissue were observed in the estrogen-treated group on posttransplantation d 1 and 2. On d 4–7, bromodeoxyuridine-positive cells were detected in the blood vessels of ectopic tissue, indicating that angiogenesis was initiated in this stage. Angiogenesis also occurred in ectopic tissue in the vehicle-treated group. Profound infiltration of neutrophils in ectopic tissue occurred on d 1–4, when the number of neutrophils and levels of macrophage inflammatory protein (MIP)-1 and MIP-2 chemokines in peritoneal fluids also reached their peak. Peritoneal macrophage numbers did not change, but secretions of TNF, IL-6, MIP-1, and MIP-2 from macrophages isolated on d 2 were higher than on d 0. In vitro studies showed that peritoneal neutrophils and macrophages secreted vascular endothelial growth factor, which was up-regulated by TNF and IL-6. Our results suggest that neutrophils and macrophages may promote angiogenesis in the early stage of endometriosis and that chemokines and cytokines amplify the angiogenic signal for the growth of endometriotic tissue.
Introduction
ENDOMETRIOSIS, WHICH IS characterized by the growth of uterine tissue outside the uterine cavity, is one of the most common gynecological diseases at reproductive age. It seldom occurs in prepubescent or menopausal women. The progression of endometriosis is estrogen dependent (1, 2, 3). Therefore, using ovariectomy or GnRH agonists to reduce endogenous estrogen causes the regression of endometriotic tissues (4). The implantation hypothesis is widely accepted as the etiology of endometriosis. During menstruation, the endometrium may retrograde into the peritoneal cavity and then implant and grow in ectopic sites (5). The ability of retrograde endometrium to grow in the peritoneal cavity depends on establishment and maintenance of new blood supply. The importance of neovascularization in endometriosis was demonstrated by the evidence that antiangiogenic agents inhibited the growth of endometriotic tissue in nude mice (6, 7). Increased angiogenic activity and vascular endothelial growth factor (VEGF) levels were found in peritoneal fluid of patients with endometriosis (8, 9, 10, 11). However, it is not clear yet how the angiogenesis in endometriosis is initiated.
Endometriosis is an inflammatory disease. Cytokine levels and macrophage numbers are elevated in peritoneal fluids of patients with endometriosis (12, 13, 14). Recently several studies showed that levels of IL-8 and epithelial neutrophil-activating peptide 78 in the peritoneal fluid of patients with endometriosis were elevated (15, 16). Both IL-8 and epithelial neutrophil-activating peptide 78 belong to the cysteine-X-cysteine (CXC) chemokine family. They are neutrophil chemoattractants and possess angiogenic activity (17). Recent evidence has shown that CXC chemokine-induced angiogenesis may occur through the recruitment of neutrophils that secrete VEGF (18, 19, 20, 21). It has been suggested that the VEGF produced by infiltrated neutrophils in the human endometrium and endometrial intravasculature may regulate angiogenesis during the menstrual cycle (22, 23). There is no available information about the contribution of neutrophils to the pathogenesis of endometriosis.
Animal models are important for elucidating the mechanisms underlying endometriosis. Mice lack a menstrual cycle and do not develop spontaneous endometriosis. To induce endometriosis in mice, endometrial tissue is transplanted into the peritoneal cavity using several methods (24, 25, 26, 27). To create an autologous model of mouse endometriosis, portions of uterine or endometrial tissue are surgically transplanted on the peritoneum, in which they develop into endometriotic-like lesions (24, 26). Although mouse endometriosis may not be exactly the same as human endometriosis, their endometriotic lesions develop in some ways, similarly to human endometriotic lesions. Mouse endometriotic tissue is estrogen responsive: it grows under estrogen stimulation and regresses after the withdrawal this hormone (24, 25). In the present study, we used a surgically induced endometriosis mouse model to investigate immune responses and angiogenesis during the development of endometriosis. Our results showed that neutrophils infiltrated into endometriotic tissue and peritoneal cavity during the early stage of endometriosis development. Meanwhile, peritoneal macrophages were activated. Both neutrophils and macrophages secrete angiogenic factors and cytokines. The secretion of macrophage inflammatory protein (MIP)-1, MIP-2, IL-6, TNF, and VEGF by neutrophils and macrophages may create a positive loop to amplify the signal for the establishment and growth of endometriotic tissue in ectopic sites.
Materials and Methods
Animals
Mature (8–10 wk old) female C57BL/6NCrj mice were purchased from the Animal Center at the College of Medicine, National Cheng Kung University. The mice were maintained on standard chow and water, which were available ad libitum, in animal facilities illuminated between 0600 and 1800 h. All procedures were performed in accordance with the Guidelines of the National Cheng Kung University Animal Center for the handling of laboratory animals.
Mouse model for endometriosis and hormone treatment
Endometriosis was surgically induced as previously described (24). In brief, the mice were anesthetized with sodium pentobarbital and their left uterine horn was removed, spliced longitudinally, and cut into approximately 0.4-cm squares that were placed in a petri dish containing DMEM supplemented with penicillin (100 U/ml) and streptomycin (100 μg/ml). The tissue explants were sutured to the peritoneum in the peritoneal cavity. On the day of surgery, the mice were ovariectomized and implanted sc with SILASTIC implants (Dow Corning, Midland, MI) containing vehicle or estradiol-17 (E2, 25 μg/ml dissolved in sesame oil) according to procedures described elsewhere (28). For the surgery, mice at stages other than proestrus were used according to their vaginal smears. For the control group, ovariectomized mice received only sutures on the peritoneum. After different time intervals, the mice were killed; their eutopic and ectopic uterine tissue was removed and fixed for paraffin embedding or directly embedded in optimal cutting temperature (OCT) compound for cryostat sections.
Immunohistochemistry
The procedures for 5-bromo-2'-deoxyuridine (BrdU) detection were as described previously (29). The mice were injected (ip) with BrdU labeling reagent (Amersham Pharmacia Biotech Inc., Piscataway, NJ) 2 h before killing. The eutopic and ectopic uterine tissue was fixed in 10% buffered formalin, dehydrated, and embedded in paraffin. Five-micrometer-thick tissue sections were deparaffinized, rehydrated, and then microwaved (640 W microwave) with 10 mM citrate buffer (pH 6) for 10 min. After they had been blocked first with 3% H2O2 and next with 10% goat serum, the tissue sections were incubated at room temperature for 60 min with anti-5-bromo-2'deoxyuridine antibody and then for 30 min with peroxidase antimouse IgG2. Color development was done using an aminoethyl carbazole substrate kit (Zymed Laboratories, Inc., San Francisco, CA) and counterstained with Mayer’s hematoxylin. For factor VIII staining, tissue sections were first treated with 0.1% trypsin for 60 min at 37 C. After they had been blocked first with H2O2 and then with 10% goat serum, the tissue sections were incubated with rabbit polyclonal antibody for factor VIII (Dako Corp., Carpinteria, CA) at 4 C overnight. After being washed in PBS, the tissue sections were incubated with biotinylated antirabbit IgG (Vector Laboratories, Inc., Burlingame, CA) for 60 min at room temperature and then visualized using an avidin-biotin complex (Vector Laboratories) and then 3–3' diaminobenzidine (Vector Laboratories).
For neutrophils and macrophages staining, cryostat sections were fixed in 3.7% paraformaldehyde and acetone. After the sections had been blocked with 3% H2O2 and washed, the tissue slides were incubated at 4 C overnight with rat antimouse Gr-1 antibody (BD PharMingen) for neutrophils, rat antimouse F4/80 antibody (Serotec Ltd., Oxford, UK) for macrophages, rat antimouse CD4 or CD8 antibody (BD PharMingen) for T cells. After they had been incubated with antirat IgG at 4 C overnight, the tissue sections were visualized using an aminoethyl carbazole substrate kit. For double immunofluorescence staining, the tissues sections were incubated with mouse antihuman VEGF (Ab-3; Neomarkers; Lab Vision Corp., Fremont, CA) overnight at 4 C, and then with antimouse IgG conjugated with fluorescent dye (Alexa Fluor 488; Molecular Probes; Invitrogen Corp., Carlsbad, CA) at room temperature for 2 h. After the sections had been washed, they were incubated with rat antimouse Mac3 (BD PharMingen; Becton, Dickinson and Co., Franklin Lakes, NJ) for macrophages or antimouse Gr-1 (BD PharMingen, San Diego, CA) for neutrophils, respectively. After they had been incubated with antirat IgG conjugated with Alexa Fluor 546 (Molecular Probes, Eugene, OR), the sections were viewed under a microscope (Leica Mikrosysteme Vertrieb GmbH, Bensheim, Germany) equipped for fluorescence with a 100-W UV lamp.
Collection of peritoneal fluid
The mice were asphyxiated using CO2, and peritoneal fluid was collected by irrigating the abdominal cavity with 1 ml cold PBS plus 0.02% EDTA. The peritoneal fluid was centrifuged at 4 C, and the supernatant was collected for cytokine assay.
Flow cytometry of immune cells in the peritoneal cavity
The exudates from the peritoneal cavity were collected by repeating the irrigation with PBS plus 0.02% EDTA, and then they were centrifuged. Cell pellets were resuspended with RBC lysis buffer [144 mM NH4Cl in 17 mM Tris buffer (pH 7.2)] for 3 min at room temperature and washed twice in cold PBS. The viability of exuded peritoneal cells was greater than 95% (determined using trypan blue dye exclusion). The isolated peritoneal exuded cells (1 x 105) were incubated with a 1:100 dilution of fluorescein isothiocyanate (FITC)-conjugated F4/80, FITC-conjugated Gr-1, FITC-conjugated CD4, or FITC-conjugated CD8 monoclonal antibody (PharMingen) for 40 min at 4 C. In addition, tubes containing FITC-conjugated rat IgG2b (PharMingen) alone were used as isotype controls. Flow-cytometric measurements were done (FACScan; Becton Dickinson, Mountain View, CA) and analyzed using Cell Quest software (Becton Dickinson).
Culture of peritoneal neutrophils and macrophages
Peritoneal neutrophils and macrophages were purified by centrifuging them on Percoll gradient. Isolated exuded peritoneal cells were mixed with a Percoll solution in a 10-ml ultracentrifuge tube (Beckman Coulter, Inc., Fullerton, CA) and centrifuged for 20 min at 60,650 x g at 4 C. The neutrophil-rich median layer and the macrophage-rich upper layer of suspension were collected. The neutrophil and macrophage suspension to washed twice in RPMI 1640 medium (Life Technologies, Inc.-BRL, Rockville, MD) containing 5% fetal bovine serum. The purity of peritoneal neutrophils and macrophages was greater than 95% (flow cytometry).
The purified neutrophils and macrophages were resuspended in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM glutamine, 50 μM 2-mercaptoethanol, 25 mM HEPES, penicillin (100 U), and streptomycin (100 μg/ml). Cells at the density of 1 x 106 cells/well were cultured in 24-well plates. After it had been stimulated using 15 ng/ml IL-6 (CytoLab Ltd., Rehovot, Israel), 50 ng/ml TNF (CytoLab), and 100 ng/ml Escherichia coli-derived lipopolysaccharide (LPS) (Sigma Chemical Co., St. Louis, MO) for 20 h, the cultured medium was removed, centrifuged at 4 C, and stored at –80 C for further cytokine assays.
ELISA of cytokines and chemokines
Concentrations of murine VEGF, IL-6, TNF, MIP-1, and MIP-2 were measured using a commercially available ELISA kit (R&D Systems Europe, Abingdon, UK) according to the manufacturer’s instructions. All data are expressed as means ± SEM. The sensitivity of the ELISA kit was less than 5.0 pg/ml.
Statistical analysis
Values are expressed as means ± SEM of at least four mice. The statistical significance of differences between respective groups was evaluated using ANOVA and then Tukey’s test. Statistical significance was set at P < 0.05.
Results
Cell proliferation in eutopic and ectopic tissue
Estrogen stimulates cell proliferation in the uterus and endometriotic tissue (24, 29). Figure 1 shows the time-course changes of cell proliferation in eutopic and ectopic tissues after uterine tissue was autotransplanted onto peritoneum of mice that received estrogen or vehicle treatment. In eutopic uterine tissue, ovariectomy caused cell death and regression of the endometrium consistent with previous reports (30, 31). No BrdU-positive cells were seen (Fig. 1, A–D). E2 treatment stimulated cell proliferation, as shown by profound BrdU-positive cells in eutopic endometrium on d 1–7 (Fig. 1, E–H). In contrast, very few BrdU-positive cells were detected in ectopic endometriotic tissue in first 1 or 2 d after its transplantation, even in E2-treated tissue (Fig. 1, M and N). Meanwhile, dead cells were observed at these periods. On d 5–7, more BrdU-positive cells were seen in the endometriotic tissue of E2-treated mice (Fig. 1, O and P). The changes in ectopic tissue of vehicle-treated mice were similar to those in E2-treated group at d 1 and 2. Dead cells instead of BrdU-positive cells appeared (Fig. 1, I and J). Without E2 stimulus, the epithelium of ectopic tissues became thinner, and only a few BrdU-positive cells were observed at d 5–7 (Fig. 1, K and L).
Angiogenesis in ectopic uterine tissue
To investigate whether proliferation in ectopic tissue is related to the establishment of new blood vessels, the changes in blood vessels in transplanted tissue were examined. Immunohistochemical staining with an endothelial cell marker (factor VIII) showed that the blood vessels in the uterine explant tissue before transplantation had intact endothelium and few BrdU-positive cells (Fig. 2, A and B). One to two days after transplantation, however, the endothelium of many blood vessels in ectopic tissue started to disintegrate (Fig. 2, C–E). From d 4 to 7, there were numerous blood vessels with intact endothelium in the ectopic tissue (Fig. 2, F–K). Furthermore, there was a significant number of BrdU-positive cells in the blood vessels, particularly on d 4 and 5 (Fig. 2, I and J), which indicated angiogenesis was occurring. The ectopic tissue of ovariectomized mice without E2 treatment showed similar results (Fig. 2, L–S).
Infiltration of immune cells into ectopic uterine tissue and the peritoneal cavity
To understand the involvement of immune cells in endometriosis, different markers of immune cells were used to identify neutrophils, macrophages, and T cells. There were few macrophages and neutrophils in uterine tissue explants on d 0 of transplantation (Fig. 3). Neutrophils in ectopic uterine tissue increased dramatically from d 1 to 5 and then declined on d 6 and 7. There was also a significant infiltration of macrophages in the ectopic tissues on d 2–4. The similar pattern was observed in the ectopic tissue of vehicle-treated mice (data not shown). Compared with profound changes in neutrophils and macrophages, only a few CD4 and CD8 immunoreactive cells appeared in the ectopic tissue, and no particular changes were seen after the tissue transplantation.
Immune cells also infiltrated into the peritoneal cavity. Figure 4 shows the time-course changes of immune cells in peritoneal cavity. In E2-treated mice with uterine tissue transplantation, total immune cell number increased significantly from d 2 to 6 and then declined to their original levels on d 14 (Fig. 4A). On d 0, macrophages were the major immune cells in the peritoneal cavity, and neutrophils and lymphocytes were the minor ones. After tissue transplantation, the number of macrophages did not change much. On the contrary, neutrophils peaked on d 2–4 after tissue transplantation and decreased thereafter (Fig. 4B). Meanwhile, the levels of the chemokines MIP-2 and MIP-1 in peritoneal fluid also peaked on d 2–4 and then declined, parallel to the changes in neutrophils (Fig. 4, C and D). In E2-untreated mice, the infiltration of neutrophils also peaked on d 2–4 (data not shown). There was no difference in peritoneal levels of MIP-1 and MIP-2 in E2-untreated and -treated mice (Table 1). In the suture-only group, inflammatory responses occurred transiently. Neutrophil infiltration and chemokine levels peaked on d 2 and then declined (Fig. 4, E–H).
Secretion of VEGF by neutrophils and macrophages
To investigate the function of infiltrated leukocytes, peritoneal neutrophils and macrophages were cultured and the VEGF level in the conditioned medium was analyzed. Neutrophils and macrophages isolated from the peritoneal cavity of E2-treated mice with uterine tissue implants secreted VEGF (Fig. 5). Treatment with IL-6, TNF, and LPS further stimulated these leukocytes to secrete more VEGF (Fig. 5, A and B). Similarly, TNF and LPS treatment caused the secretion of VEGF by peritoneal neutrophils and macrophages from vehicle-treated mice with tissue implants (Fig. 5, C and D) and from the suture-only group (Fig. 5, E and F). E2 treatment also caused them to secrete VEGF. Double-immunohistochemical staining confirmed the expression of VEGF in neutrophils and macrophages that had infiltrated ectopic endometriotic tissue (Fig. 6).
Secretion of cytokines and chemokines by neutrophils and macrophages
Peritoneal neutrophils and macrophages also secreted cytokines and chemokines. Due to the low levels of neutrophils in the peritoneal cavities of mice on d 0, neutrophils were isolated only from mice with an endometriotic transplantation on d 2, and secretion of IL-6, TNF, MIP-1, and MIP-2 was evaluated (Fig. 7). TNF and LPS treatment increased the basal secretion of IL-6 from neutrophils (Fig. 7A), and IL-6 induced these cells to secrete TNF (Fig. 7B), which indicated that there was a positive loop controlling cytokine secretion in the peritoneal cavity. IL-6, TNF, and LPS treatment also stimulated neutrophils to secrete MIP-1 and MIP-2 (Fig. 7, C and D). Although macrophage numbers did not change, the endogenous secretions of IL-6, TNF, MIP-1, and MIP-2 from peritoneal macrophages of mice with uterine tissue implants on d 2 were much higher than those on d 0. Treatment with IL-6, TNF, and LPS increased the basal secretion of cytokines from peritoneal macrophages in endometriotic tissue (Fig. 8).
Discussion
Using a mouse model of endometriosis, we showed that estrogen stimulated cell proliferation in endometriotic-like tissue primarily after the growth of new blood vessels at the implanted sites. Ectopic uterine tissue underwent cell death shortly after tissue transplantation in the peritoneum. After angiogenesis during d 4–6, estrogen-treated ectopic tissue responded with profound cell proliferation; however, without estrogen treatment, ectopic tissue with neovasculature responded with minimal growth. These results suggest that estrogen is not involved in the initial stage of endometriosis. This is consistent with a previous study in monkeys (3) showing that the establishment of ectopic endometriotic tissue in the peritoneum did not require estrogen.
Estrogen stimulates the growth of uterus and endometriotic tissue (3, 24, 29, 30, 31). In addition, cytokines and growth factors produced by endometriotic implants and macrophages may also be involved in the growth of endometriotic tissue (32, 33). In vitro studies have demonstrated that TNF, IL-8, and hepatocyte growth factor, which are significantly elevated in the peritoneal fluids of patients with endometriosis, stimulated proliferation of endometriotic stromal cells (32, 33, 34). In our study, profoundly BrdU-positive cells were seen in the epithelium and stroma in the ectopic tissue of estrogen-treated mice, and only some proliferative cells occurred in vehicle-treated tissue. However, in estrogen-treated eutopic tissue, BrdU-positive cells were seen mostly in the epithelium, not in the stroma. It is possible that cytokines produced by macrophages and neutrophils that have infiltrated the ectopic tissues and peritoneal cavity stimulate the growth of ectopic tissue. It is also possible that ectopic tissue produces cytokines that contribute to its own growth.
Increases in macrophages and inflammatory cytokines have been reported in the peritoneal fluid of women with endometriosis (12, 13, 14). Retrograde menstruation has been proposed as a source of this (35, 36). This possibility is further supported by evidence that the intrapelvic injection of endometrium in monkeys increased peritoneal leukocyte concentrations and the number of TNF-positive cells and caused endometriosis (35, 36). Mice do not menstruate, but the ip injection of endometrial cells also induced an inflammatory response and an increase in peritoneal levels of MCP-1, IL-1, and IL-6 (37). In the present study, we demonstrated that the number of leukocytes and the levels of the chemokines MIP-2 and MIP-1 in peritoneal fluid in mice increased after uterine tissue was implanted into the peritoneum. In the peritoneal cavities of control mice, there was a significant number of macrophages but not of neutrophils. After the transplantation of uterine tissue, macrophages were activated, as indicated by a higher basal secretion of TNF, IL-6, MIP-1, and MIP-2 than in healthy control mice. The activation of macrophages has been demonstrated in humans: peritoneal macrophages in women with endometriosis released higher levels of MCP-1 under basal and LPS-stimulated conditions than in healthy women (12). Interestingly, we also found a profound infiltration of neutrophils that reached its peak on d 2–4 and then declined. This infiltration was parallel to the changes of chemokines in the peritoneal fluid. The secretion of MIF-1 and MIP-2 by neutrophils and macrophages was increased by IL-6 and TNF. It seems that the autologous transplantation of uterine tissue into the peritoneum induces inflammatory responses. These cytokines and chemokines form a positive regulatory network that may subsequently help the establishment of endometriosis. Therefore, the interference of pelvic inflammatory network may prevent the development of endometriotic lesions. Indeed, in a baboon model of endometriosis, the neutralization of TNF activity by recombinant human TNFRSF1A, the soluble form of TNF receptor type I, interfered with the establishment of endometriosis (38).
VEGF and other angiogenic factors are found in the peritoneal fluid of patients with endometriosis (9, 10, 11, 15). Macrophages as well as ectopic and eutopic endometrial tissue are potential sources of VEGF (9, 10, 11, 39). We demonstrated in the present study that, after the implantation of uterine tissue into the peritoneum of mice, neutrophils infiltrated, macrophages were activated, and both secreted VEGF in response to the stimuli of TNF and IL-6. The infiltration and activation of neutrophils and macrophages occurred on d 1–4, and actively proliferating blood vessels were seen on d 4 and 5. The proceeding of leukocyte infiltration to the angiogenesis in implanted uterine tissue suggests that, in addition to being phagocytes to clear damaged cells, neutrophils and macrophages may be important for establishing a new blood supply in ectopic endometriotic tissue.
Evidence has shown that estrogen is important for angiogenesis in normal endometrium and in endometriosis. Estrogen treatment increased angiogenesis in mouse endometrium (40). It also stimulated the secretion of VEGF by peritoneal macrophages isolated from patients with endometriosis (9). However, in the present study, angiogenesis occurred in ectopic uterine tissue in estrogen-treated mice and -untreated mice. Our in vitro studies demonstrated that peritoneal neutrophils and macrophages secreted VEGF in response to TNF and IL-6 as well as estrogen. It seems that the implantation of ectopic uterine tissue in the peritoneal cavity induced an inflammatory response. Neutrophils and macrophages are recruited and activated, which produce VEGF and subsequently lead to angiogenesis in ectopic tissue. It is therefore possible that the inflammatory angiogenesis rather than estrogen-induced angiogenesis is more important at the early stage of endometriosis This is supported by a report that nonsteroidal antiinflammatory drugs reduced endometriotic lesions in a mouse model regardless of the presence of estrogen (41).
Acknowledgments
We thank Dr. Yi-Ling Chen and Mr. Wen-Wei Chang for the technical support for leukocyte culture and cytokine assays. We also thank Dr. Ming. T. Lin and Dr. Yi-Sin Lin for suggestions of the manuscript.
Footnotes
This work was supported by Grants NSC 88-2314-006-072 and NSC 93-2320-B006-002 from the National Science Council, Taiwan (to L.-Y.C.W.).
The authors have no conflict of interest.
First Published Online November 23, 2005
Abbreviations: BrdU, 5-Bromo-2'-deoxyuridine; E2, estradiol-17; FITC, fluorescein isothiocyanate; LPS, lipopolysaccharide; MIP, macrophage inflammatory protein; VEGF, vascular endothelial growth factor.
Accepted for publication November 16, 2005.
References
Strathy JH, Molgaard CA, Coulam CB, Melton 3rd LJ 1982 Endometriosis and infertility: a laparoscopic study of endometriosis among fertile and infertile women. Fertil Steril 38:667–672
Ryan IP, Taylor RN 1997 Endometriosis and infertility: new concepts. Obstet Gynecol Surv 52:365–371
Dizerega GS, Barber DL, Hodgen GD 1980 Endometriosis: role of ovarian steroids in initiation, maintenance, and suppression. Fertil Steril 33:649–653
Agarwal SK 1998 Comparative effects of GnRH agonist therapy. Review of clinical studies and their implications. J Reprod Med 43:293–298
van der Linden PJ 1996 Theories on the pathogenesis of endometriosis. Hum Reprod 11(Suppl 3):53–65
Hull ML, Charnock-Jones DS, Chan CL, Bruner-Tran KL, Osteen KG, Tom BD, Fan TP, Smith SK 2003 Antiangiogenic agents are effective inhibitors of endometriosis. J Clin Endocrinol Metab 88:2889–2899
Nap AW, Griffioen AW, Dunselman GA, Bouma-Ter Steege JC, Thijssen VL, Evers JL, Groothuis PG 2004 Antiangiogenesis therapy for endometriosis. J Clin Endocrinol Metab 89:1089–1095
Oosterlynck DJ, Meuleman C, Sobis H, Vandeputte M, Koninckx PR 1993 Angiogenic activity of peritoneal fluid from women with endometriosis. Fertil Steril 59:778–782
McLaren J, Prentice A, Charnock-Jones DS, Millican SA, Muller KH, Sharkey AM, Smith SK 1996 Vascular endothelial growth factor is produced by peritoneal fluid macrophages in endometriosis and is regulated by ovarian steroids. J Clin Invest 98:482–489
Maas JW, Calhaz-Jorge C, ter Riet G, Dunselman GA, de Goeij AF, Struijker-Boudier HA 2001 Tumor necrosis factor- but not interleukin-1 or interleukin-8 concentrations correlate with angiogenic activity of peritoneal fluid from patients with minimal to mild endometriosis. Fertil Steril 75:180–185
Mahnke JL, Dawood MY, Huang JC 2000 Vascular endothelial growth factor and interleukin-6 in peritoneal fluid of women with endometriosis. Fertil Steril 73:166–170
Akoum A, Kong J, Metz C, Beaumont MC 2002 Spontaneous and stimulated secretion of monocyte chemotactic protein-1 and macrophage migration inhibitory factor by peritoneal macrophages in women with and without endometriosis. Fertil Steril 77:989–994
Keenan JA, Chen TT, Chadwell NL, Torry DS, Caudle MR 1994 Interferon- (IFN-) and interleukin-6 (IL-6) in peritoneal fluid and macrophage-conditioned media of women with endometriosis. Am J Reprod Immunol 32:180–183
Rana N, Braun DP, House R, Gebel H, Rotman C, Dmowski WP 1996 Basal and stimulated secretion of cytokines by peritoneal macrophages in women with endometriosis. Fertil Steril 65:925–930
Arici A, Tazuke SI, Attar E, Kliman HJ, Olive DL 1996 Interleukin-8 concentration in peritoneal fluid of patients with endometriosis and modulation of interleukin-8 expression in human mesothelial cells. Mol Hum Reprod 2:40–45
Suzumori N, Katano K, Suzumori K 2004 Peritoneal fluid concentrations of epithelial neutrophil-activating peptide-78 correlate with the severity of endometriosis. Fertil Steril 81:305–308
Rossi D, Zlotnik A 2000 The biology of chemokines and their receptors. Annu Rev Immunol 18:217–242
Benelli R, Morini M, Carrozzino F, Ferrari N, Minghelli S, Santi L, Cassatella M, Noonan DM, Albini A 2002 Neutrophils as a key cellular target for angiostatin: implications for regulation of angiogenesis and inflammation. FASEB J 16:267–269
Scapini P, Morini M, Tecchio C, Minghelli S, Di Carlo E, Tanghetti E, Albini A, Lowell C, Berton G, Noonan DM, Cassatella MA 2004 CXCL1/macrophage inflammatory protein-2-induced angiogenesis in vivo is mediated by neutrophil-derived vascular endothelial growth factor-A. J Immunol 172:5034–5040
McCourt M, Wang JH, Sookhai S, Redmond HP 1999 Proinflammatory mediators stimulate neutrophil-directed angiogenesis. Arch Surg 134:1325–1331; discussion 1331–1332
Jablonska E, Piotrowski L, Jablonski J, Grabowska Z 2002 VEGF in the culture of PMN and the serum in oral cavity cancer patients. Oral Oncol 38:605–609
Mueller MD, Lebovic DI, Garrett E, Taylor RN 2000 Neutrophils infiltrating the endometrium express vascular endothelial growth factor: potential role in endometrial angiogenesis. Fertil Steril 74:107–112
Gaudry M, Bregerie O, Andrieu V, El Benna J, Pocidalo MA, Hakim J 1997 Intracellular pool of vascular endothelial growth factor in human neutrophils. Blood 90:4153–4161
Cummings AM, Metcalf JL 1995 Induction of endometriosis in mice: a new model sensitive to estrogen. Reprod Toxicol 9:233–238
Grummer R, Schwarzer F, Bainczyk K, Hess-Stumpp H, Regidor PA, Schindler AE, Winterhager E 2001 Peritoneal endometriosis: validation of an in vivo model. Hum Reprod 16:1736–1743
Rossi G, Somigliana E, Moschetta M, Santorsola R, Cozzolino S, Filardo P, Salmaso A, Zingrillo B 2000 Dynamic aspects of endometriosis in a mouse model through analysis of implantation and progression. Arch Gynecol Obstet 263:102–107
Hirata T, Osuga Y, Yoshino O, Hirota Y, Harada M, Takemura Y, Morimoto C, Koga K, Yano T, Tsutsumi O, Taketani Y 2005 Development of an experimental model of endometriosis using mice that ubiquitously express green fluorescent protein. Hum Reprod 20:2092–2096
Cohen PE, Milligan SR 1993 Silastic implants for delivery of oestradiol to mice. J Reprod Fertil 99:219–223
Lai MD, Jiang MJ, Wing LY 2002 Estrogen stimulates expression of p21Waf1/Cip1 in mouse uterine luminal epithelium. Endocrine 17:233–239
Lai MD, Lee LR, Cheng KS, Wing LY 2000 Expression of proliferating cell nuclear antigen in luminal epithelium during the growth and regression of rat uterus. J Endocrinol 166:87–93
Sato T, Fukazawa Y, Kojima H, Ohta Y, Iguchi T 2003 Multiple mechanisms are involved in apoptotic cell death in the mouse uterus and vagina after ovariectomy. Reprod Toxicol 17:289–297
Iwabe T, Harada T, Tsudo T, Nagano Y, Yoshida S, Tanikawa M, Terakawa N 2000 Tumor necrosis factor- promotes proliferation of endometriotic stromal cells by inducing interleukin-8 gene and protein expression. J Clin Endocrinol Metab 85:824–829
Khan KN, Masuzaki H, Fujishita A, Kitajima M, Sekine I, Matsuyama T, Ishimaru T 2005 Estrogen and progesterone receptor expression in macrophages and regulation of hepatocyte growth factor by ovarian steroids in women with endometriosis. Hum Reprod 20:2004–2013
Khan KN, Masuzaki H, Fujishita A, Hamasaki T, Kitajima M, Hasuo A, Miyamura Y, Ishimaru T 2002 Association of interleukin-6 and estradiol with hepatocyte growth factor in peritoneal fluid of women with endometriosis. Acta Obstet Gynecol Scand 81:764–771
D’Hooghe TM, Bambra CS, Raeymaekers BM, De Jonge I, Lauweryns JM, Koninckx PR 1995 Intrapelvic injection of menstrual endometrium causes endometriosis in baboons (Papio cynocephalus and Papio anubis). Am J Obstet Gynecol 173:125–134
D’Hooghe TM, Bambra CS, Xiao L, Peixe K, Hill JA 2001 Effect of menstruation and intrapelvic injection of endometrium on inflammatory parameters of peritoneal fluid in the baboon (Papio anubis and Papio cynocephalus). Am J Obstet Gynecol 184:917–925
Cao X, Yang D, Song M, Murphy A, Parthasarathy S 2004 The presence of endometrial cells in the peritoneal cavity enhances monocyte recruitment and induces inflammatory cytokines in mice: implications for endometriosis. Fertil Steril 82(Suppl 3):999–1007
D’Hooghe TM, Nugent NP, Cuneo S, Chai DC, Deer F, Debrock S, Kyama CM, Mihalyi A, Mwenda JM2006 Recombinant human TNFRSF1A (r-hTBP1) inhibits the development of endometriosis in baboons: a prospective, randomized, placebo- and drug-controlled study. Biol Reprod 74:131–136
Fukuda J, Kawano Y, Nasu K, Nishida M, Narahara H, Miyakawa I 2004 Differential effects of interferon- and - on the secretion of vascular endothelial growth factor by eutopic and ectopic endometrial stromal cells. Fertil Steril 82:749–751
Heryanto B, Lipson KE, Rogers PA 2003 Effect of angiogenesis inhibitors on oestrogen-mediated endometrial endothelial cell proliferation in the ovariectomized mouse. Reproduction 125:337–346
Efstathiou JA, Sampson DA, Levine Z, Rohan RM, Zurakowski D, Folkman J, D’Amato RJ, Rupnick MA 2005 Nonsteroidal antiinflammatory drugs differentially suppress endometriosis in a murine model. Fertil Steril 83:171–181(Yiu-Jiuan Lin, Ming-Derg Lai, Huan-Yao L)
Department of Physical Therapy (M.-D.L.), Shu-Zen College of Medicine and Management, Kaohsiung 82144, Taiwan
Abstract
Substantial evidence suggests that inflammatory cytokines, immune cells, and angiogenesis are important for endometriosis. In this study, we investigated the role of the sequential events in the development of endometriosis in a mouse model. Uterine tissue was transplanted into the peritoneum of ovariectomized mice and then supplemented with estrogen or vehicle. On different days after transplantation, cell proliferation, angiogenesis, and infiltrated immune cells in ectopic tissue were examined using immunochemical staining. Many disintegrated blood vessels but no bromodeoxyuridine-positive cells in ectopic tissue were observed in the estrogen-treated group on posttransplantation d 1 and 2. On d 4–7, bromodeoxyuridine-positive cells were detected in the blood vessels of ectopic tissue, indicating that angiogenesis was initiated in this stage. Angiogenesis also occurred in ectopic tissue in the vehicle-treated group. Profound infiltration of neutrophils in ectopic tissue occurred on d 1–4, when the number of neutrophils and levels of macrophage inflammatory protein (MIP)-1 and MIP-2 chemokines in peritoneal fluids also reached their peak. Peritoneal macrophage numbers did not change, but secretions of TNF, IL-6, MIP-1, and MIP-2 from macrophages isolated on d 2 were higher than on d 0. In vitro studies showed that peritoneal neutrophils and macrophages secreted vascular endothelial growth factor, which was up-regulated by TNF and IL-6. Our results suggest that neutrophils and macrophages may promote angiogenesis in the early stage of endometriosis and that chemokines and cytokines amplify the angiogenic signal for the growth of endometriotic tissue.
Introduction
ENDOMETRIOSIS, WHICH IS characterized by the growth of uterine tissue outside the uterine cavity, is one of the most common gynecological diseases at reproductive age. It seldom occurs in prepubescent or menopausal women. The progression of endometriosis is estrogen dependent (1, 2, 3). Therefore, using ovariectomy or GnRH agonists to reduce endogenous estrogen causes the regression of endometriotic tissues (4). The implantation hypothesis is widely accepted as the etiology of endometriosis. During menstruation, the endometrium may retrograde into the peritoneal cavity and then implant and grow in ectopic sites (5). The ability of retrograde endometrium to grow in the peritoneal cavity depends on establishment and maintenance of new blood supply. The importance of neovascularization in endometriosis was demonstrated by the evidence that antiangiogenic agents inhibited the growth of endometriotic tissue in nude mice (6, 7). Increased angiogenic activity and vascular endothelial growth factor (VEGF) levels were found in peritoneal fluid of patients with endometriosis (8, 9, 10, 11). However, it is not clear yet how the angiogenesis in endometriosis is initiated.
Endometriosis is an inflammatory disease. Cytokine levels and macrophage numbers are elevated in peritoneal fluids of patients with endometriosis (12, 13, 14). Recently several studies showed that levels of IL-8 and epithelial neutrophil-activating peptide 78 in the peritoneal fluid of patients with endometriosis were elevated (15, 16). Both IL-8 and epithelial neutrophil-activating peptide 78 belong to the cysteine-X-cysteine (CXC) chemokine family. They are neutrophil chemoattractants and possess angiogenic activity (17). Recent evidence has shown that CXC chemokine-induced angiogenesis may occur through the recruitment of neutrophils that secrete VEGF (18, 19, 20, 21). It has been suggested that the VEGF produced by infiltrated neutrophils in the human endometrium and endometrial intravasculature may regulate angiogenesis during the menstrual cycle (22, 23). There is no available information about the contribution of neutrophils to the pathogenesis of endometriosis.
Animal models are important for elucidating the mechanisms underlying endometriosis. Mice lack a menstrual cycle and do not develop spontaneous endometriosis. To induce endometriosis in mice, endometrial tissue is transplanted into the peritoneal cavity using several methods (24, 25, 26, 27). To create an autologous model of mouse endometriosis, portions of uterine or endometrial tissue are surgically transplanted on the peritoneum, in which they develop into endometriotic-like lesions (24, 26). Although mouse endometriosis may not be exactly the same as human endometriosis, their endometriotic lesions develop in some ways, similarly to human endometriotic lesions. Mouse endometriotic tissue is estrogen responsive: it grows under estrogen stimulation and regresses after the withdrawal this hormone (24, 25). In the present study, we used a surgically induced endometriosis mouse model to investigate immune responses and angiogenesis during the development of endometriosis. Our results showed that neutrophils infiltrated into endometriotic tissue and peritoneal cavity during the early stage of endometriosis development. Meanwhile, peritoneal macrophages were activated. Both neutrophils and macrophages secrete angiogenic factors and cytokines. The secretion of macrophage inflammatory protein (MIP)-1, MIP-2, IL-6, TNF, and VEGF by neutrophils and macrophages may create a positive loop to amplify the signal for the establishment and growth of endometriotic tissue in ectopic sites.
Materials and Methods
Animals
Mature (8–10 wk old) female C57BL/6NCrj mice were purchased from the Animal Center at the College of Medicine, National Cheng Kung University. The mice were maintained on standard chow and water, which were available ad libitum, in animal facilities illuminated between 0600 and 1800 h. All procedures were performed in accordance with the Guidelines of the National Cheng Kung University Animal Center for the handling of laboratory animals.
Mouse model for endometriosis and hormone treatment
Endometriosis was surgically induced as previously described (24). In brief, the mice were anesthetized with sodium pentobarbital and their left uterine horn was removed, spliced longitudinally, and cut into approximately 0.4-cm squares that were placed in a petri dish containing DMEM supplemented with penicillin (100 U/ml) and streptomycin (100 μg/ml). The tissue explants were sutured to the peritoneum in the peritoneal cavity. On the day of surgery, the mice were ovariectomized and implanted sc with SILASTIC implants (Dow Corning, Midland, MI) containing vehicle or estradiol-17 (E2, 25 μg/ml dissolved in sesame oil) according to procedures described elsewhere (28). For the surgery, mice at stages other than proestrus were used according to their vaginal smears. For the control group, ovariectomized mice received only sutures on the peritoneum. After different time intervals, the mice were killed; their eutopic and ectopic uterine tissue was removed and fixed for paraffin embedding or directly embedded in optimal cutting temperature (OCT) compound for cryostat sections.
Immunohistochemistry
The procedures for 5-bromo-2'-deoxyuridine (BrdU) detection were as described previously (29). The mice were injected (ip) with BrdU labeling reagent (Amersham Pharmacia Biotech Inc., Piscataway, NJ) 2 h before killing. The eutopic and ectopic uterine tissue was fixed in 10% buffered formalin, dehydrated, and embedded in paraffin. Five-micrometer-thick tissue sections were deparaffinized, rehydrated, and then microwaved (640 W microwave) with 10 mM citrate buffer (pH 6) for 10 min. After they had been blocked first with 3% H2O2 and next with 10% goat serum, the tissue sections were incubated at room temperature for 60 min with anti-5-bromo-2'deoxyuridine antibody and then for 30 min with peroxidase antimouse IgG2. Color development was done using an aminoethyl carbazole substrate kit (Zymed Laboratories, Inc., San Francisco, CA) and counterstained with Mayer’s hematoxylin. For factor VIII staining, tissue sections were first treated with 0.1% trypsin for 60 min at 37 C. After they had been blocked first with H2O2 and then with 10% goat serum, the tissue sections were incubated with rabbit polyclonal antibody for factor VIII (Dako Corp., Carpinteria, CA) at 4 C overnight. After being washed in PBS, the tissue sections were incubated with biotinylated antirabbit IgG (Vector Laboratories, Inc., Burlingame, CA) for 60 min at room temperature and then visualized using an avidin-biotin complex (Vector Laboratories) and then 3–3' diaminobenzidine (Vector Laboratories).
For neutrophils and macrophages staining, cryostat sections were fixed in 3.7% paraformaldehyde and acetone. After the sections had been blocked with 3% H2O2 and washed, the tissue slides were incubated at 4 C overnight with rat antimouse Gr-1 antibody (BD PharMingen) for neutrophils, rat antimouse F4/80 antibody (Serotec Ltd., Oxford, UK) for macrophages, rat antimouse CD4 or CD8 antibody (BD PharMingen) for T cells. After they had been incubated with antirat IgG at 4 C overnight, the tissue sections were visualized using an aminoethyl carbazole substrate kit. For double immunofluorescence staining, the tissues sections were incubated with mouse antihuman VEGF (Ab-3; Neomarkers; Lab Vision Corp., Fremont, CA) overnight at 4 C, and then with antimouse IgG conjugated with fluorescent dye (Alexa Fluor 488; Molecular Probes; Invitrogen Corp., Carlsbad, CA) at room temperature for 2 h. After the sections had been washed, they were incubated with rat antimouse Mac3 (BD PharMingen; Becton, Dickinson and Co., Franklin Lakes, NJ) for macrophages or antimouse Gr-1 (BD PharMingen, San Diego, CA) for neutrophils, respectively. After they had been incubated with antirat IgG conjugated with Alexa Fluor 546 (Molecular Probes, Eugene, OR), the sections were viewed under a microscope (Leica Mikrosysteme Vertrieb GmbH, Bensheim, Germany) equipped for fluorescence with a 100-W UV lamp.
Collection of peritoneal fluid
The mice were asphyxiated using CO2, and peritoneal fluid was collected by irrigating the abdominal cavity with 1 ml cold PBS plus 0.02% EDTA. The peritoneal fluid was centrifuged at 4 C, and the supernatant was collected for cytokine assay.
Flow cytometry of immune cells in the peritoneal cavity
The exudates from the peritoneal cavity were collected by repeating the irrigation with PBS plus 0.02% EDTA, and then they were centrifuged. Cell pellets were resuspended with RBC lysis buffer [144 mM NH4Cl in 17 mM Tris buffer (pH 7.2)] for 3 min at room temperature and washed twice in cold PBS. The viability of exuded peritoneal cells was greater than 95% (determined using trypan blue dye exclusion). The isolated peritoneal exuded cells (1 x 105) were incubated with a 1:100 dilution of fluorescein isothiocyanate (FITC)-conjugated F4/80, FITC-conjugated Gr-1, FITC-conjugated CD4, or FITC-conjugated CD8 monoclonal antibody (PharMingen) for 40 min at 4 C. In addition, tubes containing FITC-conjugated rat IgG2b (PharMingen) alone were used as isotype controls. Flow-cytometric measurements were done (FACScan; Becton Dickinson, Mountain View, CA) and analyzed using Cell Quest software (Becton Dickinson).
Culture of peritoneal neutrophils and macrophages
Peritoneal neutrophils and macrophages were purified by centrifuging them on Percoll gradient. Isolated exuded peritoneal cells were mixed with a Percoll solution in a 10-ml ultracentrifuge tube (Beckman Coulter, Inc., Fullerton, CA) and centrifuged for 20 min at 60,650 x g at 4 C. The neutrophil-rich median layer and the macrophage-rich upper layer of suspension were collected. The neutrophil and macrophage suspension to washed twice in RPMI 1640 medium (Life Technologies, Inc.-BRL, Rockville, MD) containing 5% fetal bovine serum. The purity of peritoneal neutrophils and macrophages was greater than 95% (flow cytometry).
The purified neutrophils and macrophages were resuspended in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM glutamine, 50 μM 2-mercaptoethanol, 25 mM HEPES, penicillin (100 U), and streptomycin (100 μg/ml). Cells at the density of 1 x 106 cells/well were cultured in 24-well plates. After it had been stimulated using 15 ng/ml IL-6 (CytoLab Ltd., Rehovot, Israel), 50 ng/ml TNF (CytoLab), and 100 ng/ml Escherichia coli-derived lipopolysaccharide (LPS) (Sigma Chemical Co., St. Louis, MO) for 20 h, the cultured medium was removed, centrifuged at 4 C, and stored at –80 C for further cytokine assays.
ELISA of cytokines and chemokines
Concentrations of murine VEGF, IL-6, TNF, MIP-1, and MIP-2 were measured using a commercially available ELISA kit (R&D Systems Europe, Abingdon, UK) according to the manufacturer’s instructions. All data are expressed as means ± SEM. The sensitivity of the ELISA kit was less than 5.0 pg/ml.
Statistical analysis
Values are expressed as means ± SEM of at least four mice. The statistical significance of differences between respective groups was evaluated using ANOVA and then Tukey’s test. Statistical significance was set at P < 0.05.
Results
Cell proliferation in eutopic and ectopic tissue
Estrogen stimulates cell proliferation in the uterus and endometriotic tissue (24, 29). Figure 1 shows the time-course changes of cell proliferation in eutopic and ectopic tissues after uterine tissue was autotransplanted onto peritoneum of mice that received estrogen or vehicle treatment. In eutopic uterine tissue, ovariectomy caused cell death and regression of the endometrium consistent with previous reports (30, 31). No BrdU-positive cells were seen (Fig. 1, A–D). E2 treatment stimulated cell proliferation, as shown by profound BrdU-positive cells in eutopic endometrium on d 1–7 (Fig. 1, E–H). In contrast, very few BrdU-positive cells were detected in ectopic endometriotic tissue in first 1 or 2 d after its transplantation, even in E2-treated tissue (Fig. 1, M and N). Meanwhile, dead cells were observed at these periods. On d 5–7, more BrdU-positive cells were seen in the endometriotic tissue of E2-treated mice (Fig. 1, O and P). The changes in ectopic tissue of vehicle-treated mice were similar to those in E2-treated group at d 1 and 2. Dead cells instead of BrdU-positive cells appeared (Fig. 1, I and J). Without E2 stimulus, the epithelium of ectopic tissues became thinner, and only a few BrdU-positive cells were observed at d 5–7 (Fig. 1, K and L).
Angiogenesis in ectopic uterine tissue
To investigate whether proliferation in ectopic tissue is related to the establishment of new blood vessels, the changes in blood vessels in transplanted tissue were examined. Immunohistochemical staining with an endothelial cell marker (factor VIII) showed that the blood vessels in the uterine explant tissue before transplantation had intact endothelium and few BrdU-positive cells (Fig. 2, A and B). One to two days after transplantation, however, the endothelium of many blood vessels in ectopic tissue started to disintegrate (Fig. 2, C–E). From d 4 to 7, there were numerous blood vessels with intact endothelium in the ectopic tissue (Fig. 2, F–K). Furthermore, there was a significant number of BrdU-positive cells in the blood vessels, particularly on d 4 and 5 (Fig. 2, I and J), which indicated angiogenesis was occurring. The ectopic tissue of ovariectomized mice without E2 treatment showed similar results (Fig. 2, L–S).
Infiltration of immune cells into ectopic uterine tissue and the peritoneal cavity
To understand the involvement of immune cells in endometriosis, different markers of immune cells were used to identify neutrophils, macrophages, and T cells. There were few macrophages and neutrophils in uterine tissue explants on d 0 of transplantation (Fig. 3). Neutrophils in ectopic uterine tissue increased dramatically from d 1 to 5 and then declined on d 6 and 7. There was also a significant infiltration of macrophages in the ectopic tissues on d 2–4. The similar pattern was observed in the ectopic tissue of vehicle-treated mice (data not shown). Compared with profound changes in neutrophils and macrophages, only a few CD4 and CD8 immunoreactive cells appeared in the ectopic tissue, and no particular changes were seen after the tissue transplantation.
Immune cells also infiltrated into the peritoneal cavity. Figure 4 shows the time-course changes of immune cells in peritoneal cavity. In E2-treated mice with uterine tissue transplantation, total immune cell number increased significantly from d 2 to 6 and then declined to their original levels on d 14 (Fig. 4A). On d 0, macrophages were the major immune cells in the peritoneal cavity, and neutrophils and lymphocytes were the minor ones. After tissue transplantation, the number of macrophages did not change much. On the contrary, neutrophils peaked on d 2–4 after tissue transplantation and decreased thereafter (Fig. 4B). Meanwhile, the levels of the chemokines MIP-2 and MIP-1 in peritoneal fluid also peaked on d 2–4 and then declined, parallel to the changes in neutrophils (Fig. 4, C and D). In E2-untreated mice, the infiltration of neutrophils also peaked on d 2–4 (data not shown). There was no difference in peritoneal levels of MIP-1 and MIP-2 in E2-untreated and -treated mice (Table 1). In the suture-only group, inflammatory responses occurred transiently. Neutrophil infiltration and chemokine levels peaked on d 2 and then declined (Fig. 4, E–H).
Secretion of VEGF by neutrophils and macrophages
To investigate the function of infiltrated leukocytes, peritoneal neutrophils and macrophages were cultured and the VEGF level in the conditioned medium was analyzed. Neutrophils and macrophages isolated from the peritoneal cavity of E2-treated mice with uterine tissue implants secreted VEGF (Fig. 5). Treatment with IL-6, TNF, and LPS further stimulated these leukocytes to secrete more VEGF (Fig. 5, A and B). Similarly, TNF and LPS treatment caused the secretion of VEGF by peritoneal neutrophils and macrophages from vehicle-treated mice with tissue implants (Fig. 5, C and D) and from the suture-only group (Fig. 5, E and F). E2 treatment also caused them to secrete VEGF. Double-immunohistochemical staining confirmed the expression of VEGF in neutrophils and macrophages that had infiltrated ectopic endometriotic tissue (Fig. 6).
Secretion of cytokines and chemokines by neutrophils and macrophages
Peritoneal neutrophils and macrophages also secreted cytokines and chemokines. Due to the low levels of neutrophils in the peritoneal cavities of mice on d 0, neutrophils were isolated only from mice with an endometriotic transplantation on d 2, and secretion of IL-6, TNF, MIP-1, and MIP-2 was evaluated (Fig. 7). TNF and LPS treatment increased the basal secretion of IL-6 from neutrophils (Fig. 7A), and IL-6 induced these cells to secrete TNF (Fig. 7B), which indicated that there was a positive loop controlling cytokine secretion in the peritoneal cavity. IL-6, TNF, and LPS treatment also stimulated neutrophils to secrete MIP-1 and MIP-2 (Fig. 7, C and D). Although macrophage numbers did not change, the endogenous secretions of IL-6, TNF, MIP-1, and MIP-2 from peritoneal macrophages of mice with uterine tissue implants on d 2 were much higher than those on d 0. Treatment with IL-6, TNF, and LPS increased the basal secretion of cytokines from peritoneal macrophages in endometriotic tissue (Fig. 8).
Discussion
Using a mouse model of endometriosis, we showed that estrogen stimulated cell proliferation in endometriotic-like tissue primarily after the growth of new blood vessels at the implanted sites. Ectopic uterine tissue underwent cell death shortly after tissue transplantation in the peritoneum. After angiogenesis during d 4–6, estrogen-treated ectopic tissue responded with profound cell proliferation; however, without estrogen treatment, ectopic tissue with neovasculature responded with minimal growth. These results suggest that estrogen is not involved in the initial stage of endometriosis. This is consistent with a previous study in monkeys (3) showing that the establishment of ectopic endometriotic tissue in the peritoneum did not require estrogen.
Estrogen stimulates the growth of uterus and endometriotic tissue (3, 24, 29, 30, 31). In addition, cytokines and growth factors produced by endometriotic implants and macrophages may also be involved in the growth of endometriotic tissue (32, 33). In vitro studies have demonstrated that TNF, IL-8, and hepatocyte growth factor, which are significantly elevated in the peritoneal fluids of patients with endometriosis, stimulated proliferation of endometriotic stromal cells (32, 33, 34). In our study, profoundly BrdU-positive cells were seen in the epithelium and stroma in the ectopic tissue of estrogen-treated mice, and only some proliferative cells occurred in vehicle-treated tissue. However, in estrogen-treated eutopic tissue, BrdU-positive cells were seen mostly in the epithelium, not in the stroma. It is possible that cytokines produced by macrophages and neutrophils that have infiltrated the ectopic tissues and peritoneal cavity stimulate the growth of ectopic tissue. It is also possible that ectopic tissue produces cytokines that contribute to its own growth.
Increases in macrophages and inflammatory cytokines have been reported in the peritoneal fluid of women with endometriosis (12, 13, 14). Retrograde menstruation has been proposed as a source of this (35, 36). This possibility is further supported by evidence that the intrapelvic injection of endometrium in monkeys increased peritoneal leukocyte concentrations and the number of TNF-positive cells and caused endometriosis (35, 36). Mice do not menstruate, but the ip injection of endometrial cells also induced an inflammatory response and an increase in peritoneal levels of MCP-1, IL-1, and IL-6 (37). In the present study, we demonstrated that the number of leukocytes and the levels of the chemokines MIP-2 and MIP-1 in peritoneal fluid in mice increased after uterine tissue was implanted into the peritoneum. In the peritoneal cavities of control mice, there was a significant number of macrophages but not of neutrophils. After the transplantation of uterine tissue, macrophages were activated, as indicated by a higher basal secretion of TNF, IL-6, MIP-1, and MIP-2 than in healthy control mice. The activation of macrophages has been demonstrated in humans: peritoneal macrophages in women with endometriosis released higher levels of MCP-1 under basal and LPS-stimulated conditions than in healthy women (12). Interestingly, we also found a profound infiltration of neutrophils that reached its peak on d 2–4 and then declined. This infiltration was parallel to the changes of chemokines in the peritoneal fluid. The secretion of MIF-1 and MIP-2 by neutrophils and macrophages was increased by IL-6 and TNF. It seems that the autologous transplantation of uterine tissue into the peritoneum induces inflammatory responses. These cytokines and chemokines form a positive regulatory network that may subsequently help the establishment of endometriosis. Therefore, the interference of pelvic inflammatory network may prevent the development of endometriotic lesions. Indeed, in a baboon model of endometriosis, the neutralization of TNF activity by recombinant human TNFRSF1A, the soluble form of TNF receptor type I, interfered with the establishment of endometriosis (38).
VEGF and other angiogenic factors are found in the peritoneal fluid of patients with endometriosis (9, 10, 11, 15). Macrophages as well as ectopic and eutopic endometrial tissue are potential sources of VEGF (9, 10, 11, 39). We demonstrated in the present study that, after the implantation of uterine tissue into the peritoneum of mice, neutrophils infiltrated, macrophages were activated, and both secreted VEGF in response to the stimuli of TNF and IL-6. The infiltration and activation of neutrophils and macrophages occurred on d 1–4, and actively proliferating blood vessels were seen on d 4 and 5. The proceeding of leukocyte infiltration to the angiogenesis in implanted uterine tissue suggests that, in addition to being phagocytes to clear damaged cells, neutrophils and macrophages may be important for establishing a new blood supply in ectopic endometriotic tissue.
Evidence has shown that estrogen is important for angiogenesis in normal endometrium and in endometriosis. Estrogen treatment increased angiogenesis in mouse endometrium (40). It also stimulated the secretion of VEGF by peritoneal macrophages isolated from patients with endometriosis (9). However, in the present study, angiogenesis occurred in ectopic uterine tissue in estrogen-treated mice and -untreated mice. Our in vitro studies demonstrated that peritoneal neutrophils and macrophages secreted VEGF in response to TNF and IL-6 as well as estrogen. It seems that the implantation of ectopic uterine tissue in the peritoneal cavity induced an inflammatory response. Neutrophils and macrophages are recruited and activated, which produce VEGF and subsequently lead to angiogenesis in ectopic tissue. It is therefore possible that the inflammatory angiogenesis rather than estrogen-induced angiogenesis is more important at the early stage of endometriosis This is supported by a report that nonsteroidal antiinflammatory drugs reduced endometriotic lesions in a mouse model regardless of the presence of estrogen (41).
Acknowledgments
We thank Dr. Yi-Ling Chen and Mr. Wen-Wei Chang for the technical support for leukocyte culture and cytokine assays. We also thank Dr. Ming. T. Lin and Dr. Yi-Sin Lin for suggestions of the manuscript.
Footnotes
This work was supported by Grants NSC 88-2314-006-072 and NSC 93-2320-B006-002 from the National Science Council, Taiwan (to L.-Y.C.W.).
The authors have no conflict of interest.
First Published Online November 23, 2005
Abbreviations: BrdU, 5-Bromo-2'-deoxyuridine; E2, estradiol-17; FITC, fluorescein isothiocyanate; LPS, lipopolysaccharide; MIP, macrophage inflammatory protein; VEGF, vascular endothelial growth factor.
Accepted for publication November 16, 2005.
References
Strathy JH, Molgaard CA, Coulam CB, Melton 3rd LJ 1982 Endometriosis and infertility: a laparoscopic study of endometriosis among fertile and infertile women. Fertil Steril 38:667–672
Ryan IP, Taylor RN 1997 Endometriosis and infertility: new concepts. Obstet Gynecol Surv 52:365–371
Dizerega GS, Barber DL, Hodgen GD 1980 Endometriosis: role of ovarian steroids in initiation, maintenance, and suppression. Fertil Steril 33:649–653
Agarwal SK 1998 Comparative effects of GnRH agonist therapy. Review of clinical studies and their implications. J Reprod Med 43:293–298
van der Linden PJ 1996 Theories on the pathogenesis of endometriosis. Hum Reprod 11(Suppl 3):53–65
Hull ML, Charnock-Jones DS, Chan CL, Bruner-Tran KL, Osteen KG, Tom BD, Fan TP, Smith SK 2003 Antiangiogenic agents are effective inhibitors of endometriosis. J Clin Endocrinol Metab 88:2889–2899
Nap AW, Griffioen AW, Dunselman GA, Bouma-Ter Steege JC, Thijssen VL, Evers JL, Groothuis PG 2004 Antiangiogenesis therapy for endometriosis. J Clin Endocrinol Metab 89:1089–1095
Oosterlynck DJ, Meuleman C, Sobis H, Vandeputte M, Koninckx PR 1993 Angiogenic activity of peritoneal fluid from women with endometriosis. Fertil Steril 59:778–782
McLaren J, Prentice A, Charnock-Jones DS, Millican SA, Muller KH, Sharkey AM, Smith SK 1996 Vascular endothelial growth factor is produced by peritoneal fluid macrophages in endometriosis and is regulated by ovarian steroids. J Clin Invest 98:482–489
Maas JW, Calhaz-Jorge C, ter Riet G, Dunselman GA, de Goeij AF, Struijker-Boudier HA 2001 Tumor necrosis factor- but not interleukin-1 or interleukin-8 concentrations correlate with angiogenic activity of peritoneal fluid from patients with minimal to mild endometriosis. Fertil Steril 75:180–185
Mahnke JL, Dawood MY, Huang JC 2000 Vascular endothelial growth factor and interleukin-6 in peritoneal fluid of women with endometriosis. Fertil Steril 73:166–170
Akoum A, Kong J, Metz C, Beaumont MC 2002 Spontaneous and stimulated secretion of monocyte chemotactic protein-1 and macrophage migration inhibitory factor by peritoneal macrophages in women with and without endometriosis. Fertil Steril 77:989–994
Keenan JA, Chen TT, Chadwell NL, Torry DS, Caudle MR 1994 Interferon- (IFN-) and interleukin-6 (IL-6) in peritoneal fluid and macrophage-conditioned media of women with endometriosis. Am J Reprod Immunol 32:180–183
Rana N, Braun DP, House R, Gebel H, Rotman C, Dmowski WP 1996 Basal and stimulated secretion of cytokines by peritoneal macrophages in women with endometriosis. Fertil Steril 65:925–930
Arici A, Tazuke SI, Attar E, Kliman HJ, Olive DL 1996 Interleukin-8 concentration in peritoneal fluid of patients with endometriosis and modulation of interleukin-8 expression in human mesothelial cells. Mol Hum Reprod 2:40–45
Suzumori N, Katano K, Suzumori K 2004 Peritoneal fluid concentrations of epithelial neutrophil-activating peptide-78 correlate with the severity of endometriosis. Fertil Steril 81:305–308
Rossi D, Zlotnik A 2000 The biology of chemokines and their receptors. Annu Rev Immunol 18:217–242
Benelli R, Morini M, Carrozzino F, Ferrari N, Minghelli S, Santi L, Cassatella M, Noonan DM, Albini A 2002 Neutrophils as a key cellular target for angiostatin: implications for regulation of angiogenesis and inflammation. FASEB J 16:267–269
Scapini P, Morini M, Tecchio C, Minghelli S, Di Carlo E, Tanghetti E, Albini A, Lowell C, Berton G, Noonan DM, Cassatella MA 2004 CXCL1/macrophage inflammatory protein-2-induced angiogenesis in vivo is mediated by neutrophil-derived vascular endothelial growth factor-A. J Immunol 172:5034–5040
McCourt M, Wang JH, Sookhai S, Redmond HP 1999 Proinflammatory mediators stimulate neutrophil-directed angiogenesis. Arch Surg 134:1325–1331; discussion 1331–1332
Jablonska E, Piotrowski L, Jablonski J, Grabowska Z 2002 VEGF in the culture of PMN and the serum in oral cavity cancer patients. Oral Oncol 38:605–609
Mueller MD, Lebovic DI, Garrett E, Taylor RN 2000 Neutrophils infiltrating the endometrium express vascular endothelial growth factor: potential role in endometrial angiogenesis. Fertil Steril 74:107–112
Gaudry M, Bregerie O, Andrieu V, El Benna J, Pocidalo MA, Hakim J 1997 Intracellular pool of vascular endothelial growth factor in human neutrophils. Blood 90:4153–4161
Cummings AM, Metcalf JL 1995 Induction of endometriosis in mice: a new model sensitive to estrogen. Reprod Toxicol 9:233–238
Grummer R, Schwarzer F, Bainczyk K, Hess-Stumpp H, Regidor PA, Schindler AE, Winterhager E 2001 Peritoneal endometriosis: validation of an in vivo model. Hum Reprod 16:1736–1743
Rossi G, Somigliana E, Moschetta M, Santorsola R, Cozzolino S, Filardo P, Salmaso A, Zingrillo B 2000 Dynamic aspects of endometriosis in a mouse model through analysis of implantation and progression. Arch Gynecol Obstet 263:102–107
Hirata T, Osuga Y, Yoshino O, Hirota Y, Harada M, Takemura Y, Morimoto C, Koga K, Yano T, Tsutsumi O, Taketani Y 2005 Development of an experimental model of endometriosis using mice that ubiquitously express green fluorescent protein. Hum Reprod 20:2092–2096
Cohen PE, Milligan SR 1993 Silastic implants for delivery of oestradiol to mice. J Reprod Fertil 99:219–223
Lai MD, Jiang MJ, Wing LY 2002 Estrogen stimulates expression of p21Waf1/Cip1 in mouse uterine luminal epithelium. Endocrine 17:233–239
Lai MD, Lee LR, Cheng KS, Wing LY 2000 Expression of proliferating cell nuclear antigen in luminal epithelium during the growth and regression of rat uterus. J Endocrinol 166:87–93
Sato T, Fukazawa Y, Kojima H, Ohta Y, Iguchi T 2003 Multiple mechanisms are involved in apoptotic cell death in the mouse uterus and vagina after ovariectomy. Reprod Toxicol 17:289–297
Iwabe T, Harada T, Tsudo T, Nagano Y, Yoshida S, Tanikawa M, Terakawa N 2000 Tumor necrosis factor- promotes proliferation of endometriotic stromal cells by inducing interleukin-8 gene and protein expression. J Clin Endocrinol Metab 85:824–829
Khan KN, Masuzaki H, Fujishita A, Kitajima M, Sekine I, Matsuyama T, Ishimaru T 2005 Estrogen and progesterone receptor expression in macrophages and regulation of hepatocyte growth factor by ovarian steroids in women with endometriosis. Hum Reprod 20:2004–2013
Khan KN, Masuzaki H, Fujishita A, Hamasaki T, Kitajima M, Hasuo A, Miyamura Y, Ishimaru T 2002 Association of interleukin-6 and estradiol with hepatocyte growth factor in peritoneal fluid of women with endometriosis. Acta Obstet Gynecol Scand 81:764–771
D’Hooghe TM, Bambra CS, Raeymaekers BM, De Jonge I, Lauweryns JM, Koninckx PR 1995 Intrapelvic injection of menstrual endometrium causes endometriosis in baboons (Papio cynocephalus and Papio anubis). Am J Obstet Gynecol 173:125–134
D’Hooghe TM, Bambra CS, Xiao L, Peixe K, Hill JA 2001 Effect of menstruation and intrapelvic injection of endometrium on inflammatory parameters of peritoneal fluid in the baboon (Papio anubis and Papio cynocephalus). Am J Obstet Gynecol 184:917–925
Cao X, Yang D, Song M, Murphy A, Parthasarathy S 2004 The presence of endometrial cells in the peritoneal cavity enhances monocyte recruitment and induces inflammatory cytokines in mice: implications for endometriosis. Fertil Steril 82(Suppl 3):999–1007
D’Hooghe TM, Nugent NP, Cuneo S, Chai DC, Deer F, Debrock S, Kyama CM, Mihalyi A, Mwenda JM2006 Recombinant human TNFRSF1A (r-hTBP1) inhibits the development of endometriosis in baboons: a prospective, randomized, placebo- and drug-controlled study. Biol Reprod 74:131–136
Fukuda J, Kawano Y, Nasu K, Nishida M, Narahara H, Miyakawa I 2004 Differential effects of interferon- and - on the secretion of vascular endothelial growth factor by eutopic and ectopic endometrial stromal cells. Fertil Steril 82:749–751
Heryanto B, Lipson KE, Rogers PA 2003 Effect of angiogenesis inhibitors on oestrogen-mediated endometrial endothelial cell proliferation in the ovariectomized mouse. Reproduction 125:337–346
Efstathiou JA, Sampson DA, Levine Z, Rohan RM, Zurakowski D, Folkman J, D’Amato RJ, Rupnick MA 2005 Nonsteroidal antiinflammatory drugs differentially suppress endometriosis in a murine model. Fertil Steril 83:171–181(Yiu-Jiuan Lin, Ming-Derg Lai, Huan-Yao L)