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,1
* Department of Surgery, Vascular Biology Program, Children’s Hospital, and
Department of Obstetrics and Gynecology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA
1 Correspondence: Vascular Biology Program, Children’s Hospital Boston, Harvard Medical School, 300 Longwood Ave., Boston, MA 02115, USA. E-mail: ofer.fainaru{at}childrens.harvard.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: Lewis lung carcinoma • melanoma
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The condition is associated with pelvic pain and infertility (2)
. Despite its high prevalence and incapacitating symptoms, the pathogenesis of endometriosis remains unresolved.
Retrograde menstruation with implantation of shed viable endometrial tissue in the abdominal cavity is currently the prevailing accepted theory; however, other proposed hypotheses include metaplasia of coelomic epithelium, hematogenic and lymphogenic spread, and remnants of the Müllerian duct (3)
. Impaired immune surveillance has also been implicated in the local attachment and invasion of endometrial tissue at ectopic sites (4
, 5)
. In addition, it has been recently suggested that angiogenesis (the formation and sprouting of new blood vessels) plays an essential role in the growth and survival of endometriosis lesions (6
, 7)
. Increased levels of proangiogenic cytokines have been detected in the peritoneal fluid, blood, and endometriosis tissue of these women (6
, 8
, 9)
. Moreover, our laboratory and others have demonstrated (10
11
12
13
14)
that antiangiogenic therapy is effective in suppressing the development of endometriosis lesions in rodent models. This dependency of endometrial implants on new blood vessel formation is reminiscent of tumor angiogenesis as tumors require a blood supply for expansive growth beyond the oxygen diffusion limit in the range of 1.5 mm (15)
. Of importance, new blood vessel formation depends in part on stromal supporting cells, either resident cells within the tissue, or on an influx of bone marrow-derived CD45+ hematopoietic cells (16)
.
Dendritic cells (DCs) are bone marrow-derived hematopoietic cells specialized in the initiation and modulation of the adaptive immune response (17)
. Detection of tissue damage or pathogen-associated molecular patterns, such as bacterial lipopolysaccharide (LPS) by tissue-resident DCs, initiates their maturation and migration to lymph nodes. This maturation is associated with up-regulation of major histocompatability complex (MHC) class II (MHCII) and other costimulatory molecules (17)
. Once matured, the DCs lose their ability to capture antigens and switch into the role of antigen presentation to T helper cells, thereby inducing an immune response and serving as the "bridge" between innate and adaptive immunity. Recently, tumor-infiltrating DCs have been observed at a periendothelial location and were shown to contribute to tumor neovascularization (18)
. Hence, it was hypothesized that these DCs secrete proangiogenic molecules, assemble into neovessels, and/or transdifferentiate into pericytes (18)
.
In this study we analyzed the role of DCs in both the pathogenesis of endometriosis and of tumorigenesis by using in vivo mouse models of these conditions. In addition, we investigated the specific role of DCs in angiogenesis by examining their effect on migration of endothelial cells in vitro.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Under isoflurane anesthesia a midline abdominal incision was made, and the uterine horns were removed, leaving the ovaries in situ. In a Petri dish containing Dulbecco’s modified Eagle’s medium F-12 (Life Technologies, Inc., Grand Island, NY, USA) supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin (Life Technologies, Inc.), the uterine horns were opened longitudinally with scissors. Tissue samples were obtained using a 2-mm dermal biopsy punch (Miltex, Bethpage, NY, USA). Four biopsies were sutured to the peritoneal wall using a 7-0 braided silk suture (Ethicon, Somerville, NJ, USA). The abdominal incision was closed with a continuous 5-0 braided silk suture (Ethicon). After a designated time (1–4 wk), mice were euthanized, their abdomens were opened through the same incision, and lesions were measured and analyzed.
Intraperitoneal inoculation and growth of tumors in vivo
Lewis lung carcinoma cells and B16-F10 melanoma cells (American Type Culture Collection, Manassas, VA, USA), were harvested from subconfluent cultures by trypsination (0.01% trypsin-5 mM EDTA) and then washed and resuspended in PBS. The final concentration was adjusted to 1 x 107 cells/ml, and mice were injected intraperitoneally with 2 x 106 cells in 0.2 ml of PBS.
Bone marrow-derived dendritic cell (BMDC) cultures and transplantation
BMDCs were prepared as described (19)
. Briefly, mice were euthanized, and bone marrow was extracted from femurs and tibiae by flushing the shafts with 5 ml of RPMI 1640. The cells thus obtained were seeded into nonadhesive Petri dishes at a density of 1 x 106 cells/ml in medium (RPMI 1640, 5% fetal calf serum (FCS), 5x10–5 M 2-mercaptoethanol, and penicillin/streptomycin) containing 10 ng/ml murine recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) (Peprotech, Rocky Hill, NJ, USA). The medium was replenished every 3 days, and the loosely adherent DCs were collected at the designated times and used for further studies. For BMDC maturation, the cells were treated overnight with 1 µg/ml LPS (L2654; Sigma-Aldrich, St. Louis, MO, USA).
For transplantation into the endometriosis- or tumor-bearing mice, BMDCs were cultured for at least 9 days. Every 48 h BMDCs were labeled with carboxyfluorescein diacetate, succinimidyl ester (CFSE) (Molecular Probes, Invitrogen, Carlsbad, CA, USA) and prepared for injection. Cells were washed twice in PBS and incubated in 5 µM CFSE at room temperature for 8 min. The reaction was blocked with 2% FCS-PBS, and cells were washed twice with PBS; 1 x 106 CFSE-labeled BMDCs were then injected intraperitoneally. Control mice were injected with PBS. After 8 days mice were euthanized, and lesions were measured and analyzed as described below.
Flow cytometry
Endometriosis lesions and tumor specimens were treated with collagenase (Liberase Blendzyme 3; Roche Diagnostics Corp., Indianapolis, IN) at 37°C for 30 min. Digested tissue was then filtered through a 40 µM cell strainer and resuspended in fluorescence-activated cell sorting buffer (PBS, 5 mM EDTA, and 1% BSA/0.05% sodium azide). Immunostaining (1–2x106 cells) was performed in the presence of rat anti-mouse Fc 
receptor III/II (FcRIII/II) (CD16/32; Pharmingen, San Diego, CA, USA), by incubating the cells with monoclonal antibodies for 30 min on ice (100 µl/1x106 cells). Flow cytometry was performed with a FACSCalibur (Becton Dickinson, Mountain View, CA, USA) and CellQuest software (Becton Dickinson). Staining reagents included anti-CD11c allophycocyanin (APC)/fluorescein isothiocyanate (FITC), anti-IA/IE FITC, anti-vascular endothelial growth factor receptor (VEGFR) 2-phycoerythrin, and anti-CD31 APC, rat anti-mouse 33D1 (Pharmingen), anti-CD205 FITC (Serotec, Oxford, UK), and Alexa Fluor goat anti-rat (Invitrogen, Eugene, OR, USA).
Histology
Formalin-fixed tissue specimens were embedded in paraffin, cut into 4-µm sections, and stained with Harris’ hematoxylin and eosin (Fisher Scientific, Pittsburgh, PA, USA). The sections were examined microscopically for the presence of histological hallmarks of endometriosis such as endometrial glands, stroma, and fluid-filled cysts lined by epithelial cells (14)
.
Whole-mount immunostaining and confocal microscopy
For vital staining of vessel endothelium (20)
, after anesthesia (intraperitoneal Avertin 0.25%, 400 µl) 200 µl of tetramethylrhodamine B isothiocyanate-labeled BS-1 (Bandeiraea simplicifolia) (Sigma-Aldrich) was injected intravenously through the retro-orbital plexus. After 30 min mice were euthanized, and endometriosis lesions or tumor tissue were resected. After a 1-h fixation in 2% paraformaldehyde, the tissue was washed in PBS and blocked with PBS, 2% BSA, and 0.3% Triton. Immunostaining with CD11c APC (eBioscience, San Diego, CA, USA) was performed in the presence of rat anti-mouse FcRIII/II (CD16/32; Pharmingen). Mounting was performed on glass slides with Vectashield HardSet Mounting Media with 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA).
Optical sections were scanned using a Leica TCS SP2 AOBS confocal system fitted to a DM IRE2 inverted microscope with a x40 objective and 488-nm argon, 543-nm HeNe, 633-nm HeNe, and 405-nm diode lasers. Images were scanned sequentially to avoid fluorescence crossover, and z stacks were produced by scanning optical sections every 366 nm.
Endothelial cell migration assays
Endothelial cell migration was performed as described previously (21)
. Briefly, primary human microvascular endothelial cells (HMVECs) (Cambrex/BioWhittaker, San Diego, CA, USA), passages 4–7, were grown to subconfluence. Cell migration assays were performed by using modified Boyden chambers (6.5-mm diameter, 10-µm thickness, and 8-µm pores; Transwell-Costar Corp., Cambridge, MA, USA) that were coated with 10 µg/ml fibronectin in PBS overnight at 4°C and rinsed with PBS. HMVECs were trypsinized (0.01% trypsin-5 mM EDTA), neutralized with trypsin neutralization solution (Cascade Biologics Inc., Portland, OR, USA), washed, and resuspended in endothelial basal medium (Clonetics, Walkersville, MD, USA) with 0.1% BSA. Then, after being maintained in suspension for 30 min, cells (200,000 in 0.2 ml) were added to the top of each migration chamber and allowed to migrate for 4 h in the presence or absence of 106 BMDCs in the lower chamber that were or were not pretreated with 1 µg/ml LPS for 16 h to induce maturation (22)
. Adherent endothelial cells were fixed and stained with the Hema-3 Stain System (Fisher Diagnostics, Middletown, VA, USA), following the manufacturer’s instructions. Nonmigratory cells were removed with a cotton swab, and the number of migratory cells per membrane was captured by using a bright-field microscope (x40 magnification) connected to a Spot digital camera (Diagnostic Instruments, Sterling Heights, MI, USA). Migrated cells from the captured image were counted by using NIH Image (National Institutes of Health, Bethesda, MD, USA) software.
Statistical analysis
Both endometriosis and tumor lesions were measured with calipers in two perpendicular diameters (D1 and D2), and the lesion area was calculated using the formula for an ellipse (D1xD2x
/4). Lesion size data and migration assay data were reported as mean ± SD for each group. Unpaired two-tailed Student’s t tests were performed to compare lesion sizes and numbers of migrated cells. P < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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that significant angiogenesis can be detected as early as the first week after surgical induction of endometriosis, we analyzed the endometriosis lesions 1 week after surgery and compared them with Lewis lung carcinoma 1 week after intraperitoneal inoculation. We stained the blood vessels by intravenous injection of BS-1-lectin (see Materials and Methods) 30 min before euthanizing the mice. We then excised the lesions and used whole-mount immunostaining for CD11c+ cells (Fig. 1
). In tumors, DCs (CD11c+ cells) are distributed around newly formed blood capillaries (Fig. 1A, B
). A strikingly similar perivascular distribution of DCs was observed in the endometriosis lesions (Fig. 1C
). As a positive control for the DC staining, we subjected intestinal Peyer’s patches to an identical staining protocol (Fig. 1D
) and observed resident DCs, as reported previously (23)
, at both the subepithelial dome and T cell areas.
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Endometriosis DCs are immature and express VEGFR2
Analysis of single-cell suspensions prepared from the endometriosis lesions revealed that CD11c+ DCs comprise 11–14% of the cell population (Fig. 2
A), a 10-fold increase over DCs populating the unmanipulated uterine horn. Because VEGFR2 expression on DCs was shown to be critical for their proangiogenic phenotype (18)
, we analyzed its expression on the endometriosis DCs (Fig. 2B
). Almost all CD11c+ DCs (gating in Fig. 2A
) infiltrating the endometriosis lesions expressed VEGFR2, whereas only 
50% of uterine horn DCs expressed this receptor. Interestingly, only 1–7% of DCs obtained from the mouse spleen expressed VEGFR2. To confirm the hematopoietic origin of the infiltrating DCs we stained the cells for CD45 expression (Fig. 2C
). Endometriosis DCs expressed CD45, in contrast with CD31+ endothelial cells from the same lesions that did not express this hematopoietic marker, as expected (18)
. In contrast with splenic DCs, which highly express MHCII (Fig. 2D
), endometriosis DCs express low amounts of this molecule, indicating an immature phenotype of the latter. We further analyzed the expression of DC-specific markers, 33D1 and DEC-205, in the endometriosis lesions. These markers identify two DC subsets (24)
: CD205+ DCs that are biased for MHC class I (MHCI) cross-presentation and 33D1+ DCs that are biased for MHCII presentation. Of the CD11c+ DCs infiltrating endometriosis lesions 80% expressed 33D1 (Fig. 2D
). Similarly, 70% of cultured bone marrow-derived DCs (culture day 7) also expressed this DC marker. Conversely, only 4% of the same CD11c+ DC population expressed DEC-205, whereas 40% of the bone marrow-derived DC population expressed this marker.
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We next analyzed DCs infiltrating abdominal tumors. One week after injecting B16 melanoma cells into the abdominal cavity, we observed one large vascularized tumor mass and several small (<1 mm) avascular tumors (C. Chen, unpublished observations). CD11c+ DCs extracted from the large angiogenic lesions (Fig. 2E
) were immature (MHCIIlow) and expressed VEGFR2, whereas those extracted from the nonangiogenic tumors were mature (MHCIIbright) and expressed VEGFR2 to a lesser extent.
DCs augment endometriosis formation
We next tested the effect of DC transplantation on growth of the endometriosis lesions. To this end we have grown BMDCs by treating bone marrow cells with GM-CSF (19)
. We used cultured cells derived from day 9 onwards, yielding a population of >90% CD11c+ DCs, which were mostly of immature phenotype (i.e., MHCIIlow) (
Fig. 4F
). To label the transplanted BMDCs we incubated them with CFSE before injection. Starting 2 days after surgical induction of endometriosis 106 CFSE+ BMDCs or PBS was injected into the peritoneal cavity every 48 h. Control mice were injected with the vehicle only. On day 8, mice were euthanized, and the lesions were removed and analyzed (Fig. 3
). BMDC-treated mice grew large vascularized (red) lesions, whereas the control mice grew small white lesions (Fig. 3A-C
). Histological sections of the lesions (Fig. 3D
, F) showed an increased stromal element and engorged blood vessels in the BMDC-treated mice compared with the controls (Fig. 3E
, G). We also analyzed CD31+ vessel density; however, because of the high variability in the counts, the differences were not significant (data not shown). When analyzing single-cell suspensions derived from the endometriosis lesions by flow cytometry (Fig. 4
A), we observed that CFSE+CD11c+ DCs incorporated into the lesion. We did not detect any CFSE+CD11c– cells, indicating that only DCs incorporated into the lesions and not contaminating cells (<10%) present in the cultured BMDCs. The endometriosis lesions were significantly larger in the BMDC-injected mice compared with the vehicle-injected controls (5.1±2.5 vs. 1.5±0.5 mm2, n=4 and 4, P<0.0001) (Fig. 4B
).
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DCs promote mouse melanoma growth
We compared these results to those achieved by transplanting CFSE+ BMDCs into a melanoma tumor model. Mice were injected intraperitoneally with 106 B16 mouse melanoma cells and with either CFSE+ DCs or vehicle, following a protocol similar to that for the endometriosis experiment. At day 8 mice were killed, and the abdominal tumors were removed and analyzed. Incorporation of the transplanted CFSE+ DCs into the tumor was demonstrated by flow cytometry (Fig. 4C
). No CFSE+CD11c– contaminating cells were incorporated into the tumors. Lesion area (Fig. 4D
) was significantly larger in the BMDC-injected mice compared with vehicle-injected controls (67.6±15.1 vs. 22.7±14.6 mm2, n=5 and 7, P=0.0004).
DCs induce endothelial cell migration
DCs extracted from large angiogenic tumors (Fig. 2E
) and from vascularized endometriosis lesions (Fig. 2D
) were immature (MHCIIlow) and expressed VEGFR2, whereas those extracted from the nonangiogenic tumors were mature (MHCIIbright) and expressed VEGFR2 to a lesser extent. We therefore hypothesized that the maturity state of the DC would determine its effect on angiogenesis. To test this hypothesis we analyzed the effect of DCs on endothelial cell migration. Human microvascular endothelial cells (Fig. 4E
) were allowed to migrate toward medium only, toward immature BMDCs (MHCIIlow), and toward LPS-matured BMDCs (MHCIIbright). Immature DCs induced a 2.5-fold increase in endothelial cell migration compared with both mature DCs and medium only in the bottom chamber (219±51 vs. 93±32, P=0.02 vs. 82±23, P=0.01; migrated cells, respectively). Of importance, mature DCs did not affect endothelial cell migration compared with their basal migration to medium only. Accordingly, immature MHCIIlow BMDCs expressed VEGFR2, indicating an angiogenic phenotype, whereas LPS-matured MHCIIhigh BMDCs lost their expression (Fig. 4F
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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that DCs regulate lymph node angiogenesis and are located at perivascular sites. Our observations differ, however, from the report of Conejo-Garcia et al. (26)
, who demonstrated DC incorporation into the vessel wall in tumors of mice inoculated with ovarian carcinoma cell lines.
Nevertheless, regardless of the exact location of the infiltrating DCs vis-à-vis the vessel wall, their abundance at sites of angiogenesis suggests that these cells may serve as key players in supporting angiogenesis. We have shown here that DCs infiltrating both endometriosis lesions and angiogenic tumors express VEGFR2. This receptor was expressed to a much lesser extent in splenic or normal uterine DCs. This finding is in agreement with Fernandez Pujol et al. (27)
, who have shown that DCs grown in an "angiogenic milieu" undergo a process of "endothelization," in which they acquire endothelial cell markers such as VEGFR2. We ruled out contamination by endothelial cells that normally express VEGFR2, by showing that the VEGFR2+ DCs express CD45, a hematopoietic marker not expressed on endothelial cells. This observation suggests that DCs infiltrating tumors or endometriosis lesions become responsive to VEGF, which may in turn lead to a switch in role from presenting antigen to supporting angiogenesis. We have also shown that the CD11c+ DCs in endometriosis express 33D1 and do not express DEC205. This subclass of DCs has been shown (24)
to be biased toward MHCII presentation rather than MHCI cross-presentation. The functional importance of these findings warrants further studies. Tumor-infiltrating DCs are of an immature phenotype and as such are thought to promote immune tolerance to tumor antigens (28)
. We have also observed that DCs infiltrating both the angiogenic tumors and the endometriosis lesions are immature (i.e., MHCIIlow). Furthermore, we have demonstrated that immature DCs (MHCIIlow) and not mature DCs (MHCIIhigh) enhance endothelial cell migration in vitro. This finding points to immature DCs as potential key players in angiogenesis, as the migration and redistribution of endothelial cells from existing vessels enable vascular sprouting and elongation even without endothelial cell proliferation (29
, 30)
. Moreover, this finding supports the notion that immature DCs promote angiogenesis by secreting proangiogenic factors rather than by incorporating into vessel walls. Taken together the CD45+CD11c+VEGFR2+MHCIIlow phenotype of endometriosis-infiltrating DCs and its close resemblance to tumor-infiltrating DCs suggest that DCs play a role in endometriosis similar to their role in tumor angiogenesis.
We next tried to determine whether immature DCs play a functional role in endometriosis angiogenesis. Culturing bone marrow cells in the presence of GM-CSF (19)
yields CD11c+ BMDCs exhibiting all of the characteristics of canonical DCs, including maturation induced by bacterial LPS in vitro and induction of antigen-specific T cell proliferation. We injected CFSE-labeled DCs into mice with induced endometriosis or with inoculated tumors. Analysis of these cells by flow cytometry proved that the injected cells were CD11c+VEGFR2+MHCIIlow, namely immature DCs expressing the receptor for VEGF. Of importance, we showed that after intraperitoneal injection, only CFSE+CD11c+ DCs incorporated into the lesions. BMDC-treated mice grew large vascularized (red) endometriosis lesions and larger tumors, whereas the control mice grew small white endometriosis lesions and smaller tumors. To rule out the possibility that the differences in lesion size we observed resulted from nonspecific irritation caused by cellular material, we have shown that transplanted immature BMDCs significantly enhanced tumor growth compared with PBS-injected controls, whereas transplanted LPS-treated mature BMDCs resulted in similar tumor size (unpublished results). A similar incorporation of injected CFSE+ BMDCs and their angiogenesis-promoting effect was observed by Webster et al. (25)
when studying lymph node angiogenesis. Moreover, Conejo-Garcia et al. (26)
also showed DC incorporation, promotion of tumor growth, and decreased survival in ovarian cancer-inoculated mice.
Histological sections of the endometriosis lesions showed engorged blood vessels in the BMDC-treated mice. However, using CD31 staining we did not find differences in vessel density between the groups. As previously discussed (31)
there is a poor correlation between microvascular density and the appearance of endometriosis lesions or their size. Lesion size may be augmented with BMDC treatment by increasing the absolute number of vessels without altering their density. To exclude the possibility that DC injection altered the ovarian cycle and thereby influenced our results, we analyzed daily vaginal cytology during the estrous cycle (14)
. The mice in both the CFSE+ BMDC injected- and vehicle injected groups repeatedly passed through the various stages of the estrous cycle (data not shown). Accordingly, we found equal numbers of corpora lutea in histological sections of ovaries of both groups of mice (data not shown), confirming the fact that DC injections did not affect reproductive function. Taken together, our results indicate a role for DCs in promoting both angiogenesis and lesion growth in endometriosis, similar to that observed in tumor angiogenesis. In both models, immature DCs enhanced tumor size and expressed VEGFR2, the receptor for the major angiogenic cytokine—VEGF. The mechanism for such support awaits further studies.
In summary, the pathogenesis of endometriosis remains unresolved; however, evidence points to its dependency on angiogenesis. We describe here a novel putative role for DCs as promoters of angiogenesis in endometriosis and enhancers of lesion growth in a mouse model of this disease. We show that this effect bears striking similarity to tumor angiogenesis and tumor growth in mice. Therefore, blocking DC function or interfering with the invasion of DCs into lesions may provide a novel therapeutic approach for endometriosis.
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ACKNOWLEDGMENTS |
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Received for publication May 22, 2007. Accepted for publication August 16, 2007.
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REFERENCES |
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+, and double-negative Peyer’s patch dendritic cells. J. Immunol. 166,4884-4890This article has been cited by other articles:
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