Endometriosis is characterized by the presence of elevated proinflammatory cytokines such as tumor necrosis factor (TNF) α in the peritoneal cavity. Blocking interaction of TNFα with its receptor by the addition of excess TNFα-binding protein (TBP)-1 (a soluble form of TNF receptor-1) was effective in animal models of endometriosis. Recently, a novel, high-affinity inhibitor of TNFα, TNF-soluble high-affinity receptor complex (TNF-SHARC), was created by fusing TBP to both the α and β subunits of inactive human chorionic gonadotropin. This dimeric protein was effective in inhibiting collagen-induced arthritis in mice. In the present study, the efficacy of TNF-SHARC in cellular and in vivo models of endometriosis was examined. TBP and TNF-SHARC dose-dependently inhibited TNFα-induced secretion of interleukin (IL)-6, IL-8, granulocyte macrophage–colony-stimulating factor, and monocyte chemoattractant protein-1 in immortalized human endometriotic cells. An in vivo mouse model of experimentally induced endometriosis using cycling C57BL/6 mice was established. Antide treatment (0.5 mg/kg), used as positive control, initiated 7 days after the establishment of the disease, reduced the weight of the lesions compared with control. TNF-SHARC at 3 mg/kg was not effective in inhibiting the disease, whereas at 9 mg/kg there was reduction in the lesion weight. In addition, antide and TNF-SHARC treatment in vivo increased in vitro natural killer cell activity compared with untreated animals. Thus, we provide evidence for supporting the development of TNF-SHARC as a therapeutic candidate for treating endometriosis in human.
Endometriosis is a disease characterized by the implantation and growth of endometrial tissue at sites outside of the uterus, most frequently the peritoneal cavity. It is a relatively common disease affecting approximately 6 to 10% of women of reproductive age (Fedele et al., 2008). Endometriosis causes severe pelvic pain and infertility. The prevalence of endometriosis approaches 35 to 50% in women with pelvic pain, infertility, or both (Giudice and Kao, 2004). Although the exact cause of endometriosis is, as of yet, undetermined, the leading theory suggests retrograde menstruation as a potential cause (Sampson, 1927). This hypothesis is supported by the demonstration that injection or seeding of endometrial tissue into the peritoneal cavity of both rodents and baboons can lead to the development of disease (Story and Kennedy, 2004).
Although retrograde menstruation is a likely factor in the establishment of disease, it is clearly not the only factor because the majority of women with retrograde menstruation do not develop disease (Seli et al., 2003). Once endometrial tissue is introduced into the peritoneal cavity it must attach, invade deep into the mesothelium, establish a vascular supply, and proliferate. It seems likely therefore that either the endometrium or the peritoneal environment of women with the disease is abnormal and thus promotes establishment and maintenance of disease (Seli and Arici, 2003). The potential involvement of the immune system in endometriosis has been debated for the past several years (Sidell et al., 2002). There is ample evidence that although there is an increase in cytokines and activation of peritoneal macrophages in women with endometriosis, these macrophages in fact have decreased phagocytic activity (Lebovic et al., 2001). These reports suggest a reduced ability to clear ectopic endometrial tissue in those women with disease. In addition, several groups have demonstrated a reduced NK cytotoxicity in women with endometriosis (Oosterlynck et al., 1991), possibly due to an increased expression of inhibitory receptors and an increased expression of human leukocyte antigen-E on endometriotic tissue (Galandrini et al., 2008).
Several studies have demonstrated an increase in a number of inflammatory cytokines, including IL-6, IL-8, MCP-1 [CCL2, chemokine (C-C motif) ligand 2], and regulated on activation normal T cell expressed and secreted, in the peritoneal fluid of women with disease compared with those without (Gazvani and Templeton, 2002). The proinflammatory cytokine TNFα has also been shown to be elevated not only in the peritoneal fluid but also in the serum of women with the disease. Indeed, there is a positive correlation between peritoneal levels of TNFα and the size and number of active lesions (Harada et al., 1999). In addition to its proinflammatory functions, TNFα also stimulates the expression of matrix metalloproteinases in endometrial tissue (Braundmeier and Nowak, 2006). Matrix metalloproteinases are known to play a role in tissue remodeling and invasion of endometriotic lesions (Bruner-Tran et al., 2002). Taken together, these data suggest that TNFα may influence the establishment and progression of disease and that an antagonist of TNFα may be effective in treating patients with endometriosis.
The effects of TNFα are transmitted through two different membrane receptors, TNF receptor-1 and TNF receptor-2. Soluble forms of these receptors can form high-affinity complexes with TNFα and inhibit its binding to the receptor, thereby acting as an antagonist. The soluble form of human TNFα receptor-1, called TNF-binding protein (TBP)-1, was effective in inhibiting endometriosis in both rat and baboon animal models of endometriosis (D'Antonio et al., 2000; D'Hooghe et al., 2006). We recently described the development of novel TNFα antagonist by fusing TBP-1 to both the α and β subunits of an inactive hCG (McKenna et al., 2007). Removal of the last five amino acids from the C-terminal region of the α-subunit renders the hCG formed an inactive protein (McKenna et al., 2007). The resulting fusion protein, called TNF-soluble high-affinity receptor complex (TNF-SHARC), was described to have a prolonged pharmacokinetic profile compared with TBP-1 (McKenna et al., 2007). This novel fusion protein, due to the dimeric nature of TNFα binding showed increased efficacy in vitro and was effective in inhibiting collagen-induced arthritis in mice (McKenna et al., 2007). Engineering Fc-fusion proteins can also increase the half-life of newly formed proteins; however, they have the potential to lead to immunoglobulin-mediated effector functions such as antibody dependent cell cytotoxicity. Therefore, developing novel molecules with an alternative scaffold to generate a long-acting version of TBP-1 would be desirable for clinical development. In the current study, we examine the effectiveness of TNF-SHARC to regress established lesions in a mouse model of endometriosis as well as its ability to inhibit TNFα-induced inflammatory cytokine expression in immortalized human endometriotic cells.
Materials and Methods
Cells, Animals, Cytokines, and Inhibitors.
YAC-1, a murine lymphoma cell line, was obtained from American Type Culture Collection (Manassas, VA). The human endometriotic cell line 12Z was obtained by immortalization of endometriotic epithelial cells with simian virus 40 (Zeitvogel et al., 2001). Culture media, antibiotics, Vybrant DiO cell-labeling solution, and serum were obtained from Invitrogen (Carlsbad, CA). TNFα was purchased from R&D Systems (Minneapolis, MN). TBP-1/Onercept and TNF-SHARC were produced at EMD Serono Research Institute Inc. (Rockland, MA) (McKenna et al., 2007). Antide was obtained from Bachem (Bubendorf, Switzerland). PMSG and PKH67 dye were procured from Sigma-Aldrich (St. Louis, MO). Propidium iodide was purchased from Calbiochem (Gibbstown, NJ).
All in vivo studies were approved by the Institutional Animal Care and Use committees and the respective governmental regulatory agencies. Six- to eight-week-old female C57BL/6 mice (Charles River Laboratories, Inc., Wilmington, MA) were housed under the following environmental conditions: 22 ± 2°C, 55 ± 10% RH, and a 16:8 h light/dark cycle. A maximum of five animals were housed in one cage. Animals were provided with food pellets and water ad libitum. Before initiating the experiments, animals were allowed to acclimate to these conditions for 6 to 8 days.
Measurement of Secreted Cytokines by Multiplex Assay.
The simian virus 40 T-antigen-transformed human ectopic endometrial epithelial cell line 12Z was maintained in Dulbecco's modified Eagle's medium/Ham's F-12 supplemented with 10% fetal calf serum and penicillin-streptomycin at 37°C and 5% CO2 (Zeitvogel et al., 2001; Grund et al., 2008). For stimulation experiments, cells (20,000/well) were seeded in 96-well plates; the next day, cells were washed once with phosphate-buffered saline (PBS) and serum-free medium was added. Cells were stimulated with TNFα either in the presence or absence of varying doses of TBP-1 or TNF-SHARC in serum-free medium for 24 h as described previously (Grund et al., 2008). Culture supernatant was used for determination of cytokine levels. Electrochemiluminescence assays were performed on biological triplicate samples using capture antibody precoated 96-well MULTI-SPOT plates from Meso Scale Discovery (Gaithersburg, MD). Multiplex assay of four cytokines (GMCSF, IL-8, IL-6, and MCP-1) were measured. These assays are very sensitive, with a larger dynamic range over the conventional assays. More information on this technology platform can be obtained from the company website (http://www.mesoscale.com). In brief, assay plates coated with four different antibodies in four distinct spots were used for cytokine measurement. Samples or calibrators (25 μl/well) were added to each well and incubated with shaking for 1 h at room temperature. After incubation, plates were washed three times with Tris-buffered saline (137 mM sodium chloride and 20 mM Tris) and 0.05% Tween 20. Specific detection antibody (25 μl/well of 1 μg/ml of each antibody) labeled with Meso Scale Discovery SULFO-TAG reagent was added, and the sample was incubated with shaking for 1 h at room temperature. The plates were then washed three times, and 150 μl of 2× read buffer was added. Plates were immediately read using the SECTOR Imager 6000 (Meso Scale Discovery), and data were analyzed using Discovery Workbench and SoftMax PRO 4.0 software (Molecular Devices, Sunnyvale, CA).
Establishment of Endometriosis.
Animals were randomly assigned to two groups, donor or recipient. Donor mice were injected intraperitoneally with 10 IU PMSG to induce similar estrogen levels between various animals. The animals were euthanized 41 h later by progressive CO2 asphyxia. The uterus was removed through a midline incision and washed in PBS (2.7 mM potassium chloride, 1.8 mM potassium phosphate, 137 mM sodium chloride, and 10.1 mM sodium phosphate) before extrauterine tissue, including ovary and oviduct, was removed under a dissecting microscope. A longitudinal incision was made from one horn to the other. Tissue was then transferred to a 1.5-ml centrifuge tube containing fresh PBS and minced with dissecting scissors. Minced tissue from all donors was pooled, and the volume was adjusted to the equivalent of one uterus/500 μl of PBS.
Recipient mice were injected intraperitoneally with the equivalent of tissue from one uterus in 500 μl of PBS (1:1 donor/recipient ratio) along the midventral line using an 18-gauge needle. The tissue was injected intraperitoneally to allow the development of lesion in the intraperitoneal region. The disease was allowed to establish for 7 days. Recipient mice were then randomly assigned to the following treatment groups: PBS control, antide (a GnRH antagonist) used as positive control (0.5 mg/kg), TNF-SHARC (3 mg/kg), or TNF-SHARC (9 mg/kg). Animals received a series of three subcutaneous injections on days 7, 1, and 12 after tissue injection and were euthanized by progressive CO2 asphyxia on day 14. To assess treatments, the peritoneal cavities of the recipient mice were inspected, and in some instances they were photographed. The number and appearance of lesions were documented, and then the lesions were carefully removed. Lesions were then measured and weighed, and in some cases, they were fixed in formalin.
Labeling of Endometrial Cell Preparation with the Fluorescent Lipophilic Dye DiO.
Uterine tissue obtained from the donor mice treated with PMSG as described above was subjected to a labeling step before injecting into the recipient mice. Tissue was minced in RPMI 1640 medium, centrifuged, and then the supernatant was removed. DiO cell-labeling solution was prepared by diluting the Vybrant DiO solution 1:100 in RPMI 1640 medium. The minced uterine tissue pellet was then resuspended in the DiO cell-labeling solution for 2.5 h at 37°C. The DiO-labeled tissue suspension was washed three times with PBS and resuspended in PBS (adjusted to 2.5 uteri/500 μl of PBS). Then, 500 μl of this tissue suspension was injected intraperitoneally into recipient mice. The mice were euthanized after 7 days, and the lesions were examined and photographed with an SMZ1500 stereomicroscope (Nikon Inc., Melville, NY), and the fluorescent images were captured with an epifluorescence illuminator for green fluorescent protein (Nikon Inc.).
The formalin-fixed endometriotic-like foci were embedded in paraffin blocks, sectioned at 5 μm in thickness, and stained with hematoxylin and eosin. The stained sections were then examined with an Eclipse TE2000-S inverted microscope (Nikon Inc.).
NK Cell Cytotoxicity Assay.
Flow cytometry-based NK cell cytotoxicity assay was established according to Hatam et al. (1994). This assay was used to measure the percentage of cytotoxicity of NK cells isolated from spleens of endometriotic mice after their treatment. In brief, spleens were removed aseptically from treated and control endometriotic mice. Spleen cells were gently teased apart in 50 ml of PBS, and the resulting cell suspension was passed through a 40-μm sterile cell strainer to remove any large tissue fragments. The cell suspension was then centrifuged for 5 min at 1500 rpm and resuspended in 5 ml of ACK lysis buffer (8.29 g/l NH4Cl, 1.00 g/l KHCO3, and 0.0372 g/l disodium EDTA·2H2O) to lyse erythrocytes. After 5 min, 40 ml of PBS was added, and the cell suspension was passed through a 40-μm sterile cell strainer and centrifuged once more for 5 min at 1500 rpm. The splenocyte population was resuspended in complete medium (RPMI 1640 medium with 10% FBS) at a final concentration of 5 × 106cells/ml. YAC-1, a murine T-lymphoma cell line that is sensitive to NK-cell cytotoxicity was used as target cells. The YAC-1 cells were cultured in RPMI 1640 media with 10% fetal bovine serum (FBS), washed several times with serum-free RPMI 1640 medium, and then suspended in the same medium at a final concentration of 106 cells/ml. The cells were then incubated in an iso-osmotic solution (diluent C) with 1 μM PKH67 green fluorescent dye. After 2 min, an equal volume of FBS was added, and the cells were incubated another minute. After two washes in complete medium, the PKH67-labeled cells were adjusted to 105cells/ml. These cells were used as target cells for testing the NK cell activity of the splenocytes isolated from the endometriotic mice after the completion of their treatment with antide or TNF-SHARC. The splenocytes and the YAC-1 cells were then incubated at effector/target cell ratios of 50:1, 25:1, and 12.5:1. After 2 h of incubation at 37°C, PI solution was added to 15 μM final concentration. The cell suspensions were then analyzed using an FC500 MPL flow cytometer (Beckman Coulter, Fullerton, CA). The number of PI-positive YAC-1 cells within the PKH67-positive cell population was considered as lysed by cytotoxic NK cells and graphed accordingly.
Student's t test was used to compare the efficacy of treatment against the control group. P < 0.05 is considered as significant.
TNF-SHARC Inhibits the TNFα-Induced Response in 12Z Human Endometriotic Cell Line.
In the first set of experiments, the ability of TNF-SHARC to block TNFα-induced cytokine production was examined in 12Z cells, an immortalized human endometriotic cell line with invasive properties. This inhibitory effect was compared with TBP-1. Upon incubation of this cell line with human TNFα for a period of 24 h, a significant increase in the levels of inflammatory cytokines IL-8, MCP-1, GMCSF, and IL-6 was observed (Fig. 1A). When either TBP-1 or TNF-SHARC was added to the cells, a basal level of protein secretion was observed with all four cytokines (Fig. 1, B–E). In the presence of 15 ng/ml TNFα, both TNF-SHARC and TBP-1 decreased the secretion of all four cytokines in a dose-dependent manner (Fig. 1, B–E). TBP-1 effectively inhibited GMCSF, IL-6, and IL-8 at similar concentrations, whereas for complete inhibition of MCP-1, a very high concentration of TBP-1 was needed (Fig. 1C; Table 1). Conversely, TNF-SHARC inhibited the secretion all four cytokines at similar concentrations (Fig. 1; Table 1). The IC50 value of TNF-SHARC to block cytokine secretion is much smaller compared with TBP-1 (Table 1). Thus, the potency of TNF-SHARC to inhibit GMCSF, IL-6, IL-8, and MCP-1 compared with TBP-1 was 91-, 26-, 96-, and 1325-fold, respectively (Table 1).
Establishment and Verification of Endometriosis Model.
Endometriosis was established by intraperitoneal injection of uterine tissue suspension into female mice. The effectiveness of this model was analyzed by two methods. In the first method, the uterine tissue suspension was first labeled with a fluorescent dye to demonstrate that the lesions that developed were in fact formed by the injected tissue suspension. For this experiment, the fluorescently labeled tissue minces were injected intraperitoneally, and recipient mice were sacrificed 7 days later. The establishment of the disease was confirmed by intraperitoneal lesion formation (Fig. 2A). The presence of well developed vasculature could be observed in the lesion (white arrows in Fig. 2A). When the intraperitoneal cavity of these mice was analyzed by fluorescent microscopy, only the lesions in the cavity were fluorescent, with no background from recipient tissue (Fig. 2B). The size of the lesions ranged from 5 to 12 mm (Fig. 2C). Effectiveness of this model was also demonstrated by histological examination of the lesions. Lesions were carefully removed, fixed, sectioned, and stained to determine their morphology (Fig. 3). Histologically, the murine lesions demonstrated hallmarks of human pathology. Endometrial glands and stroma were clearly present along with epithelial cells lining the lumen. In addition, many glands were observed within the endometriotic lesions (Fig. 3A). Large cuboidal or cylindrical epithelial cells formed a line around these endometrial glands (Fig. 3, B and C). Stromal cells were found to be interspersed between glands within the lesion (Fig. 3C). The lesions were highly vascularized as observed by gross morphological observation (white arrows in Fig. 3D).
TNF-SHARC Decreases the Endometriotic Lesion Weight in a Mouse Endometriosis Model.
To determine the effectiveness of TNF-SHARC in reduction of endometrial lesion weight, animals in which disease had been established for 7 days were treated with either PBS as negative control, antide as a positive control, or two different doses of TNF-SHARC. As expected, antide treatment significantly decreased the lesion weight compared with control (7.05 ± 3.69 mg/animal compared with 71.86 ± 27.45 mg/animal in the control group) (Fig. 4). High dose (9 mg/kg) of TNF-SHARC reduced the lesion weight to 17.44 ± 10.94 mg/animal, although this reduction was not statistically significant (Fig. 4). The experiment also showed that the effect of TNF-SHARC was dose-dependent, because a lower dose (3 mg/kg) resulted in smaller reduction in weight of the observed lesions (44.04 ± 16.29 mg/animal; Fig. 4).
NK Cell Cytotoxicity Increases with TNF-SHARC Treatment.
A nonradioactive flow cytometry-based cytotoxicity assay was used to assess the activity of NK cells in the disease-bearing animals. Using YAC-1 target cells, the cytotoxicity of the primary NK cells in the splenocyte population was determined (Fig. 5). Cells from control animals showed a steady increase in cytotoxicity when the effector/target cell ratio was increased from 12.5:1 to 50:1 (Fig. 5). Cells isolated from antide-treated animals showed elevated cytotoxicity, up to 5% more than cells from control-treated animals even at a lower effector/target cell ratio (Fig. 5). In comparison, cells from TNF-SHARC-treated animals had even higher lytic activity. NK cell cytotoxic activity with the initial effector/target ratio of 12.5:1 showed 8% increase in the TNF-SHARC-treated group, which was 2.5 times the amount of cytotoxic activity compared with the cells isolated from control animals (Fig. 5).
The therapeutic potential of TBP-1 and etanercept, a fusion protein consisting of human recombinant soluble TNF receptor-2 (p75) conjugated to a human Fc antibody subunit, have previously been demonstrated in primate models of endometriosis (Barrier et al., 2004; D'Hooghe et al., 2006). The present study explores the efficacy of TNF-SHARC as an effective therapy to treat endometriosis. The primary findings from this study can be summarized in three important points. First, TNF-SHARC is very potent in inhibiting TNFα-induced secretion of IL-6, IL-8, MCP-1, and GMCSF production from endometriotic epithelial cells. Second, TNF-SHARC is capable of inhibiting the growth of established lesions in the rodent model similar to GnRH antagonist antide in an in vivo model of endometriosis established in normal cycling mice. Third, the reduced NK cell activity in “endometriotic animals” was reversed by treatment with TNF-SHARC or antide.
Endometriosis is a heterogeneous disease with a significant degree of inflammation, and progesterone resistance (Ozkan et al., 2008). In general, TNFα and other inflammatory-mediated pathways represent one of the major mechanisms involved in progression of this disease. Peritoneal fluid from women with endometriosis contains elevated levels of several cytokines (Gazvani and Templeton, 2002; Seli and Arici, 2003). Endometriotic epithelial cells have been shown to produce IL-6, IL-8, IL-12, IL-13, and TNFα (Kagan et al., 2008). Results from our previous studies clearly demonstrate an effect of TNFα, a primary effector of inflammatory responses, to increase expression of cytokines GMCSF, MCP-1, IL-6, and IL-8 from 12Z endometriotic cells. Furthermore, we had demonstrated that neutralization of TNFα with TBP-1 suppresses production of these cytokines (Grund et al., 2008). In the current study, we demonstrate that TNF-SHARC is capable of inhibiting TNF-induced cytokines with greater efficacy than TBP-1.
Endometriotic cells respond to TNFα with increased secretion of MCP-1 (Akoum et al., 1995). In addition, the levels of MCP-1 were found to be increased in peritoneal fluid of patients with endometriosis (Akoum et al., 1996). It still remains unclear whether immunological abnormalities in the pelvis of patients with endometriosis are the cause of disease or a consequence of disease. Several anti-inflammatory modulators such as cyclooxygenase 2 inhibitors (Matsuzaki et al., 2004), peroxisome proliferator-activated receptor-γ agonist (Lebovic et al., 2007), and TNFα inhibitors (Barrier et al., 2004; D'Antonio et al., 2000) were effective in animal models of endometriosis. Overall, data emerging from these anti-inflammatory treatments are extremely promising and suggest potential for targeting the immune system to treat patients with endometriosis.
Testing the efficacy of novel therapeutics in an in vivo animal model is a crucial step to further the development of a therapeutic. A nonhuman primate such as a baboon would be the ideal choice for studying pathogenesis and spontaneous evolution of endometriosis and endometriosis associated infertility. However, the cost and the nonavailability of the animals prohibit its use for general testing of molecules. Thus, the rodent model of endometriosis could be used as a convenient alternative to the nonhuman primate (Story and Kennedy, 2004). Within the past few years, noninvasive models of endometriosis have been developed using endometrial tissue infected with green fluorescent protein-expressing adenovirus (Hirata et al., 2005) or from luciferase-expressing transgenic mice (Becker et al., 2006). These models may be suited for testing efficacy of novel therapies; however, they require transgenic animals for the donor tissue. Instead, we focused our efforts in establishing a disease model in normal animals. Uterine tissue obtained from syngenic C57BL/6 mice was injected into normal cycling females. By labeling the injected tissue with fluorescent dye, we successfully demonstrated that the lesions formed in these animals were, indeed, from the injected donor tissue.
Further histochemical studies of the established lesions demonstrated the presence of endometrial gland, epithelial cells, and stromal cells, all characteristic features of ectopic lesions found in human disease (Giudice and Kao, 2004). Lesions were highly vascularized and localized in the peritoneum or attached to organs, including intestine and uterus, as well as adipose tissue. Surgery represents the first line of treatment options for women with endometriosis (Giudice and Kao, 2004). Surgical removal of ectopic lesions provides pain relief and increases chances of pregnancy. However spontaneous pregnancy rates are generally below 50%, and the pelvic pain recurs within 2 years after the surgery (Fedele et al., 2008). In line with these observations, medical treatments currently available are aimed to prevent or treat recurrence. The primary therapy for treating endometriosis has been suppression of the hypothalamic-pituitary axis with GnRH analogs creating a hypoestrogenic state, or the use of oral contraceptive pills or progestins (Fedele et al., 2008). The success achieved with these therapies is unsatisfactory due to high recurrence rate and inability to improve fertility rate of women affected (Crosignani et al., 2006).
Antide, a GnRH antagonist, and TBP-1 have been shown to regress endometriosis in rodent as well as baboon models of endometriosis (D'Antonio et al., 2000; D'Hooghe et al., 2006). Although antide treatment led to almost 100% regression, TBP-1 was only approximately 65% effective in reducing disease in the rat model (D'Antonio et al., 2000). In the present study, we demonstrated that TNF-SHARC at higher dose can reduce lesion weight, but to a lesser extent than antide, which is similar to the observations made in the rat model (D'Antonio et al., 2000). The dosing regime of TNF-SHARC (9 mg/kg) in the present study was once every 3 days, so TNF-SHARC appears to be more potent than TBP-1 used in the rat study in which it was administered twice a day (10 mg/kg/day). However, in baboons, TBP-1 was as effective as antide in partially preventing the development of endometriotic lesions and in fully preventing the establishment of pelvic adhesions in ovary, fallopian tube, and cul-de-sac (D'Hooghe et al., 2006). Furthermore, etanercept can effectively reduce the amount of spontaneously occurring endometriosis in baboon (Barrier et al., 2004).
Effectiveness of TNF-SHARC in inhibiting disease progression can be attributed to its role in altering the immunological response/activity in treated animals. Thus, as we had demonstrated in 12Z cells, inflammatory cytokines in the peritoneum could be reduced due to the treatment. In addition, we demonstrated that treatments with antide and TNF-SHARC increased NK cell activity similar to the human studies (Umesaki et al., 1999). Clinical data show reduced NK cell activity in women with endometriosis (Tanaka et al., 1992; Quaranta et al., 2006), which correlates with the severity of the disease (Wilson et al., 1994). Recent studies propose the decreased NK cell activity in women with endometriosis could be due to an increased proportion of NK cells expressing CD94/NKG2A (Galandrini et al., 2008). In the rat model of endometriosis, both antide and TBP-1 treatment showed no effect on NK cell activity (D'Antonio et al., 2000), in contrast to the present study, where these molecules were effective in increasing the NK cell activity.
In a very recent clinical trial, infliximab, a chimeric monoclonal antibody against TNFα, did not appear to affect pain associated with deep endometriosis (Koninckx et al., 2008). This recent study only included women with deep endometriosis, whereas its effect on other types of endometriosis is not known. Although infliximab and etanercept have similar TNFα binding characteristics, they differ significantly in their effectiveness in controlling Crohn's disease and psoriasis (Scallon et al., 2002). Furthermore, clinical findings using different TNFα antagonists in many autoimmune diseases clearly demonstrate differential efficacy despite the fact all these molecules effectively inhibit a TNFα-mediated effect (Licastro et al., 2009). Therefore, there is still room to improve the potential of new TNFα antagonist. The present molecule TNF-SHARC, with a different scaffold increased efficacy and better pharmacokinetic properties compared with TBP-1, could be a better therapeutic for treating women with endometriosis.
The results of the current investigation demonstrate that TNF-SHARC, a fusion protein of TBP-1 with inactivated hCG, is capable of inhibiting secretion of inflammatory cytokines in response to TNFα in immortalized endometriotic cells. Furthermore, it was demonstrated that TNF-SHARC can inhibit established lesion progression in a rodent model of endometriosis. This, in part, may be achieved by increasing NK cell activity. In conclusion, the present study provides evidence to develop TNF-SHARC as a novel therapy to treat women with endometriosis.
We acknowledge the members of molecular biology and protein expression group for efforts in providing TNF-SHARC protein for this study. We also appreciate the help of Dr. Gilman-Sachs (Rosalind Franklin University, North Chicago, IL) in establishing the NK cell cytotoxicity assay. The administrative help by Nancy Towle is highly appreciated.
This work was supported by EMD Serono Research Institute Inc.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- natural killer
- monocyte chemoattractant protein
- tumor necrosis factor
- tumor necrosis factor α-binding protein
- human chorionic gonadotropin
- tumor necrosis factor-soluble high-affinity receptor complex
- pregnant mare serum gonadotropin
- phosphate-buffered saline
- gonadotropin-releasing hormone
- fetal bovine serum
- propidium iodide
- granulocyte macrophage–colony-stimulating factor.
- Received January 27, 2010.
- Accepted April 29, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics