BIOLOGY OF REPRODUCTION 73, 1211–1218 (2005) Published online before print 10 August 2005. DOI 10.1095/biolreprod.105.044131
Experimental Intestinal Endometriosis Is Characterized by Increased Levels of Soluble TNFRSF1B and Downregulation of Tnfrsf1a and Tnfrsf1b Gene Expression1 Carmencita Rojas-Cartagena,3 Caroline B. Appleyard,4 Olga I. Santiago,4 and Idhaliz Flores2,3 Departments of Microbiology3 and Physiology,4 Ponce School of Medicine, Ponce, Puerto Rico 00732-7004 ABSTRACT Endometriosis is commonly associated with symptoms similar to those of gastrointestinal diseases, such as inflammatory bowel disease (IBD), leading to erroneous diagnosis and inappropriate management. The role of tumor necrosis factor alpha (TNF) in IBD is well established, but its role in endometriosis—also characterized by the activation of inflammatory mechanisms—is still under study. Furthermore, little is known about the involvement of TNF receptors. Intestinal endometriosis was surgically induced in female Sprague-Dawley rats (n ¼ 10). Control rats (n ¼ 10) received sutures with no implants. Samples of tissue and fluids were collected 60 days after surgery. Endometriotic implants were classified in grades, and the gastrointestinal tract was examined for damage. A significant increase was observed in protein levels of TNF and soluble TNFRSF1B in the peritoneal fluid of experimental rats compared to controls. Expression of Tnf mRNA was significantly increased both in peritoneal leukocytes and in intestinal segments associated with implants in experimental animals. Bioactivity of TNF in tissues was confirmed by overexpression of Icam1, Sele, Vegfa, Flt1 and Kdr. Gene expression of Tnfrsf1a and Tnfrsf1b was downregulated in colon and small intestine of experimental animals, possibly as a mechanism of protection against TNF cytotoxicity. Significant overexpression of genes encoding TNF receptor-associated factors that have been linked to activation of antiapoptotic pathways also was observed. Overexpression of TNF and target genes, underexpression of TNF-receptor genes, and increased shedding of TNFRSF1B in this animal model provide further evidence for involvement of the TNF system in the pathogenesis of endometriosis. female reproductive tract
INTRODUCTION Tumor necrosis factor alpha (TNF), a multifunctional cytokine produced primarily by activated monocytes/macrophages, plays a crucial role in inflammation, angiogenesis, apoptotic cell death and proliferation [1]. TNF induces the expression of a wide variety of inflammatory factors, including interleukin-8, intracellular adhesion molecule 1 (ICAM1), Eselectin (SELE), and key molecules involved in the process of angiogenesis (e.g., vascular endothelial growth factor [VEG1
Supported by NIGMS F31-GM68392 (C.R.C.) and S06-GM08239 (C.B.A. and I.F.) from the National Institutes of Health. Also supported by RCMI Grant 2 G12 RR03050–18 through the RCMI Publications office and the Molecular Biology Core. 2 Correspondence: Idhaliz Flores, Department of Microbiology, Ponce School of Medicine, 395 Zona Ind Reparada 2, Ponce, PR 00716-2347. FAX: 787 290 0876; e-mail:
[email protected] Received: 26 May 2005 First decision: 22 June 2005 Accepted: 10 August 2005 Ó 2005 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org
FA] and its two receptors, FLT1 and KDR) [2–5]. Although normal endometrium has been shown to express both TNF receptors [6] and TNF throughout the menstrual cycle [7–9], abnormal production of this inflammatory cytokine has been implicated in the pathogenesis of endometriosis [10]. Several lines of evidence support the involvement of TNF as an important factor in the development of this disease. First, levels of TNF are increased in the peritoneal fluid of patients with endometriosis and can be correlated with disease severity [11]. Second, TNF stimulates the proliferation of endometriotic stromal cells [5, 12]. Third, anti-TNF therapy inhibits the development of experimentally induced endometriosis in an animal model [13]. It has been speculated that TNF may facilitate the implantation and growth of endometrium in ectopic sites, such as the intestines, by increasing the expression of adhesion molecules, inducing angiogenesis, augmenting invasiveness, and inducing cell proliferation [14]. This hypothesis is based on the following key observations: 1) enhanced expression of ICAM1 and VEGFA has been reported in patients with endometriosis [15]; 2) TNF has been associated with increased angiogenic activity of the peritoneal fluid in patients with endometriosis [16]; and 3) TNF induces the activation of prometalloproteinase 2 [17]. Despite all the evidence pointing to TNF as a logical culprit in this condition, the underlying mechanims whereby this cytokine mediates its effects in endometriosis have not been fully elucidated. Both TNFRSF1A and TNFRSF1B are coexpressed on virtually all cells and initiate distinct signal transduction pathways by interacting with different signaling factors [18]. The 55-kDa receptor (i.e., TNFRSF1A, TNF-R55, CD120a) contains an intracellular cell death domain, which is required for signaling of apoptosis and activation of the proinflammatory transcription factor NFKB1 [19]. Ligand binding to TNFRSF1A can lead to either apoptotic or antiapoptotic cascades, depending on the recruitment of different cellular factors that bind to the intracellular death domain, including TRAF2, TRADD, and FADD [20, 21]. On the other hand, TNFRSF1B (i.e., TNF-R75, CD120b) usually is associated to activation of NFKB1 and antiapoptotic cell signaling cascades, mediated by binding to TRAF1/TRAF2 heterodimers and cellular inhibitor of apoptosis proteins 1 and 2 (cIAP1/cIAP2). Recently, a role of TNFRSF1B as modulator of TNFRSF1Amediated apoptotic mechanisms has been proposed [22]. Also, TNFRSF1B has been proposed to play a key role in chronic inflammatory disorders [23]. Women with mild endometriosis (stages I and II) have been shown to have deficient expression of TNFRSF1B [24]; however, little is known about the pathophysiological effects, if any, of the differential expression of TNF receptors in endometriosis. Both TNF receptors can be downregulated by TNF in a time- and dose-dependent manner through internalization and/or shedding [25]. Membrane-bound receptors can be cleaved proteolytically to yield soluble proteins that retain the capacity to bind TNF [26, 27]. Soluble TNF receptors have
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been shown to be increased in several chronic inflammatory and autoimmune disorders, including endometriosis; women with endometriosis have increased peritoneal levels of both TNFRSF1A and TNFRSF1B [28]. The main source of soluble TNF receptors is activated monocytes, but the physiological consequences of their production remain controversial [29]. It appears that depending on the concentration of soluble receptors and the biological system studied, binding of TNF by soluble receptors may either neutralize or increase its biological activity [30, 31]. In any case, soluble TNF receptors are increasingly considered to be an important regulatory mechanism in inflammatory disorders and key targets for therapy. To date, six distinct TRAF molecules (TRAF1 to TRAF6) have been identified in mammalian species [32]. In general, TRAFs modulate the ability of receptors to trigger distinct signaling pathways that lead to activation of protein kinases and, subsequently, to activation of proinflammatory transcription factors, including NFKB1 [33]. It has been speculated that the differential modulation of the downstream events that either keep cells alive or trigger apoptosis may depend on differences in the expression levels of TRAFs [21]. For instance, TRAF1 interacts only with TNFRSF1B, whereas TRAF2 can bind both receptors. The TRAF1/TRAF2 heterodimer recruits cIAP1 and cIAP2 to the TNFRSF1B, which leads to activation of proinflammatory gene expression. On the other hand, TRAF2 is a key player in mediating NFKB1 activation by both TNFRSF1A and TNFRSF1B and also can block TNFRSF1Adependent apoptotic pathways by binding to TRADD [34]. To our knowledge, whether differences exist in the expression of these accessory molecules, which could explain the physiological effects of TNF in endometriosis, has not been investigated. The central hypothesis of the present study is that dysregulation of TNF-receptor expression and function plays a critical role in endometriosis. Specifically, we hypothesized that the expression of TNF receptors may be modulated as a mechanism of protection, leading to the survival of endometriotic cells at ectopic sites. Also, it may be possible that the levels of expression of TNF receptors and TRAFs are specific for the peritoneal inflammation associated with endometriosis and could result in resistance to apoptosis and cell proliferation. To test these hypotheses, expression levels of TNF, TNF receptors, TRAFs, ICAM1, SELE, VEGFA, and the VEGFA receptors 1 (FLT1) and 2 (KDR) were determined in a rat model of intestinal endometriosis. MATERIALS AND METHODS The experiments reported herein were performed in accordance with the principles described in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (Publication DHMS 86–23).
Animal Model and Collection of Tissues Studies were performed with female Sprague-Dawley rats weighing 275– 300 g (Southern Veterinary Service). All animals were maintained in restrictedaccess rooms with a controlled temperature (238C) and a 12L:12D photoperiod. Standard laboratory chow and drinking water were provided ad libitum. All experimental procedures involving animals were approved by the Animal Care and Use Committee at Ponce School of Medicine. Intestinal endometriosis was induced surgically in mature female rats under pentobarbital anesthesia based on the method described by Vernon and Wilson [35]. Briefly, the distal 1 cm of the right uterine horn was removed and immersed in warm (378C), sterile culture medium. The endometrium was exposed by opening it lengthwise with a pair of sterile scissors, and four pieces of uterine horn measuring 2 3 2 mm were cut. Four implants of uterine tissue were sutured next to the mesenteric vessels of the small intestine in the experimental group (n ¼ 10). In the control group (n ¼ 10), four silk sutures were attached to the mesentery of the intestine
without implants, and the uterine horn was massaged with fingertips for 2 min. All animals were allowed to recover for 60 days before they were killed. Vaginal cytologic smears were carried out for all rats before and after surgical intervention to monitor their reproductive cyclicity. A laparotomy was performed, and the peritoneal cavity was opened and examined systematically for implants and the original sutures. Peritoneal fluid was inspirated aseptically using a sterile micropipette, taking care not to avoid contamination with blood. A Wright-stained smear was prepared for quantification of cells, and the remainder was stored at 808C for RNA analyses. The number of white blood cells in the peritoneal fluid was determined microscopically (high-power field, 3400). The classification of implants in terms of grades of growth was done as described previously [36]. Tissue samples (e.g., small intestine near the suture site, colon), peritoneal fluid, and serum were collected. Tissues were immediately immersed in RNAlater reagent (Ambion) and stored in tightly closed containers. Both tissues and serum were stored at 808C. Blood samples were taken by cardiac puncture and centrifuged for 2 min at 14 000 3 g; plasma was removed and stored at 808C until analysis.
Macroscopic and Microscopic Damage The whole colon was removed and examined for macroscopic damage (ulceration, adhesions, colon thickness, and diarrhea) using an established, previously well-defined scoring system [37]. Tissue segments were fixed in 10% formalin. After routine processing, sections were stained with hematoxylin-eosin to determine the extent of inflammatory infiltration and the appearance of the underlying muscle layers. Histological assessment of damage was performed using previously published criteria [37]. Briefly, we evaluated the loss of mucosal architecture, muscle thickness, neutrophil infiltration, crypt abscess formation, and goblet cell depletion.
Immunoassays Total protein was extracted from the colon and small intestine tissues after homogenization using Trizol Reagent (Gibco BRL), following the manufacturer’s specifications. To determine TNF protein levels in tissue homogenates, and those of TNF and soluble TNF receptors in peritoneal fluid, aliquots of each were assayed by sandwich ELISA using commercial kits (R&D Systems), following the manufacturer’s protocol. The detection limit was less than 5 pg/ ml; intra- and interassay variations were less than 10% for TNF. For both soluble TNFRSF1A and TNFRSF1B assays, the detection limit was 5 pg/ml, and intra- and interassay variations were less than 8%.
Relative and Quantitative Gene Expression The RNA was isolated using the Trizol protocol as described above. Quality of the extracted RNA was evaluated by formamide/agarose electrophoresis, and quantity was determined by spectrophotometry at OD260 in a GeneQuant DNA/RNA calculator (Pharmacia). Before RT-PCR, RNA from serum and peritoneal white blood cells was amplified as described previously using a commercial kit (MessageAmp; Ambion, Inc.) [38, 39]. Quantitative real-time RT-PCR was conducted following manufacturer’s specifications using the QuantiTect SYBR Oregon Green RT-PCR kit (Qiagen). The PCR amplification included an RT step (508C for 20 min), an activation step (958C for 15 min), and 50 cycles of 948C for 15 sec, genespecific annealing temperature for 30 seconds, and 728C for 1 min. Annealing temperatures were determined empirically for each gene: 568C for Tnf, 598C for Tnfrsf1a, and 538C for Tnfrsf1b. Construction of standards for real-time RTPCR was performed as described elsewhere [40]. For gene-specific relative RT-PCR, first-strand synthesis was performed with the RETROscript First Strand Synthesis Kit following the manufacturer’s specifications (Ambion). Briefly, 1 lg of total RNA was incubated with random decamers for 3 min at 808C. After incubation, dNTPs, buffer, RNase inhibitor (Ambion), and reverse transcriptase were added and the reaction incubated at 448C for 1 h. This was followed by a 10-min incubation at 928C. The single-stranded cDNA obtained was amplified by PCR as described below. All reactions used 1.5 mM MgCl2, except for Sele and Kdr, for which 2.0 mM MgCl2 was used, and included 4 ll of primer/competimer (2:8 ratio) for the 18S internal control. The primers and conditions used for the amplification of Traf1 [41], Traf2 [42], Traf3 [43], Traf5 [44], Traf6 [45], Icam1 and Sele [46], and Vegfa, Flt1, and Kdr [47] have been described elsewhere. All primers were synthesized at the Molecular Resource Facility, New Jersey Medical School, Newark, NJ.
Statistical Analysis Data analysis was carried out using the SPSS Base 8.0.1 statistical package (SPSS Inc.). Values are presented as the mean 6 SEM. Differences between
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ROLE OF TNF RECEPTORS IN INTESTINAL ENDOMETRIOSIS TABLE 1. Microscopic damage in colon and small intestine. Mean score Colona Criteria
Control
Loss of mucosal architecture Cellular infiltration Muscle thickening Goblet cell depletion Crypt abscess formation
1.30 1.40 1.10 0.70 0.40
Total damage score
4.90 6 0.86
a
6 6 6 6 6
Intestinea Experimental
0.34 0.31 0.28 0.15 0.16
2.60 2.30 2.20 0.40 0.00
6 6 6 6 6
Control
0.16** 0.21* 0.29* 0.16 0.00
1.90 1.40 0.30 0.10 0.00
7.50 6 0.48*
6 6 6 6 6
Experimental
0.23 0.34 0.21 0.10 0.00
2.60 2.00 0.90 0.30 0.30
3.70 6 0.56
6 6 6 6 6
0.22* 0.26 0.31 0.15 0.15
6.10 6 0.46**
Values are means 6 SEM of 10 animals per group; signficantly different from corresponding control group (*P , 0.05; **P , 0.01).
the means of each group were analyzed using the Student t-test. Correlations between variables were analyzed using the Pearson correlation. The level of significance was fixed at 0.05 for all statistical tests.
RESULTS Animal Model After the animals were killed, classification of the implants was performed as described by Ingelmo et al. [36]. Briefly, vesicles at the suture sites (four sutures per rat) were classified as grade 1 through grade 4 (grade 1, no vesicle; grade 2, vesicle 2 mm; grade 3, vesicle 2 mm but , 4.5 mm; grade 4, vesicle 4.5 mm). The experimental (implanted) rats developed a vesicle in 57.89% of their sutures. Of these, 86.37% had a diameter larger than 2 mm (grade 3 or 4). Of the 10 experimental (implanted) rats, only one did not develop a vesicle at any suture. None of the control rats (n ¼ 10) developed a vesicle at the site of the suture. No difference was observed in stage of the estrous cycle between the two groups at the time of sacrifice. Macroscopic and Microscopic Damage Macroscopic damage was evaluated in the colon of the experimental and control animals. To determine the extent of underlying tissue damage, histological preparations were performed for colon and small intestine samples. Implanted rats exhibited significantly higher macroscopic colonic damage (1.96 6 0.28, P , 0.05) than controls (1.03 6 0.15). The total microscopic damage was significantly increased in the colon (7.50 6 0.48, P , 0.05) and in the small intestine (6.10 6 0.46, P , 0.01) of the experimental rats compared to the controls (4.90 6 0.86 and 3.70 6 0.56, respectively) (Table 1). Cellular infiltration, a marker for inflammation, was significantly higher in the colon of rats with experimental endometriosis (2.30 6 0.21, P , 0.05) compared to controls (1.40 6 0.31).
Peritoneal White Blood Cell Number The number of white blood cells in the peritoneal fluid was measured to determine the presence of peritoneal inflammation in the experimental and control rats. Rats with experimental endometriosis showed a significantly higher number of white blood cells (17.56 6 3.40 cells per high-power field, P , 0.01) than controls showed (6.28 6 1.33) (Fig. 1). A significant correlation was found between the grade of the vesicle and the number of peritoneal white blood cells (r2 ¼ 0.55, P , 0.05) (Fig. 2). Protein Levels of Soluble TNF, TNFRSF1A, and TNFRSF1B Peritoneal fluid and plasma samples were collected to determine protein levels of TNF and its soluble receptors (Table 2). The protein levels of TNF in peritoneal fluid were 2.5-fold higher in experimental animals than in controls (P , 0.01). In addition, soluble TNFRSF1B protein levels in the peritoneal fluid of experimental rats were 2.5-fold higher than those in controls (P , 0.05). Significant correlations were found between the protein levels of peritoneal fluid TNF and plasma TNFRSF1B of the experimental animals (r2 ¼ 0.83, P , 0.01) (Fig. 3). Also, TNF protein levels in the peritoneal fluid significantly correlated with the number of peritoneal white blood cells (r2 ¼ 0.78, P , 0.01) (Fig. 4). TNF Protein Levels Levels of TNF protein were measured by ELISA in the colon and small intestine of experimental and controls rats. Protein levels of TNF in colon and small intestine were not significantly different between the experimental (colon, 23.98 6 3.55 pg/ml; small intestine, 38.43 6 3.84 pg/ml) and control rats (colon, 23.98 6 2.36 pg/ml; small intestine, 29.55 6 3.03 pg/ml). However, a significant correlation was found between protein levels of TNF in the colon of experimental rats and the colonic macroscopic damage score (r2 ¼ 0.60, P , 0.01) (Fig. 5).
TABLE 2. Protein levels (pg/ml) of TNF and soluble TNF receptors in plasma and peritoneal fluid. Plasmaa Protein TNF Soluble TNFRSF1A Soluble TNFRSF1B a
Peritoneal fluida
Control
Experimental
Control
Experimental
144.83 6 14.55 696.51 6 171.29 390.81 6 60.29
186.90 6 52.74 1129.50 6 418.96 518.10 6 72.22
114.10 6 9.00 1376.51 6 96.52 260.39 6 13.88
285.40 6 21.37** 1460.68 6 68.23 660.18 6 123.76*
Values are means 6 SEM of 10 animals per group; signficantly different from corresponding control group (*P , 0.05; **P , 0.01).
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FIG. 1. Number of white blood cells in the peritoneal fluid. The number of white blood cells in the peritoneal fluid of implanted rats was increased compared to the number of controls. Values are presented as the mean 6 SEM of 10 animals per group. **P , 0.01 between experimental and controls.
FIG. 3. Correlation between protein levels of TNF in the peritoneal fluid and soluble TNFRSF1B in plasma. A significant correlation was found between peritoneal fluid levels of TNF and plasma levels of soluble TNFRSF1B (r2 ¼ 0.83, P , 0.01) in the experimental animals.
Tnf, Tnfrsf1a, and Tnfrsf1b Gene Expression
Traf Gene Expression
The mRNA levels of Tnf, Tnfrsf1a, and Tnfrsf1b were determined using quantitative real-time RT-PCR. The mRNA copy number for each gene was assessed in colon, small intestine, plasma, and peritoneal white blood cells (Fig. 6). Expression of Tnf was significantly increased in colon (10-fold, P , 0.01) and small intestine (28-fold, P , 0.01) of the implanted rats compared to controls. Expression of Tnf also was significantly increased in the peritoneal white blood cells (15-fold, P , 0.01) of the experimental rats. In contrast, Tnfrsf1a mRNA levels were significantly decreased in the small intestine (P , 0.01) of the experimental rats versus the controls, whereas Tnfrsf1b expression was significantly decreased in both the colon (P , 0.001) and the small intestine (P , 0.001) of the experimental rats. No significant differences were found in mRNA levels of Tnfrsf1a and Tnfrsf1b in plasma or peritoneal white blood cells of the experimental animals compared to controls.
Gene-specific relative RT-PCR was performed to determine the expression of genes encoding TRAFs in both the colon and the small intestine (Table 3). We observed that the expressions of Traf1 and Traf2 were significantly increased in small intestine (Traf1, 1.6-fold, P , 0.01; Traf2, 2.8-fold, P , 0.001) of experimental rats compared to those in controls. Expression of Traf5, on the other hand, was significantly increased in both colon (2.8-fold, P , 0.01) and small intestine (2-fold, P , 0.001) of the rats with intestinal endometriosis. No significant difference was observed in the mRNA levels of Traf3 in either colon or small intestine between groups, and Traf6 mRNA levels were undetectable.
FIG. 2. Correlation between number of white blood cells and implant grade. A significant correlation was found between the number of peritoneal white blood cells and the grade of the vesicle or implant (r2 ¼ 0.55, P , 0.05) in the experimental animals.
FIG. 4. Correlation between TNF protein levels in the peritoneal fluid and the number of peritoneal white blood cells. Significant correlations were found between peritoneal fluid levels of TNF and the number of white blood cells (r2 ¼ 0.78, P , 0.01) in the experimental animals.
Icam1 and Sele Gene Expression Gene-specific relative RT-PCR was performed to determine the level of expression of Icam1 and Sele in colon and small intestine (Table 4). Gene expression levels of both adhesion molecules were increased significantly by 1.8- and 2.3-fold,
ROLE OF TNF RECEPTORS IN INTESTINAL ENDOMETRIOSIS
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FIG. 5. Correlation between TNF protein levels in colon and colonic macroscopic damage. A significant correlation was found between levels of TNF in colon and macroscopic colonic damage (r2 ¼ 0.60, P , 0.01) in the experimental animals.
respectively, in small intestine of the experimental rats compared to controls (P , 0.05). No differences were found in the expression of these genes in colon between the groups. Vegfa, Flt1, and Kdr Gene Expression Expression levels of Vegfa, Flt1, and Kdr, as measured by relative RT-PCR, were significantly increased in small intestine (Vegfa, 1.6-fold, P , 0.01; Flt1, 2.3-fold, P , 0.001; Kdr, 1.6fold, P , 0.05) of the implanted rats compared to controls. No differences in gene expression in colon were observed between the groups (Table 4). DISCUSSION In agreement with observations made in patients, peritoneal fluid protein levels of TNF were significantly elevated by 2.5fold in rats with experimental endometriosis as compared to control animals [48–50]. The source of TNF likely is peritoneal white blood cells, because production of this cytokine is mostly restricted to activated macrophages and T cells [1, 51]. In support of this, we observed a 15-fold increased gene expression of Tnf in peritoneal white blood cells of rats with intestinal endometriosis. It is interesting to note that disease severity was correlated with the number of peritoneal white blood cells, which highlights the important role of these inflammatory cells in endometriosis. In fact, peritoneal macrophages in women with endometriosis are reported to produce more TNF compared to those obtained in women without the condition [52]. Because activated macrophages infiltrate endometriotic implants, locally produced TNF may, in fact, enhance the adhesion of stromal cells to the intestine and promote cell proliferation [14, 53, 54]. We also observed increased gene expression of Tnf locally in colon (10-fold) and small intestine (28-fold) of the experimental rats, although only a higher level of gene expression was able to result in a modest increase in protein synthesis. This discrepancy may result from the fact that TNF production is regulated at the transcriptional, posttranscriptional, and translational levels [55]. Also, because the primary product of the Tnf gene is the membrane-bound protein, it is possible that we were only measuring cell-free TNF protein in our assay. Because TNF induces its own gene expression, we speculate that the observed upregulation of Tnf
FIG. 6. TNF and TNF-receptor mRNA expression in colon, intestine, plasma, and peritoneal fluid. Values are presented as the mean 6 SEM of 10 animals per group. **P , 0.01 and ***P , 0.001 between control (hatched bars) and experimental rats (solid bars).
mRNA levels in gastrointestinal tissues is induced mainly by exogenous sources of TNF (e.g., the peritoneal fluid), although local production of this cytokine by infiltrating leukocytes also could play a role [56]. Our observations thus indicate that the peritoneal environment exerts an important role during the early stages of disease development. In addition, plasma levels of TNF did not differ between experimental and control rats, an observation that also has been made in patients [57, 58]. This may indicate that systemic immune responses are not at play in endometriosis, but others have reported the following in patients with this disease: 1) increased levels of monocyte
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TABLE 3. TRAF gene expression in colon and small intestine.a b
b
Control Gene
Colon
Intestine
Traf1 Traf2 Traf3 Traf5 Traf6
0.73 6 0.13 0.70 6 0.05 0.51 6 0.09 1.50 6 0.12 ND
0.74 6 0.05 0.77 6 0.10 0.35 6 0.05 1.59 6 0.10 ND
Experimental Colon 0.90 0.81 0.61 2.26
6 0.20 6 0.10 6 0.10 6 0.20* ND
TABLE 4. Icam1, Sele, Vegfa, Flt1, and Kdr gene expression in the colon and small intestine.a Controlb
Intestine 1.20 2.18 0.40 3.18
6 0.13* 6 0.11** 6 0.09 6 0.18** ND
a The levels of mRNA for TRAF genes are reported as optical density per lg total RNA; samples were normalized against 18s. b Values are means 6 SEM of 10 animals per group; *P , 0.01 and **P , 0.001 between control and experimental rats; ND, not detected.
chemoattractant protein 1 (CCL2 or MCP1) and TNF in serum, 2) increased CD4:CD8 ratios in peripheral blood, and 3) decreased numbers of natural killer cells in peripheral blood [59, 60]. Therefore, more studies are necessary to confirm the role of systemic responses in this disease. A significant increase in the protein levels of soluble TNFRSF1B (2.5-fold), but not those of soluble TNFRSF1A, was detected in the peritoneal fluid of the experimental rats compared to controls. Increased peritoneal levels of both receptors have been reported in women with endometriosis [28]. A possible explanation for this discrepancy is that this animal model recreates the early stages of disease development, whereas in humans, the disease usually is studied in the advanced stages. Because implants were examined at Day 60 postinduction, TNFRSF1A expression may have not been induced yet or may have already subsided. Alternatively, the discrepant results may be related to the fact that surgicallyinduced endometriosis in the rat is not an exact representation of the human disease. Despite this limitation, animal models allow controlled investigations, which are impossible to conduct with human subjects. In the present study, peritoneal fluid levels of soluble TNFRSF1B were correlated with its plasma levels, suggesting that shedding of the receptor occurs early in this model. Rapid shedding of TNFRSF1B may result in reduced cell sensitivity to TNF, because ligand presentation to TNFRSF1A may be severely impaired [61]. Consequently, the intracellular signaling leading to apoptosis may be disrupted, and cell survival mechanisms will be favored. Our results suggest that regulation of TNFRSF1B expression by shedding plays an important role during the early stages of endometriosis and favor the hypothesis that a differential regulation of TNF receptors may be taking place in this pathology. Soluble TNF receptors have been detected in patients with other chronic inflammatory disorders, such as Crohn disease, ulcerative colitis, systemic lupus erythematosus, and rheumatoid arthritis [23, 62]. In all these cases, it has been speculated that elevated levels of soluble receptors could represent a regulatory mechanism to bind and inactivate TNF, thereby preventing its cytotoxic effects. Also, it has been suggested that an imbalance between TNF and soluble TNFreceptor expression may lead to perpetuation of inflammation and worsening of symptoms [63]. The clinical implications of soluble TNF receptors in inflammatory disorders are highlighted by the success of novel drugs, such as Etanercept, a recombinant soluble TNFRSF1B:Fc fusion protein [64]. The underlying mechanisms whereby soluble receptors may be involved in causing the symptomatology associated with endometriosis certainly deserve further investigations, as does the potential use of such receptors as a new modality in the treatment of this condition.
Gene Icam1 Sele Vefga Flt1 Kdr
Colon 0.92 0.68 0.47 0.60 0.59
6 6 6 6 6
0.11 0.09 0.03 0.24 0.07
Experimentalb
Intestine 0.82 0.52 0.56 1.07 0.58
6 6 6 6 6
0.14 0.10 0.09 0.10 0.06
Colon 1.10 0.99 0.54 0.95 0.62
6 6 6 6 6
0.11 0.18 0.05 0.26 0.05
Intestine 1.45 1.21 0.90 2.42 0.90
6 6 6 6 6
0.19* 0.16* 0.08** 0.19*** 0.11*
a The levels of mRNA are reported as optical density per lg total RNA; samples were normalized against 18s. b Values are means 6 SEM of 10 animals per group. *P , 0.05; **P , 0.01; ***P , 0.001 between control and experimental rats.
Gene expression of both Tnfrsf1a and Tnfrsf1b was significantly decreased in rats with experimental intestinal endometriosis, which suggests that dysregulation of receptor expression is involved in the pathogenesis of this condition. Expression of Tnfrsf1a was considered to be constitutive in many tissues; however, in some chronic inflammatory disorders, expression of Tnfrsf1a is subject to regulation [23]. On the other hand, expression of Tnfrsf1b is under both transcriptional and posttransciptional control. The observed downregulation of TNF receptors in the small intestinal segments associated with endometrial vesicles may be a mechanism to counteract the negative effects of TNF, which include cytotoxic, antiproliferative, and apoptotic outcomes. Our results are in agreement with previous studies showing that TNF expression is correlated inversely to TNFRSF1A expression during the menstrual cycle [65] and that decreased susceptibility of endometrial cells to apoptotic death could be an important factor in endometriosis [66]. Moreover, women with endometriosis have deficient endometrial expression of TNFRSF1B [24], which supports our present findings. Taken together, these observations suggest that downregulation of the TNF receptors may make the endometrial tissue resistant to the cytotoxic effects of this cytokine, contributing significantly to a failure to eliminate endometrial cells growing ectopically. To our knowledge, this is the first report of Traf gene expression in endometriosis, showing increased levels of Traf1 and Traf2 mRNA in affected tissues. The TRAFs are cytoplasmic proteins that transduce apoptotic or mitogenic signals on ligand binding to both TNF receptors. After transient overexpression, TRAF1 and TRAF2 act in concert to inhibit TNFRSF1A-dependent caspase activation, which is essential for TNFRSF1A-induced apoptosis [67]. Furthermore, TRAF2 overexpression is itself a strong activator of NFKB1 [33, 41], which results in the upregulation of proinflammatory and antiapoptotic factors, such as TRAF1 and cIAP1 and cIAP2 [68]. Thus, in this model of endometriosis, overexpression of TRAF1 and TRAF2, together with downregulation of TNF receptors, may favor an imbalance of tissue homeostasis toward survival and proliferation, thereby promoting implantation of the uterine stroma and proliferation at ectopic sites. Overexpression of TRAF5, which also is upregulated in this model, has been shown to activate NFKB1, but mice deficient in TRAF5 did not present defects in TNF-induced NFKB1 activation [44]. Thus, TRAF5 either is not essential or plays a redundant role in NFKB1 activation by TNF. Not only does TNF activate endothelium by upregulating adhesion molecules, such as ICAM1 and SELE, it also induces the expression of cytokines and chemokines [69, 70]. Vital to any effective inflammatory or immune response is the ability of leukocytes to adhere to and migrate through the endothelium to
ROLE OF TNF RECEPTORS IN INTESTINAL ENDOMETRIOSIS
reach the site of action. We report the increased gene expression of Icam1 and Sele in the small intestine of the implanted rats, which may be responsible for the increase in cellular infiltration observed in this model. Upregulation of these adhesion molecules in the affected tissues also demonstrates that a functional TNF/TNF-receptor interaction has taken place and provides further support for involvement of the TNF system in endometriosis. In addition to leukocyte migration, it has been suggested that TNF-mediated upregulation of adhesion molecules may play a role in the initiation of the disease by facilitating the attachment of the uterine cells to the intestine and other sites [71]. A potent angiogenic factor, VEGFA is involved in both physiological and pathological angiogenesis. Sources of VEGFA include eutopic endometrium, ectopic endometriotic tissue, and peritoneal fluid macrophages. An increasing amount of evidence indicates that the VEGFA family is involved in both the etiology and maintenance of peritoneal endometriosis [72]. In addition, VEGFA has been reported to be elevated in women with endometriosis, especially during the advanced stages [73]. Similarly, expression of VEGFA and its receptors is elevated in endometrial carcinoma [74]. Significant differences in the mRNA levels of Vegfa were found in rats with experimental endometriosis compared to controls. This finding supports the view that angiogenesis plays an important role in endometriosis by facilitating the progressive growth of the implants. We also show that expression of the two known VEGFA-receptor genes, Flt1 and Kdr, was significantly increased in the implanted animals compared to controls. It is known that FLT1 functions in cellular migration, whereas VEGFA binding to KDR can result in pathological angiogenesis [75]. High expression of FLT1 and KDR in this model of intestinal endometriosis thus may facilitate proliferation and migration of endothelial cells during the implantation of uterine tissue in the intestine, which facilitates survival. Based on these findings, the VEGFA system may play an important role in the initiation and establishment of the implants in the animal model of intestinal endometriosis. In summary, the documented downregulation of TNF receptors, increased expression of antiapoptotic TRAF1 and TRAF2, and increased levels of soluble TNFRSF1B seen in this endometriosis model may be important determinants of cell resistance to the cytotoxic effects of TNF, making elimination of the implanted cells difficult. Thus, this model has provided support for a key role of differential TNF-receptor expression in the pathophysiology of endometriosis and uncovered novel therapeutic targets for treatment of this disease.
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ACKNOWLEDGMENTS The authors wish to acknowledge the technical assistance of Cariluz Santiago, Karina Resto, and Jainarine Lalla. Thanks also go to the RCMI Publications office and the Molecular Biology Core.
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