Molecular Human Reproduction, Vol. 8, No. 12, 1103-1110,
December 2002
© 2002 European Society of Human Reproduction and Embryology
Molecular events in the uterus |
Distinct mechanisms regulate cyclooxygenase-1 and -2 in peritoneal macrophages of women with and without endometriosis
1 Institute of Clinical Medicine, 2 Department of Obstetrics/Gynecology, 3 Institute of Molecular Medicine and 4 Department of Physiology, National Cheng Kung University Medical College, Tainan 701, Taiwan, Republic of China
Abstract
Prostaglandin (PG) E2 has been shown to stimulate steroidogenesis in ectopic endometriotic stromal cells and may be involved in the development of endometriosis since this disorder is highly estrogen dependent. The biosynthesis of PGE2 is controlled by the rate-limiting enzyme termed cyclooxygenase (COX). The objective of the current study was to investigate the expression of COX in peritoneal macrophages isolated from women with and without endometriosis, and to explore the effects of pro-inflammatory agents on COX expression in peritoneal macrophages. Using quantitative RT–PCR and Western blot analyses, we found that expression of COX-2 was markedly increased (P < 0.05) in peritoneal macrophages isolated from women with early or severe endometriosis, whereas expression of COX-1 was elevated only in the severe stage (P < 0.05). On the contrary, monocytes/macrophages purified from peripheral blood of patients with endometriosis had minimal or undetectable levels of COX-2, and this was not different from disease-free women. Treatment with interleukin-1ß, tumour necrosis factor-
or PGE2 caused a significant increase in COX-2 (P < 0.05) but not COX-1 expression in peritoneal macrophages isolated from disease-free women. In contrast, these agents had no substantial effect on COX-1 and COX-2 expression in peritoneal macrophages from women with endometriosis. In summary, expression of COX in peritoneal macrophages was associated with the severity of endometriosis. Elevated expression of both COX-1 and COX-2 in peritoneal macrophages may contribute to the increased peritoneal fluid PGE2 concentrations and may thus play an important role in the development of endometriosis.
cyclooxygenase/endometriosis/interleukin-1ß/macrophages/prostaglandin E2
Introduction
Endometriosis is a common gynaecological disease which has a complex, multifactorial aetiology and causes severe pelvic pain and even infertility. It is considered as a polygenical disease affecting 
10% of women of reproductive age. Although retrograde menstruation has been suggested to be the crucial constituent in the development of endometriosis (Sampson, 1927
), factors allowing the implantation and propagation of endometriotic lesions are largely unclear. Critical factors leading to the development of endometriosis are aberrant production of steroids by ectopic endometriotic lesions and alteration/dysfunction of the immune system. It has been demonstrated that overproduction of estrogen in ectopic endometriotic stromal cells due to aberrant expression of steroidogenic acute regulatory protein (StAR) and aromatase may promote the development of endometriosis (Bulun et al., 1999; Tsai et al., 2001b). Prostaglandin (PG) E2 has been found to be a key stimulator in the expression of these two steroidogenic proteins (Noble et al., 1997; Tsai et al., 2001b) and may thus play an important role in the development of endometriosis. Numerous studies have attempted to measure the concentrations of PGE2 in peritoneal fluid of patients with endometriosis, but contradictory results have been documented (Halme et al., 1983; Dawood et al., 1984; Rezai et al., 1987; Syrop and Halme, 1987; Sharma et al., 1994; Karck et al., 1996; Raiter-Tenenbaum et al., 1998). Moreover, the types of cells contributing to the accumulation of peritoneal PGE2 in women with endometriosis are not at all clear.
The rate-limiting step in PGE2 biosynthesis is regulated by cyclooxygenase (COX), also known as PG G/H synthase (PGHS), which catalyses the conversion of arachidonic acid to PGH2. Two isoforms of COX exist, the constitutively expressed COX-1 and the inducible COX-2. COX-1 is expressed ubiquitously and is thought to produce PG for primary housekeeping functions such as platelet aggregation, vasodilatation in the kidney, and cytoprotection of gastric mucosa (Langenbach et al., 1995; Smith et al., 1996). On the other hand, COX-2 is normally expressed at very low amounts or is even undetectable in most tissues under physiological conditions, but is rapidly induced by cytokines, endotoxins, pro-inflammatory agents, tumour promoters, and certain hormones (Herschman, 1994; Smith et al., 1996). Therefore, PG produced by COX-2 up-regulation usually lead to pathological alteration in various tissues.
Since PG are unstable eicosanoids with very short half-lives (Ferreira and Vane, 1967), it is generally believed that they must be produced and function locally. Hence, peritoneal macrophages represent one of the most likely candidates that would contribute to the elevation of peritoneal PG (Karck et al., 1996; Raiter-Tenenbaum et al., 1998). During endometriosis development, immune cells are recruited to the peritoneal cavity. Among these immune cells, macrophages are the dominant cell type in the peritoneal cavity and are involved in phagocytosis and inflammation, especially in cleaning the retrograded endometrial debris (Haney et al., 1981; Dunselman et al., 1988). Nevertheless, hyperactivation of macrophages may play a crucial role in the pathogenesis of this disorder. Peritoneal macrophages isolated from patients with endometriosis tend to have higher capability for producing inflammatory agents, but poor phagocytotic capability (Dmowski et al., 1994). Recent data also indicate that phenotypic and functional alterations in peritoneal macrophages are associated with endometriosis (Raiter-Tenenbaum et al., 1998). Whether this alteration includes the expression pattern of COX-1 and/or COX-2 in peritoneal macrophage remains to be determined.
It is known that several cytokines such as interleukin-1 (IL-1) and tumour necrosis factor- (TNF-) are elevated in the peritoneal fluid of patients with endometriosis (Keenan et al., 1995; Cheong et al., 2002). These cytokines are polypeptides or glycoproteins acting as paracrine and/or autocrine signals to regulate immune response and inflammation. They contribute to the formation of endometriosis by influencing the establishment and proliferation of ectopic endometrial implants and stimulating further cytokine secretion by macrophages (Keenan et al., 1995). Effects of these cytokines on regulating COX-2 expression or its mRNA stability in the U937 human macrophage cell line have been reported (Arias-Negrete et al., 1995; Barrios-Rodiles and Chadee, 1998; Huang et al., 2000). However, whether these pro-inflammatory factors will differentially affect COX-1 and COX-2 expression in peritoneal macrophages of women with and without endometriosis remains unclear. The objectives of the current study were to explore the expression pattern of COX-1 and COX-2 in peritoneal macrophages obtained from normal or endometriosis patients and to investigate the effects of pro-inflammatory cytokines on the expression of COX-1 and COX-2 in peritoneal macrophages. Results from this study should allow us to unravel the functional role of peritoneal macrophages in the development of endometriosis.
Materials and methods
Patients
Forty-nine women (Table I) were recruited for this study and informed consent was obtained from each woman. The study was approved by the Clinical Research Ethics Committee at the National Cheng Kung University Medical Center. Patients were grouped by their endometriosis grade (American Society of Reproductive Medicine, 1997). All endometriosis samples were histologically confirmed. Peritoneal macrophage specimens were obtained from patients scheduled for laparotomy or laparoscopy at the Department of Obstetrics and Gynecology, National Cheng Kung University Hospital. For the control group, women undergoing laparoscopy for benign gynaecological conditions were recruited. All patients were of reproductive age with normal menstrual cycles. The patients were not receiving any endocrine therapy, such as GnRH analogue, danazol, or pseudopregnancy therapy. The following cases were excluded from the study: malignant neoplasms other than cervical carcinoma in situ, ovarian neoplasms, pelvic inflammation and pregnancy.
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Isolation of monocytes/macrophages from peritoneal fluid and peripheral blood
Peritoneal fluid was collected using a follicular aspiration needle prior to any pelvic surgery under direct vision. The pelvis was inspected in detail for signs of pelvic inflammatory disease. The peritoneal fluid, collected in a sterile manner at the time of laparoscopy, was centrifuged for 10 min at 500 g and the supernatant was transferred to a new centrifuge tube and stored at –80°C for later determination of PGE2 and PGF2
concentrations. The resulting pellet was resuspended with 6 ml phosphate-buffered saline (PBS) and then slowly layered onto 4 ml of Ficoll-Paque (Promega, Madison, WI, USA) solution. This was centrifuged at 700 g for 30 min at 4°C. The mononuclear cell layer was transferred to a clean centrifuge tube and washed twice with PBS. The pellet was resuspended with Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F-12 and cell number was counted using a haemocytometer under a light microscope. The cell viability was determined by 0.05% trypan blue dye exclusion. 2x105 live mononuclear cells were allowed to adhere onto a 30 mm Petri dish for 30 min and unattached cells were washed off using PBS. The cells attached were primary CD14 positive cells as determined by fluorescent immunostaining followed by flow cytometry (data not shown). For isolation of mononuclear cells from peripheral blood, heparinized venous blood was centrifuged at 400 g for 10 min. After removal of plasma, the cell pellet was then resuspended in PBS and mononuclear cells were isolated as described above. Viability of cells was accessed as described above.
Flow cytometry assay of monocytes/macrophages
Mononuclear cells isolated from peritoneal fluid or peripheral blood were directly analysed for CD14, CD25 and CD38 or allowed to adhere for 30 min and then analysed after washing off unattached cells. The monoclonal antibodies used in this study were anti-CD14-FITC, anti-CD14-PE, anti-CD25-PE and anti-CD38-FITC (Becton Dickinson Immunocytometry Systems, San José, CA, USA). The cells were washed in PBS and resuspended in 100 µl PBS containing EDTA, incubated with appropriate antibodies for 30 min, washed, and then fixed for flow cytometry analysis using the manufacturer’s protocol (FacSort; Becton Dickinson).
Cell culture and treatments
Purified peritoneal mononuclear cells (2x105 cells) were allowed to adhere onto the surface of 30 mm Petri dishes for 30 min in the presence of 3 ml of DMEM/F12 supplemented with 10% fetal bovine serum and antibiotics (penicillin 100 IU/ml, streptomycin sulphate 100 µg/ml and fungizone 0.625 µg/ml) and cultured in a humidified atmosphere with 5% CO2 at 37°C. The cells were subjected to various treatments after unattached cells were washed off using PBS. The cells were then directly lysed in specific lysis buffer and subjected to mRNA and proteins analyses. To address the effects of pro-inflammatory factors on expression of COX-1 and COX-2 in peritoneal macrophage from normal women or patients with endometriotiosis, cells were treated with vehicle, IL-1ß (1 ng/ml), or TNF- (1 pg/ml) for 8 h according to a previous pilot study for time effects (data not shown). In a separate experiment, normal and endometriotic peritoneal macrophages were incubated with vehicle, IL-1ß (1 ng/ml), TNF- (1 pg/ml) or PGE2 (1 µmol/l) for 12 h. Steady state concentrations of mRNA encoding for COX-1 or COX-2 were analysed using standard curve quantitative, competitive RT–PCR (QC-RT–PCR) methodology while COX-1 and COX-2 proteins were detected with specific antibodies by Western blot.
Isolation and quantification of mRNA
Total RNA and mRNA were isolated as previously described (Tsai and Wiltbank, 2001; Tsai et al., 2001a). The concentrations of mRNA were quantified using standard curve QC-RT–PCR (Tsai and Wiltbank, 1996). The generation and validation of native and competitor RNA for COX-1, COX-2, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was reported previously (Tsai et al., 2001a). Human COX-2 primers were 5'-CCACCAACTTACAATGCTGC-3' (sense) and 5'-CACCAGACCAAAGACCTCC-3' (antisense). Human COX-1 primers were 5'-CTGGCTCCGGAATTCACT-3' (sense) and 5'-CATCTGGCAACTGCTTCTTC-3' (antisense). Human GAPDH primers were 5'-TGGCGTCTTCACCACCAT-3' (sense) and 5'-CACCACCCTGTTGCTGTA-3' (antisense). Procedures for standard curve QC-RT–PCR have been validated and used routinely in our laboratory (Tsai and Wiltbank, 1998; Tsai et al., 2001a,b; Wu et al., 2002).
Western blotting
The procedure for Western blotting has been reported previously (Tsai et al., 2001a). In brief, 20 µg of proteins were loaded to each lane, separated on an 8% SDS–PAGE and transferred onto a polyvinylidene difluoride membrane (Millipore Co., Bedford, MA, USA). Mouse anti-COX-2 monoclonal antibody (Cayman, Ann Arbor, MI, USA) at 1:1000 dilution, rabbit-anti-COX-1 polyclonal antiserum (Cayman) at 1:1000 dilution, or mouse anti-ß-actin monoclonal antibody (Amersham) at 1:2500 dilution were used as primary antibodies. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Chemicon International Inc., Temecula, CA, USA) at 1:25 000 dilution and HRP-conjugated goat anti-rabbit IgG (Sigma, St Louis, MO, USA) at 1:25 000 dilution were used as secondary antibodies respectively. Signals were detected by ECL (Amersham, Life Science, Little Chalfont, UK). For each experiment, the membrane was stripped with the stripping buffer (100 mmol/l 2-mercaptoethanol, 2% SDS, and 62.5 mmol/l Tris–HCl, pH 6.7) and blotted with the second or third kinds of antibodies after the detection of first kind of protein. The X-ray films were analysed using AlphaImagerTM software (Alpha Innotech Corp., San Leandro, CA, USA) to quantify band intensity.
Prostaglandin E2 and PGF2 enzyme-linked immunoassay
Peritoneal fluid concentrations of PGE2 were analysed by Monoclonal PGE2 enzyme immunoassay kit (Cayman) according to the manufacturer’s protocol. The sensitivity of the assay (80% bound) was 15 pg/ml and the intra- and inter-assay coefficients of variation (CV) were 6.6 and 9.6% respectively. Concentrations of PGF2 were determined by a competitive enzyme-linked immunosorbent assay (ELISA) procedure as previously described (Tsai et al., 2001a). The detection limit was 0.15 ng/ml with intra- and inter-CV of 4.8 and 9.1% respectively.
Statistical analysis
Results were expressed as mean ± SEM. Differences in mRNA, protein or PG concentrations among disease groups or treatment groups were analysed by one-way analysis of variances (ANOVA) using Prism® statistical software version 3.02 (GraphPad Software, Inc.). Post-test multiple comparisons were performed using Duncan’s procedure and significant differences were accepted when two-tailed analysis yielded P < 0.05.
Results
Concentrations of PG and number of mononuclear cells in peritoneal fluid
Concentrations of PGF2 in peritoneal fluid were associated with advancing of endometriosis, with the greatest levels found in severe (stages III and IV) endometriosis (Figure 1A). Concentrations of PGE2 in peritoneal fluid were also elevated in patients with endometriosis and the pattern was similar to that of PGF2 (Figure 1B). There were no significant differences in PGE2 and PGF2 concentrations among the phases of menstrual cycle (data not shown). Therefore, data were combined and analysed as control versus early endometriosis versus severe endometriosis. The number of peritoneal macrophages in peritoneal fluid was also increased along with the severity of the disease (Figure 1C).
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Expression of COX-1 and -2 in peritoneal macrophages
Steady state concentrations of mRNA encoding for COX-2 were markedly increased in peritoneal macrophages obtained from samples of early as well as severe endometriosis (Figure 2
). In contrast, GAPDH mRNA was not changed between normal women and women with endometriosis (data not shown). Western blot analysis also confirmed the up-regulation of COX-2 expression in peritoneal macrophages isolated from patients with endometriosis with the greatest amount detected in severe stage (Figure 3A and B). In contrast, mononuclear cells isolated from peripheral blood had no detectable COX-2 expression, evident in samples obtained from both endometriosis patients and otherwise healthy women (Figure 3C). Low levels of COX-2 were detected when peripheral blood mononuclear cells were allowed to adhere for 16–18 h (data not shown).
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The expression of COX-1 mRNA was elevated in peritoneal macrophages isolated from women with severe but not early endometriosis (Figure 4A and B
). Further analysis using Western blotting confirmed the up-regulation of COX-1 protein in peritoneal macrophages isolated from women with severe (n = 8) but not early (n = 6) endometriosis (Figure 4C and D). The COX-1 protein was undetectable or barely detected in peritoneal macrophages of normal women (n = 5) or women with early stage endometriosis after 30 min adherence. The primary antibody used for detecting COX-1 (Cayman, Cat. #160108) was tested for cross-reaction with recombinant ovine COX-2 and the result was negative (Figure 7 and data not shown), demonstrating the specificity of the antibody.
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Analysis of types of mononuclear cell markers
Mononuclear cells isolated from peripheral blood expressed CD14 markers and were primarily CD38 positive but CD25 negative regardless of the status of the disease (data not shown), indicating that they were naive monocytes/macrophages. More than 98% of CD14 positive peritoneal macrophages of women without endometriosis were CD38 positive but CD25 negative (n = 4, Figure 5A and B
). In contrast, significant numbers (19.3 ± 8.2%) of CD14 positive mononuclear cells isolated from peritoneal fluid of women with endometriosis expressed CD25 cell antigen (n = 4, Figure 5C and D), indicating that they were activated macrophages.
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Effects of IL-1ß, TNF-
and PGE2 on COX-1 and COX-2 expressionThe expression of COX-2 mRNA and protein in peritoneal macrophages isolated from disease-free women was markedly induced by treatment with IL-1ß or TNF-
(Figure 6). Peritoneal macrophages isolated from women with early or severe endometriosis expressed greater amounts of COX-2 than those from disease-free women (Figure 6). Treatment with IL-1ß or TNF- did not further stimulate COX-2 expression in peritoneal macrophages obtained from women with early or severe endometriosis (Figure 6).
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To address whether COX-1 can be induced in peritoneal macrophages, IL-1ß, TNF-
, or PGE2 was administrated to peritoneal macrophages of women with or without endometriosis for 12 h. In peritoneal macrophages isolated from endometriosis-free women, the expression of COX-1 was greater in cells allowed to adhere for 12 h than in those which adhered for only 30 min (Figures 4 and 7
). Steady state concentrations of mRNA encoding for COX-1 were not different in peritoneal macrophages treated with IL-1ß, TNF- or PGE2 (data not shown). Similarly, these three agents failed to induce COX-1 protein expression but significantly stimulated COX-2 expression (as a positive control) in peritoneal macrophages isolated from women without endometriosis. In peritoneal macrophages isolated from women with endometriosis, neither COX-1 nor COX-2 was induced by any of the treatments (Figure 7).
Correlation between PGF2 concentrations and levels of COX expression in macrophages
After incubation for 30 min, macrophages obtained from women with endometriosis produced a greater amount of PGF2 than did those from normal women (Figure 8A). The concentrations of PGF2 in media were positively correlated with levels of COX-2 expression in macrophages (r = 0.923, P < 0.05). The basal level of PGF2 was greater in conditioned medium collected at 12 h after plating when compared to that collected at 30 min after plating (Figure 8A and B). Treatment with IL-1ß significantly stimulated PGF2 production by macrophages isolated from women without endometriosis, but had no substantial effect on macrophages obtained from women with endometriosis at 12 h post treatment (Figure 8B).
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Discussion
The development of pelvic endometriosis is highly estrogen dependent and ectopic endometriotic lesions overcome the cyclic decrease in this steroid by acquiring the capacity of producing it (Bulun et al., 2000). The acquisition of steroidogenic capacity may permit the ectopic endometriotic tissues to survive despite the lack of ovarian steroids during menstruation. One of the major factors that stimulate de-novo synthesis of estrogen by ectopic endometriotic stromal cells is PGE2, which induces aberrant expression of StAR and aromatase in these cells (Noble et al., 1997; Tsai et al., 2001b). It is well known that COX control the first committed step of PG production by converting arachidonic acid to PGH2. A recent study has demonstrated that eutopic endometrium and ectopic endometriotic implants of women with endometriosis express more COX-2 than endometrium of disease-free women (Ota et al., 2001), indicating that over-expression of COX-2 may be associated with this disorder. In the current report, we demonstrate that COX-1 and COX-2 were markedly increased in peritoneal macrophage of patients with endometriosis and provide evidence linking hyperactivation of peritoneal macrophages and elevation of PGE2 in peritoneal fluid of women with endometriosis.
Numerous studies have attempted to measure levels of certain PGs and numbers of macrophages in peritoneal fluid but controversial results have been found (Syrop and Halme, 1987). The discrepancy may not be due to a single reason but to several, e.g. sensitivity of assays, stages of the disease, whether or not samples were pooled, and variations between subjects. In the current study, we have demonstrated that PGE2 and PGF2 concentrations are elevated in peritoneal fluid of women with endometriosis and are associated with the severity of the disorder. This is in agreement with those reports showing increased PG in peritoneal fluid of women with endometriosis (Sharma et al., 1994; Karck et al., 1996; Raiter-Tenenbaum et al., 1998) but contrasts with others (Halme et al., 1983; Dawood et al., 1984; Rezai et al., 1987). Furthermore, we demonstrated that the elevation of peritoneal PG concentrations is associated with increased number of active macrophages (CD14+/CD25+) present in the peritoneal fluid. It is worth noting that we calculated not only total number of mononuclear cells but also the percentage of CD14+/CD25+ mononuclear cells in the peritoneal fluid. This provides support for the association between activated peritoneal macrophages and the elevation of PG in peritoneal fluid of women with endometriosis.
The expression pattern of COX in peritoneal macrophages of normal and endometriotic women as well as the relationship between COX levels and concentrations of PG in peritoneal fluid had not previously been explored. In the current report, we demonstrated for the first time that expression of COX (especially COX-2) in peritoneal macrophages is associated with concentrations of both PG in the peritoneal fluid and the severity of endometriosis. It has been proposed that dysfunction of the immune system due to genetic background may account for the prevalence of endometriosis (Dmowski et al., 1990; Gleicher, 1995; Somigliana et al., 2001; Vinatier et al., 2001). In this study, we have examined COX-2 expression patterns of mononuclear cells from both peripheral circulation and peritoneal fluid. Mononuclear cells isolated from peripheral blood of women with endometriosis appeared to be the same as those obtained from disease-free women in terms of COX-2 expression and the presence of a surface marker (CD14). In contrast, significant numbers of mononuclear cells isolated from peritoneal fluid of women with endometriosis clearly expressed an activated macrophage specific marker (CD25) whereas those isolated from disease-free women predominantly expressed monocytic cell antigen (CD38). Thus, distinct patterns of COX-2 expression in peritoneal macrophages seem unlikely to be due to genetic variation but rather to exposure of micro-environmental stimulants in the peritoneal fluid of women with endometriosis.
It is known that local, sterile inflammation occurs in the peritoneal cavity of patients with endometriosis, and that increased pro-inflammatory agents, such as IL-1ß and TNF-, in peritoneal fluid have been reported in such cases (Keenan et al., 1995; Cheong et al., 2002). These pro-inflammatory cytokines represent some likely candidates for stimulation of COX-2 expression in peritoneal macrophages. Our results clearly demonstrated that peritoneal macrophages from endometriosis-free women had no or minimal amounts of COX-2 expression, but COX-2 was markedly induced by these pro-inflammatory agents. Induction of COX-2 expression correlated with an increase in PGF2, and may also be correlated with other PG. In contrast, peritoneal macrophages isolated from women with endometriosis already expressed significantly greater amounts of COX-2 as compared to those from disease-free women. Treatment of these cells with pro-inflammatory cytokines resulted in no substantial change in COX-2 expression or PG production. Our results indicated that peritoneal macrophages in women with endometriosis have already acquired a PG-producing capacity whereas those in disease-free women have not. This was in concordance with results of the flow cytometry study showing more peritoneal macrophages expressing the activated macrophage cell marker (CD25) in endometriosis patients than those obtained from otherwise-healthy women. It is likely that pro-inflammatory factors such as IL-1ß, TNF- or even PGE2 may contribute to the acquisition of PG-producing capacity by peritoneal macrophages. However, they may not be the only factors that regulate this transition and more studies are needed before we can draw a comprehensive picture regarding factors controlling the recruitment and differentiation of peritoneal macrophages in women with endometriosis. The failure of pro-inflammatory factors to stimulate increased COX expression in peritoneal macrophages isolated from women with endometriosis may be due to desensitization of macrophages after long-term exposure to stimuli in the peritoneal fluid, as has been reported elsewhere (Karck et al., 1996). However, mechanisms responsible for this desensitization remain to be determined.
One of the most intriguing findings of this study was the marked elevation of COX-1 expression in peritoneal macrophages obtained from severe endometriosis. It is generally believed that expression of COX-1 is constitutive and primarily responsible for housekeeping functions. Nonetheless, elevation of COX-1 has been detected in numerous cancer models (Hwang et al., 1998; Bauer et al., 2000; Kirschenbaum et al., 2000; Sales et al., 2002) and in some cell types during cell differentiation (Smith et al., 1993; Bryant et al., 1998). These data suggested that COX-1 and/or its products might also be involved in pathological and/or physiological processes. In this study, we found that levels of COX-1 were much greater in peritoneal macrophages from women with severe endometriosis, indicating that prolonged exposure to stimuli in the peritoneal fluid of women with endometriosis may result in induction not only of the inducible COX-2 but also the constitutive isoform, COX-1. Our finding is in agreement with those reports showing that both COX-1 and COX-2 are elevated in certain cells (Hwang et al., 1998; Bauer et al., 2000; Kirschenbaum et al., 2000; Sales et al., 2002), though the physiological significance of these findings is not clear. It has been reported that the subcellular localization of, and preferential substrate used by, COX-1 and COX-2 are different (Morita et al., 1995; Reddy and Herschman, 1996; Spencer et al., 1998). Thus, these two isozymes may be activated in the same cell type at the same time but may exert distinct functions.
To test whether the elevation of COX-1 in peritoneal macrophage of women with severe endometriosis was due to stimulation by some known pro-inflammatory agents, cells were incubated with IL-1ß, TNF- or PGE2. Interestingly, these three pro-inflammatory mediators, although effective at inducing COX-2 expression, failed to stimulate COX-1 expression at 8 h or 12 h post treatment. The failure of IL-1ß, TNF-, and PGE2 in stimulation of COX-1 expression may be due to insufficient doses or inappropriate time of exposure. Induction of COX-1 as early as 3 h or not until 24 h post treatment has been reported (Maldve et al., 2000), thus we believe that the two time points we tested (8 and 12 h) should be able to detect the elevation of COX-1 if it was induced within this time frame. Alternatively, these three pro-inflammatory agents may not be the proper factors for stimulation of COX-1 expression in peritoneal macrophages. It is not known which factors are responsible for induction of COX-1 in peritoneal macrophages of women with severe endometriosis. The attempt to identify this/these factor(s) represents a formidable task since numerous factors are differentially elevated in the peritoneal fluid of women with endometriosis.
In conclusion, peritoneal macrophages of women with endometriosis appear to have acquired the capacity to synthesize PG owing to over-expression of COX-1 and/or COX-2, leading to elevation of PGE2 and PGF2 concentrations in the peritoneal fluid. Accumulation of PG in the peritoneal fluid may contribute to severe symptoms of endometriosis such as pelvic pain and infertility. More importantly, elevated PGE2 in peritoneal fluid due to aberrant expression of COX in peritoneal macrophages may result in unregulated production of estrogen and thus may play a critical role in the development of endometriosis. However, mechanisms responsible for COX-1 and COX-2 up-regulation in peritoneal macrophages are obviously different and further investigation is necessary to identify the cause and effect of COX elevation in peritoneal macrophages during endometriosis formation and progression.
Acknowledgements
This work was supported by grants NSC90-2320-B006-045 (to S.J.T.) and NSC902314-B006-167 (to M.H.W.) from National Science Council of Republic of China.
Notes
5 To whom correspondence should be addressed at Department of Physiology, National Cheng Kung University Medical College, 1 University Road, Tainan 701, Taiwan, Republic of China. E-mail: seantsai{at}mail.ncku.edu.tw 
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Submitted on June 5, 2002; accepted on September 26, 2002.
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