- Email: [email protected]
Change of proinflammatory cytokines follows certain patterns after induction of endometriosis in a mouse model
Change of proinflammatory cytokines follows certain patterns after induction of endometriosis in a mouse model Qiong-Hua Chen, Ph.D.,a Wei-Dong Zhou, Ph.D.,b Zhi-Ying Su, M.D.,a Qian-Sheng Huang, Ph.D.,b Jin-Na Jiang, M.D.,a and Qing-Xi Chen, Ph.D.b a Department of Obstetrics and Gynecology, Xiamen First Hospital, affiliated with Fujian Medical University; and b Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen, People’s Republic of China
Objective: To examine the change in proinflammatory cytokines in the pathologic processes of endometriosis in mice. Design: A dynamic study on a murine model of endometriosis. Setting: Medical school. Animal(s): Female BALB/c mice. Intervention(s): Endometriosis was induced by injecting endometrial fragments of syngenic mice into the peritoneal cavity of model mice; in control group, phosphate-buffered saline instead of fragments was injected. The peritoneal fluid and the endometriotic lesions were harvested 1 to 21 days after the induction. Main Outcome Measure(s): The endometriotic lesions were weighed, the gene and protein levels of some proinflammatory cytokines, including interleukin 1b, tumor necrosis factor a, vascular endothelial growth factor, and monocyte chemoattractant protein 1, were determined by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Result(s): The levels of these cytokines reached the first peak on the first day and no endometriotic lesions were found. The lesions began to appear on the second day, presenting red color during the initial 6 days, and then they turned dark-red, brown, or bluish. The adhesion took place on the 9th day, and all the lesions evolved into white or transparent cysts on the 15th day. Corresponding to these changes, the second and the third peaks were identified during the 3rd–6th day and the 12th-15th day, respectively. Conclusion(s): The change pattern of cytokines over time might bear some relationship with the development and progression of the endometriosis. (Fertil Steril 2010;93:1448–54. 2010 by American Society for Reproductive Medicine.) Key Words: Endometriosis, IL-1b, TNF-a, VEGF, MCP-1, pathogenesis
Endometriosis is characterized by the growth of endometriotic tissue outside the uterine cavity, and its pathogenesis is not fully understood (1). Recently, research effort has been directed to the role of the abnormal immunoinflammatory reaction in the pathogenesis of endometriosis (2). The cellular communication during inflammatory responses is mediated via cytokines and chemokines (3). Increasing evidence has indicated that some cytokines, such as interleukin 1b (IL-1b) (4), tumor necrosis factor a (TNF-a) (5), vascular endothelial growth factor (VEGF) (6), and monocyte chemoattractant protein 1 (MCP-1) (7), are elevated in the peritoReceived October 20, 2008; revised January 21, 2009; accepted February 5, 2009; published online April 1, 2009. Q.-H.C. has nothing to disclose. W.-D.Z. has nothing to disclose. Z.-Y.S. has nothing to disclose. Q.-S.H. has nothing to disclose. J.-N.J. has nothing to disclose. Q.-X.C. has nothing to disclose. Supported by grant no. 2007-1-43 from the Ministry of Health of Fujian Province, P.R. China, by grant 2009-CXB-49 of the medical innovation project of Fujian Province, P.R. China, and grant nos. 3502 Z 20077047 and 3502 Z 20084006 from the Science and Technology Planning Project of Xiamen City, P.R. China. Reprint requests: Qing-Xi Chen, Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen, People’s Republic of China 361005 (FAX: þ86-592-2185487; E-mail: [email protected]).
1448
neal fluid of women with endometriosis. It remains unclear whether the elevation of cytokine levels precedes or follows the emergence of the endometriotic lesions. Moreover, it is well known that the morphology of the ectopic lesions varies greatly with the stages of the condition, but it is still uncertain whether the cytokine levels are linked with morphologic change of the ectopic lesions. Owing to the limitations associated with human study, it is difficult to examine the changes of cytokines over time in the development of endometriosis, especially during the period from the beginning of the retrograde menstruation into the peritoneal cavity to the development of endometriotic lesion. In our previous study (8), we found that the phagocytotic function of peritoneal macrophages in the murine endometriosis model was enhanced, presenting certain patterns of change over time. This finding led us to investigate whether the cytokines undergo a similar change and if the change in proinflammatory cytokines is tied with the development of endometriosis. On the 1st, 2nd, 3rd, 4th, 5th, 6th, 9th, 12th, 15th, 18th, and 21st days after the induction of endometriosis in BALB/c mice, we measured the gene and protein levels of the cytokines (IL-1b, TNF-a, VEGF, and MCP-1) in the peritoneal fluid by using quantitative polymerase chain reaction (PCR) and ELISA, in an attempt
Fertility and Sterility Vol. 93, No. 5, March 15, 2010 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/10/$36.00 doi:10.1016/j.fertnstert.2009.02.013
to identify some pattern of change in the cytokine expression at the early stage of endometriosis. MATERIALS AND METHODS Chemicals The ELISA kits were purchased from R&D Systems (Minneapolis, MN); SYBR Primescript RT-PCR kit was bought from TaKaRa Co. (Kyoto, Japan); RNAspin Mini RNA isolation kit was procured from GE Healthcare (Uppsala, Sweden). Other reagents were of analytical grade. All reagents, except phosphate-buffered saline (PBS), were prepared daily before use and stored in a refrigerator at 4 C. The reagents were equilibrated at room temperature for 0.5 hour before use. Animals The BALB/c mice were provided by the Animal Research Laboratory at Xiamen University. A total of 175 female BALB/c mice (8 weeks old, 19–21 g in weight), including 110 model (recipient) mice, 55 donor mice, and 10 mice serving as control subjects, were kept on a light/dark cycle of 12/12 hours under controlled conditions in a room at a constant temperature of 21–22 C and humidity of 60 5%. The mice were caged in groups of five for a week before the onset of the experiment, with free access to a commercial balanced mouse diet and tap water. The animals were procured, maintained, and used in accordance with the ‘‘Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals’’ formulated by Xiamen University’s Animal Ethics Committee. The study was approved by the institutional review board of Xiamen University. Experimental Design Endometriosis was induced by using a previously described method with minor modifications (9). After the 55 donor mice were killed, the uterine horns were taken out and put into a dish containing PBS. The endometrium-rich fragments, obtained by peeling off the serosa and myometrium, were finely chopped using a razor blade. Fragments suspended in 1 mL PBS at 37 C were injected by means of an 18-gauge needle through the abdominal wall into the peritoneal cavity of the recipient mice, with a ratio of 1:2 (donor/recipient). For each mouse, the operation time was limited to 5 minutes. The 110 model mice were randomly divided into 11 groups, with ten animals in each group. Ten mice were killed by cervical dislocation on each observation day after the induction of endometriosis, i.e., the 1st (24 hours), 2nd, 3rd, 4th, 5th, 6th, 9th, 12th, 15th, 18th, and 21st days. Another ten noninduced mice served as a control group, designated day 0. All operations were performed under aseptic conditions by the lead author. In all of the procedures, the examiners were blind to the treatment given to each mouse. Specimen Collection After killing it, 3 mL PBS was injected into the peritoneal cavity of each mouse. After shaking the mouse, the peritoneal fluid was collected and centrifuged. The supernatant was kept Fertility and Sterility
at 80 C and the settled peritoneal cells, including peritoneal macrophages, were used for RNA extraction. Then laparotomy was performed, the number of endometriotic lesions was counted, and the lesions were morphologically observed. All lesions and uteri were quickly excised, and normal surrounding tissues, whenever possible, were removed. The lesions were put in formaldehyde solution for routine histopathologic examination and confirmed to be endometriotic lesions. Morphologically, ectopic lesions of endometriosis present as red, blue, brown, or transparent blotches, small cysts, or scars on the surface of organs or on the peritoneum of the abdominopelvic cavity, so they can be identified by naked eyes. The pathologic diagnostic criteria for endometriosis are the presence of any two of the four foregoing changes, i.e., endometrial epithelium, glands or gland-like structures, stroma, and hemorrhage (10).
RNA Extraction and Quantitative Real-Time ReverseTranscription PCR Total RNA was obtained by extraction with RNAspin mini kit (GE Healthcare), following the manufacturer’s instructions. The total RNA was reverse-transcribed in a system of 20 mL (final volume) by using SYBR Primescript RT-PCR kit (TaKaRa) in accordance with manufacturer’s instructions. The following conditions were applied to reverse transcription: 37 C for 15 minutes (reverse transcription reaction), 1 cycle; and 85 C for 5 seconds (denaturation of reverse transcriptase). The first-strand cDNA obtained from RNA samples was stored at 80 C until use. The mRNA expression of IL-1b, TNF-a, VEGF, and MCP-1 in each sample was measured by quantitative realtime PCR. Primers were synthesized by TaKaRa company (Kyoto, Japan). The primers used for real-time PCR were: b-actin: forward 50 -CATCCGTAAAGACCTCTATGCCAA C-30 , reverse 50 -ATGGAGCCACCGATCCACA-30 ; IL-1b: forward 50 -TCCAGGATGAGGACATCACATGAGCAC-30 , reverse 50 -GAACGCACCAGCAGGTTA-30 ; TNF-a: forward 50 -AAGCCTGTAGCCCACGTCGTA-30 , reverse 50 -GGCAC CACTAGTTGGTTGTCTTTG-30 ; VEGF: (forward 50 -GTG CACTGGACCCTGGCTTTA-30 , reverse 50 -GGTCTCAATC GGACGGCAGTA-30 ; and MCP-1: forward 50 -GCATCCAC GTGTTGGCTCA-30 , reverse 50 -CTCCAGCCTACTCATTG GGATCA-30 ). Quantitative real-time PCR assays were carried out with SYBR Primescript RT-PCR kit (TaKaRa). The reactions were run in 20mL mixture containing 10mL SYBR Premix EX Taq (2), 0.4mL of each primer (10mmol/mL), 1.6mL cDNA, and 7.6mL ddH2O. The PCR was performed in a Rotor-Gene 3000 (Corbett Research, Sydney, Australia), and the thermocycling parameters were as follows: 10 seconds at 95 C, 1 cycle; and 5 seconds at 95 C, 20 seconds at 60 C, 45 cycles. Fluorescence level was detected at the annealing stage of each cycle. A melting curve was generated during the reactions to check for the possibility of primer-dimer formation. The 2DDCt method was used to calculate the relative mRNA level of each gene (11). 1449
TABLE 1 Dynamic changes in lesions after endometriosis (EM) induction. Day 1 2 3 4 5 6 9 12 15 18 21
Recipient mice (n)
Mice with induced EM (n)
Lesions (n)
Weight (mean ± SD, mg)
Color
Adhesion
10 10 10 10 10 10 10 10 10 10 10
0 3 6 9 8 10 9 8 10 9 9
0 7 15 20 16 25 23 19 22 23 22
0 26.5 2.8 32.3 3.3 51.5 4.7 61.3 4.5 66.5 5.5 71.2 6.3 67.8 5.2 65.7 5.8 72.3 6.6 68.9 7.2
— red red red red dark red dark red or blue reddish or blue transparent transparent transparent
— N N N N N Y Y Y Y Y
Note: N ¼ no adhesion found in the pelvic cavity; Y ¼ adhesion developed in the pelvic cavity. Chen. Cytokine expression in endometriosis. Fertil Steril 2010.
Measurement of Cytokines Concentrations of IL-1b, TNF-a, and VEGF in peritoneal fluids were measured by using respective mouse-specific ELISA according to the manufacturer’s instructions. The intra-assay and interassay coefficients of variation were <5% in these assays. Statistical Analysis Statistical analysis was performed on a personal computer by using SPSS (version 14.0). Lilliefors-adjusted KolmogorovSmirnov test was used to test whether the variables used in the study were normally distributed. Data were expressed as mean SD for weight (normally distributed data). Bonferroni-adjusted Mann-Whitney t test was used for the analysis of the data. A P value of < .05 was considered to be statistically significant. RESULTS Animal Model In each of the 11 observation days after the induction of endometriosis, ten BALB/c mice were killed. No lesion was found on the 1st day, and the endometriotic lesions began to appear on the 2nd day, imparting bright-red color during the first 6 days after the induction. The adhesion developed on the 9th day, with the lesions presenting as dark-red, blue, brown, pink, or transparent blotches, small cysts, or scars on the surface of organs or peritoneum of the abdominopelvic cavity. All of the lesions turned white or transparent on the 15th day. The endometriotic lesions were collected by excision. The total number and weight of endometriotic lesions in each group were determined and the results compared. The total weight of endometriotic lesions from the 4th day (51.5 4.7 mg) to the 21st day (68.8 5.9 mg) was not statistically different. The results are given in Table 1 and Figure 1. 1450
Chen et al.
Cytokine expression in endometriosis
mRNA of Cytokines at Different Days The mRNA expressions of IL-1b, TNF-a,VEGF and MCP-1 from the peritoneal cavity cells in the 120 mice (110 model mice and ten control mice) were determined by quantitative real-time PCR, and the results are shown in Figure 2. The level of IL-1b mRNA reached the first peak on the 1st day, the second peak emerged on the 12th day, the level of the 2nd day to the 9th day fluctuated around a certain level, and the level of the 15th day to the 21st day fluctuated around another level. The first peak (48.00 0.40) was significantly higher than the second one (20.83 1.15; P<.01), and the average of the 2nd to 9th days (11.27 1.64) was significantly higher than that of the 15th to 21st days (7.31 1.26; P<.01), both values being higher than that of control group (1.00 0.38; P<.05). The level of TNF-a mRNA reached the first peak on the 1st day, the second peak on the 4th day, and the third peak on the 12th day, and during other time periods, the level fluctuated around a certain level. The first peak (18.02 1.28) was significantly higher than the second one (9.56 0.57) and the third one (9.30 0.26; P<0.01), and no significant difference was noted between the second and the third peaks (P>.05), with both of them being higher than the average level (3.59 0.92; P<0.01), which was higher than that of control group (1.00 0.21; P<.05). The first peak of VEGF mRNA appeared on the first day, the second peak on the 3rd day, the third peak on the 12th day. During other time periods, the VEGF mRNA level fluctuated around a certain level from the 2nd day to the 12th day, and it fluctuated around a lower level from the 15th day to the 21st day. The first peak (19.19 0.61) was significantly higher than the second (8.91 1.13) and the third (8.31 0.62) peaks (P<.01), and the second peak was not significantly different from the third one in terms of magnitude (P>.05). The levels of the 2nd day to the 12th day (6.12 1.21) were Vol. 93, No. 5, March 15, 2010
FIGURE 1 Morphologic changes of the endometriotic lesions at different days: (A) 2nd day; (B) 4th day; (C) 6th day; (D) 9th day; (E) 15th day; (F) 21st day.
Chen. Cytokine expression in endometriosis. Fertil Steril 2010.
significantly higher than those of the 15th day to the 21st day (2.81 0.55; P<.05), and the latter was still higher than that of the control group (1 0.25; P<.05). The level of MCP-1 mRNA reached a peak on the 1st day, and during other time periods the level fluctuated around a certain level. The first peak (57.00 0.40) was significantly higher than the second (16.03 1.14) and the third (11.47 1.78) peaks (P<.01), and the second peak was not significantly different from the third in terms of magnitude (P>.05). However, the magnitudes of both were higher than the average level (6.88 1.63; P<.01), and the average value was higher than that of control group (1 0.40; P<.05). Concentrations of Cytokines at Different Days The total proteins of IL-1b, TNF-a, and VEGF were determined by ELISA, and the results are given in Figure 3. Fertility and Sterility
The protein level of IL-1b showed the first peak lasting from the 1st to 3rd days, the second peak appeared on the 15th day, and during other time periods it fluctuated around a certain level. The magnitude of the first peak (0.160 0.003 ng/mL) was not significantly different from that of the second peak (0.178 0.011 ng/mL; P>.05), both of them being higher than the average level (0.105 0.010 ng/mL; P<.05), and the average level being significantly higher than that of the control group (0.053 0.005 ng/mL; P<.05). The protein level of TNF-a reached the first peak on the 1st to 2nd days and the second peak on the 6th day, and during other time periods it fluctuated around certain level. There was no obvious difference in terms of magnitude between the first peak (0.588 0.027 ng/mL) and the second peak (0.650 0.040 ng/mL; P>.05), with both being significantly higher than the average level (0.379 0.066 ng/mL; P<.05), 1451
FIGURE 2 Change in mRNA level of cytokines over time. All of them reached the first peak on the 1st day, and the second and the third peaks appeared during the time periods from the 3rd day to the 6th day and from the 12th day to the 15th day. IL-1b ¼ interleukin-1b; MCP-1 ¼ monocyte chemoattractant protein 1; TNF-a ¼ tumor necrosis factor a; VEGF ¼ vascular endothelial growth factor.
Chen. Cytokine expression in endometriosis. Fertil Steril 2010.
and the average values being significantly higher than that of the control group (0.180 0.016; P<.05). The protein level of VEGF reached at a peak which lasted from the 1st day to the 12th day, and during other time periods it fluctuated around a certain level. No significant difference was observed among the first peak (0.173 0.020 ng/mL), the second peak (0.216 0.023 ng/mL), and the average level (0.157 0.022 ng/mL; P>.05), and all of them were significantly higher than that of the control group (0.056 0.005; P<.05). DISCUSSION Although endometriosis is one of the most-investigated gynecologic disorders, a full understanding of its etiology and pathogenesis remains elusive. The current consensus is that endometriosis is associated with an abnormal local pelvic inflammatory process plus accompanying high level of proinflammatory cytokines in the peritoneum (12). The cytokines and chemokines, such as IL-1b, TNF-a, VEGF, and MCP-1, are fundamental factors involved in the endometriosis-associated intraperitoneal inflammation. Interleukin-1b is well known to play a critical role in the pathogenesis of this disor1452
Chen et al.
Cytokine expression in endometriosis
der by inducing the growth, adhesion, invasiveness, and angiogenesis of endometrial fragments outside the uterus (13). Tumor necrosis factor a can directly or indirectly promote proliferation, adhesion, and invasion of endometrial cells and the angiogenesis seen in endometriosis (14). Vascular endothelial growth factor is an important vasoactive growth factor which is involved in both the development and progression of peritoneal endometriosis (15). Our previous study demonstrated that VEGF was expressed in epithelial and stromal cells (16). A substantial body of evidence has shown that VEGF gene expression was higher in ectopic endometrium than in its eutopic counterpart (17). Monocyte chemoattractant protein 1 is a chemotactic cytokine that can influence both innate immunity, through its effects on monocytes, and adaptive immunity, via T helper cell polarization (18). An earlier study (19) demonstrated that MCP-1 could substantially increase the production of Fas ligand in cultured endometrial and stromal cells, and, interestingly, this increased production did not up-regulate the apoptosis of endometrial cells but increased the apoptosis of T lymphocytes. This might result in the development of immunotolerance by increasing the apoptosis of leukocytes and thereby helping the survival of ectopic endometrial cells (19). Vol. 93, No. 5, March 15, 2010
FIGURE 3
Therefore, animal model is a useful tool for the investigation of the pathogenesis and treatment of endometriosis.
Change in the protein level of cytokines over time. All of them arrived at the first peak on the 1st day, and the second and the third peaks appeared during the time period from the 3rd day to the 6th day and from the 12th day to the 15th day. Abbreviations as in Figure 2.
The present study revealed that the cytokines increased substantially on the 1st day although no lesions developed. This finding is of great importance, because, for a long time it has been controversial whether the elevated cytokine levels are causes or results of endometriosis. Some researchers have shown that the secretion of these proinflammatory proteins in the peritoneal microenvironment might contribute to the establishment and further progression of endometriosis (21), whereas others have suggested that these molecular alterations might be more a consequence of the disease than a cause (22). The dispute seems to fall into the ‘‘chicken-and-egg’’ dilemma. We found that the rise of the cytokines preceded the development of ectopic lesions, suggesting that the high-level cytokines might be more a cause than a result of endometriosis. This issue warrants further investigation. A question may arise concerning the origin of these cytokines. Research has shown that macrophages play key roles in endometriosis by producing and secreting cytokines, chemokines, and growth factors, which are believed to contribute significantly to the pathogenesis of the disease (23). In a previous study (8), we found that the peritoneal macrophages in the murine endometriosis model reached the first peak on the 1st day after the induction of endometriosis. Furthermore, according to Dmowski and Braun (24), the endometrial fragments, when misplaced into the pelvic cavity, might secrete a large number of cytokines to induce a local inflammatory response which further facilitates the cell invasion and promotes progression of the disease. The four cytokines arriving at peak on the 1st day supports our hypothesis that the endometrial cells that have entered the abdominal cavity can secrete cytokines by themselves or by recruiting macrophages.
Chen. Cytokine expression in endometriosis. Fertil Steril 2010.
Although the levels of IL-1b, TNF-a, VEGF, and MCP-1 have been reported to be abnormally high in the peritoneal fluid of women with endometriosis, and the role of cytokines has been intensively studied, it is unclear whether the rise of the cytokines takes place before or after the development of ectopic lesion. Moreover, it is not known if the change of cytokine levels follows any pattern. Answers to these questions are critical for the elucidation of the pathogenesis of endometriosis. One obstacle to the investigation of the disease is our inability to track the changes in cytokine levels during the initial stage of endometriosis in human beings, and animal models offer us a possibility to dynamically follow these changes. Umezawa et al, (20) demonstrated that the expressions of cytokines and chemokines in rats with endometriosis were similar to those in human endometriosis patients. Fertility and Sterility
As we know it, endometriotic lesions can be classified according to their morphology and their behaviors. The red lesions are more vascularized and have a higher epithelial mitotic index than the typical puckered black or bluish peritoneal lesions, and the vascularization and the mitotic index are still lower in the white lesions (25). Interestingly, in the present study, we found that the lesions were red during the initial 6 days. The adhesion developed on the 9th day and the lesions presented as dark red, blue, brown, or transparent blotches, small cysts, or scars on the surface of organs or on peritoneum of the abdominopelvic cavity. All of the lesions developed into white or transparent lesions on the 15th day. Thus, red lesions are thought to correspond to the first active stage of implantation of endometriotic lesions and evolve into the typical black or bluish lesions. The white or transparent lesions, which are believed to correspond to fibrotic quiescent lesions, showed less vascularization and were less active (26). It is noteworthy that, corresponding to the process of the endometriotic lesions transforming from red to white, the cytokines presented three peaks: the first peak took place on the 1st day, the second and the third peaks appeared during the time periods from the 3rd day to the 6th day and from the 12th day to the 15th day, and during other time periods the 1453
cytokines fluctuated around a certain level higher than the level of the control group. Resolution is an active process, and acute inflammatory response lasts for approximately 3–4 days (27). In the present study, the gene and protein levels of the cytokines dropped on the 2nd day but did not decline on the 3rd–4th day as natural resolution of inflammation proceeds, and instead on the 3rd-6th day it rose. The elevated cytokines might be related to the secretion of the newly developed endometriotic lesions. The occurrence of third peak might be due to the endometriotic lesions appearing on the 12th day to the 15th day, which contained more macrophages, myofibroblasts, and vascular endothelial cells (28). The pattern of change in the cytokines is similar to that of phagocytic function of macrophages, as we had previously reported (8), suggesting, from one perspective, that the two functions (secretion and phagocytosis) of macrophages complement each other to play an important role in the development and progression of endometriosis. Therefore, we support the notion that endometriosis per se might create a condition that further promotes its progression (29). Further research needs to be conducted on whether the estrous cycle of mice is related to the change in cytokines (30). In conclusion, this study, for the first time, demonstrated that cytokines (IL-1b, TNF-a, VEGF, and MCP-1) followed certain patterns from the 1st day to the 21st day, suggesting that endometriosis might be a positive feedback process. This finding is helpful in the understanding of the pathogenesis of endometriosis.
REFERENCES 1. Giudice LC, Kao LC. Endometriosis. Lancet 2004;364:1789–99. 2. Rier SE. Environmental immune disruption: a comorbidity factor for reproduction? Fertil Steril 2008;89:103–8. 3. Zhang XM, Wen J, Deng L, Lin J. Decreased levels of peritoneal interleukin-1 receptor antagonist in patients with endometriosis and disease-related dysmenorrheal. Fertil Steril 2007;88:594–9. 4. Bedaiwy MA, Falcone T, Sharma RK, Goldberg JM, Attaran M, Nelson DR, et al. Prediction of endometriosis with serum and peritoneal fluid markers: a prospective controlled trial. Hum Reprod 2002;17: 426–31. 5. Machado DE, Abrao MS, Berardo PT, Takiya CM, Nasciutti LE. Vascular density and distribution of vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 (Flk-1) are significantly higher in patients with deeply infiltrating endometriosis affecting the rectum. Fertil Steril 2008;90:148–55. 6. Ulukus M, Ulukus EC, Goker ENT, Tavmergen E, Zheng W, Arici A. Expression of interleukin-8 and monocyte chemotactic protein 1 in women with endometriosis. Fertil Steril. In Press. 7. Gazvani R, Templeton A. Peritoneal environment, cytokines and angiogenesis in the pathophysiology of endometriosis. Reproduction 2002;123:217–26. 8. Chen QH, Pu DM, Li T, Zhou WD, Wang Q, Chen QX. The changing pattern of phagocytotic function of peritoneal macrophages in the mouse endometriosis model. Chin J Histochem Cytochem 2008;17:362–8. 9. Dabrosin C, Gyorffy S, Margetts P, Ross C, Gauldie J. Therapeutic effect of angiostatin gene transfer in a murine model of endometriosis. Am J Pathol 2002;161:909–18.
1454
Chen et al.
Cytokine expression in endometriosis
10. Cao ZY. Chinese obstetrics and gynecology. In: Gao Y, ed. The pathology of endometriosis. Vol. 2. Beijing: People’s Health Publishing House, 2004:1486–8. 11. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2DDCt method. Methods 2001;25:402–8. 12. Dionyssopoulou E, Vassiliadis S, Evangeliou A, Koumantakis EE, Athanassakis I. Constitutive or induced elevated levels of l-carnitine correlate with the cytokine and cellular profile of endometriosis. J Reprod Immunol 2005;65:159–70. 13. Kyama CM, Overbergh L, Mihalyi A, Meuleman C, Mwenda JM, Mathieu C, et al. Endometrial and peritoneal expression of aromatase, cytokines, and adhesion factors in women with endometriosis. Fertil Steril 2008;89:301–10. 14. Grund EM, Kagan D, Tran CA, Zeitvogel A, Palmer S, Nataraja S. TNF-a regulates the expression of inflammatory and invasive genes via differential kinase signaling in human endometriotic epithelial cells. Fertil Steril 2006;86:S32. 15. McLaren J. Vascular endothelial growth factor and endometriotic angiogenesis. Hum Reprod Update 2000;6:45–55. 16. Chen QH, Qu JY, Zhong S, Fang QQ, Ma YH. Expression of the vascular endothelial growth factor in ectopic and eutopic endometrium in patients with endometriosis. Prog Obstet Gynecol 2004;13:279–83. 17. Bourlev V, Volkov N, Pavlovitch S, Lets N, Larsson A, Olovsson M. The relationship between microvessel density, proliferative activity and expression of vascular endothelial growth factor-A and its receptors in eutopic endometrium and endometriotic lesions. Reproduction 2006;132:501–9. 18. Gu L, Tseng S, Horner RM, Tam C, Loda M, Rollins BJ. Control of TH2 polarization by the chemokine monocyte chemoattractant protein-1. Nature 2000;404:407–11. 19. Selam B, Kayisli UA, Akbas GE, Basar M, Arici A. Regulation of FAS ligand expression by chemokine ligand 2 in human endometrial cells. Biol Reprod 2006;75:203–9. 20. Umezawa M, Sakata C, Tanaka N, Kudo S, Tabata M, Takeda K, et al. Cytokine and chemokine expression in a rat endometriosis is similar to that in human endometriosis. Cytokine 2008;43:105–9. 21. Gazvani R, Templeton A. Peritoneal environment, cytokines and angiogenesis in the pathophysiology of endometriosis. Reproduction 2002;123:217–26. 22. Seery JP. Endometriosis associated with defective handling of apoptotic cells in the female genital tract is a major cause of autoimmune disease in women. Med Hypotheses 2006;66:945–9. 23. Wu RJ, Van der Hoek KH, Ryan NK, Norman RJ, Robker RL. Macrophage contributions to ovarian function. Hum Reprod Update 2004;10: 119–33. 24. Dmowski WP, Braun DP. Immunology of endometriosis. Best Pract Res Clin Obstet Gyn 2004;18:245–63. 25. Donnez J, Squifflet J, Casanas-Roux F, Pirard C, Jadoul P, Langendonckt AV. Typical and subtle atypical presentations of endometriosis. Obstet Gyn Clin North Am 2003;30:83–93. 26. Kats R, Metz CN, Akoum A. Macrophage migration inhibitory factor is markedly expressed in active and early-stage endometriotic lesions. J Clin Endocrinol Metabol 2002;87:883–9. 27. Levy BD, Clish CB, Schmidt B, Gronert K, Serhan CN. Lipid mediator class switching during acute inflammation: signals in resolution. Nat Immunol 2001;2:612–9. 28. Hull ML, Escareno CR, Godsland JM, Doig JR, Johnson CM, Phillips SC, et al. Endometrial-peritoneal interactions during endometriotic lesion establishment. Am J Pathol 2008;173:700–15. 29. Lebovic DI, Mueller, Taylor RN. Immunobiology of endometriosis. Fertil Steril 2001;75:1–10. 30. Li YG, Pu DM, Li YL. The expression of cyclooxygenase-2 in cervical cancers and hela cells was regulated by estrogen/progestogen. J Huazhong Univ Sci Technol 2007;27:457–60.
Vol. 93, No. 5, March 15, 2010
Objective: To examine the change in proinflammatory cytokines in the pathologic processes of endometriosis in mice. Design: A dynamic study on a murine model of endometriosis. Setting: Medical school. Animal(s): Female BALB/c mice. Intervention(s): Endometriosis was induced by injecting endometrial fragments of syngenic mice into the peritoneal cavity of model mice; in control group, phosphate-buffered saline instead of fragments was injected. The peritoneal fluid and the endometriotic lesions were harvested 1 to 21 days after the induction. Main Outcome Measure(s): The endometriotic lesions were weighed, the gene and protein levels of some proinflammatory cytokines, including interleukin 1b, tumor necrosis factor a, vascular endothelial growth factor, and monocyte chemoattractant protein 1, were determined by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Result(s): The levels of these cytokines reached the first peak on the first day and no endometriotic lesions were found. The lesions began to appear on the second day, presenting red color during the initial 6 days, and then they turned dark-red, brown, or bluish. The adhesion took place on the 9th day, and all the lesions evolved into white or transparent cysts on the 15th day. Corresponding to these changes, the second and the third peaks were identified during the 3rd–6th day and the 12th-15th day, respectively. Conclusion(s): The change pattern of cytokines over time might bear some relationship with the development and progression of the endometriosis. (Fertil Steril 2010;93:1448–54. 2010 by American Society for Reproductive Medicine.) Key Words: Endometriosis, IL-1b, TNF-a, VEGF, MCP-1, pathogenesis
Endometriosis is characterized by the growth of endometriotic tissue outside the uterine cavity, and its pathogenesis is not fully understood (1). Recently, research effort has been directed to the role of the abnormal immunoinflammatory reaction in the pathogenesis of endometriosis (2). The cellular communication during inflammatory responses is mediated via cytokines and chemokines (3). Increasing evidence has indicated that some cytokines, such as interleukin 1b (IL-1b) (4), tumor necrosis factor a (TNF-a) (5), vascular endothelial growth factor (VEGF) (6), and monocyte chemoattractant protein 1 (MCP-1) (7), are elevated in the peritoReceived October 20, 2008; revised January 21, 2009; accepted February 5, 2009; published online April 1, 2009. Q.-H.C. has nothing to disclose. W.-D.Z. has nothing to disclose. Z.-Y.S. has nothing to disclose. Q.-S.H. has nothing to disclose. J.-N.J. has nothing to disclose. Q.-X.C. has nothing to disclose. Supported by grant no. 2007-1-43 from the Ministry of Health of Fujian Province, P.R. China, by grant 2009-CXB-49 of the medical innovation project of Fujian Province, P.R. China, and grant nos. 3502 Z 20077047 and 3502 Z 20084006 from the Science and Technology Planning Project of Xiamen City, P.R. China. Reprint requests: Qing-Xi Chen, Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen, People’s Republic of China 361005 (FAX: þ86-592-2185487; E-mail: [email protected]).
1448
neal fluid of women with endometriosis. It remains unclear whether the elevation of cytokine levels precedes or follows the emergence of the endometriotic lesions. Moreover, it is well known that the morphology of the ectopic lesions varies greatly with the stages of the condition, but it is still uncertain whether the cytokine levels are linked with morphologic change of the ectopic lesions. Owing to the limitations associated with human study, it is difficult to examine the changes of cytokines over time in the development of endometriosis, especially during the period from the beginning of the retrograde menstruation into the peritoneal cavity to the development of endometriotic lesion. In our previous study (8), we found that the phagocytotic function of peritoneal macrophages in the murine endometriosis model was enhanced, presenting certain patterns of change over time. This finding led us to investigate whether the cytokines undergo a similar change and if the change in proinflammatory cytokines is tied with the development of endometriosis. On the 1st, 2nd, 3rd, 4th, 5th, 6th, 9th, 12th, 15th, 18th, and 21st days after the induction of endometriosis in BALB/c mice, we measured the gene and protein levels of the cytokines (IL-1b, TNF-a, VEGF, and MCP-1) in the peritoneal fluid by using quantitative polymerase chain reaction (PCR) and ELISA, in an attempt
Fertility and Sterility Vol. 93, No. 5, March 15, 2010 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/10/$36.00 doi:10.1016/j.fertnstert.2009.02.013
to identify some pattern of change in the cytokine expression at the early stage of endometriosis. MATERIALS AND METHODS Chemicals The ELISA kits were purchased from R&D Systems (Minneapolis, MN); SYBR Primescript RT-PCR kit was bought from TaKaRa Co. (Kyoto, Japan); RNAspin Mini RNA isolation kit was procured from GE Healthcare (Uppsala, Sweden). Other reagents were of analytical grade. All reagents, except phosphate-buffered saline (PBS), were prepared daily before use and stored in a refrigerator at 4 C. The reagents were equilibrated at room temperature for 0.5 hour before use. Animals The BALB/c mice were provided by the Animal Research Laboratory at Xiamen University. A total of 175 female BALB/c mice (8 weeks old, 19–21 g in weight), including 110 model (recipient) mice, 55 donor mice, and 10 mice serving as control subjects, were kept on a light/dark cycle of 12/12 hours under controlled conditions in a room at a constant temperature of 21–22 C and humidity of 60 5%. The mice were caged in groups of five for a week before the onset of the experiment, with free access to a commercial balanced mouse diet and tap water. The animals were procured, maintained, and used in accordance with the ‘‘Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals’’ formulated by Xiamen University’s Animal Ethics Committee. The study was approved by the institutional review board of Xiamen University. Experimental Design Endometriosis was induced by using a previously described method with minor modifications (9). After the 55 donor mice were killed, the uterine horns were taken out and put into a dish containing PBS. The endometrium-rich fragments, obtained by peeling off the serosa and myometrium, were finely chopped using a razor blade. Fragments suspended in 1 mL PBS at 37 C were injected by means of an 18-gauge needle through the abdominal wall into the peritoneal cavity of the recipient mice, with a ratio of 1:2 (donor/recipient). For each mouse, the operation time was limited to 5 minutes. The 110 model mice were randomly divided into 11 groups, with ten animals in each group. Ten mice were killed by cervical dislocation on each observation day after the induction of endometriosis, i.e., the 1st (24 hours), 2nd, 3rd, 4th, 5th, 6th, 9th, 12th, 15th, 18th, and 21st days. Another ten noninduced mice served as a control group, designated day 0. All operations were performed under aseptic conditions by the lead author. In all of the procedures, the examiners were blind to the treatment given to each mouse. Specimen Collection After killing it, 3 mL PBS was injected into the peritoneal cavity of each mouse. After shaking the mouse, the peritoneal fluid was collected and centrifuged. The supernatant was kept Fertility and Sterility
at 80 C and the settled peritoneal cells, including peritoneal macrophages, were used for RNA extraction. Then laparotomy was performed, the number of endometriotic lesions was counted, and the lesions were morphologically observed. All lesions and uteri were quickly excised, and normal surrounding tissues, whenever possible, were removed. The lesions were put in formaldehyde solution for routine histopathologic examination and confirmed to be endometriotic lesions. Morphologically, ectopic lesions of endometriosis present as red, blue, brown, or transparent blotches, small cysts, or scars on the surface of organs or on the peritoneum of the abdominopelvic cavity, so they can be identified by naked eyes. The pathologic diagnostic criteria for endometriosis are the presence of any two of the four foregoing changes, i.e., endometrial epithelium, glands or gland-like structures, stroma, and hemorrhage (10).
RNA Extraction and Quantitative Real-Time ReverseTranscription PCR Total RNA was obtained by extraction with RNAspin mini kit (GE Healthcare), following the manufacturer’s instructions. The total RNA was reverse-transcribed in a system of 20 mL (final volume) by using SYBR Primescript RT-PCR kit (TaKaRa) in accordance with manufacturer’s instructions. The following conditions were applied to reverse transcription: 37 C for 15 minutes (reverse transcription reaction), 1 cycle; and 85 C for 5 seconds (denaturation of reverse transcriptase). The first-strand cDNA obtained from RNA samples was stored at 80 C until use. The mRNA expression of IL-1b, TNF-a, VEGF, and MCP-1 in each sample was measured by quantitative realtime PCR. Primers were synthesized by TaKaRa company (Kyoto, Japan). The primers used for real-time PCR were: b-actin: forward 50 -CATCCGTAAAGACCTCTATGCCAA C-30 , reverse 50 -ATGGAGCCACCGATCCACA-30 ; IL-1b: forward 50 -TCCAGGATGAGGACATCACATGAGCAC-30 , reverse 50 -GAACGCACCAGCAGGTTA-30 ; TNF-a: forward 50 -AAGCCTGTAGCCCACGTCGTA-30 , reverse 50 -GGCAC CACTAGTTGGTTGTCTTTG-30 ; VEGF: (forward 50 -GTG CACTGGACCCTGGCTTTA-30 , reverse 50 -GGTCTCAATC GGACGGCAGTA-30 ; and MCP-1: forward 50 -GCATCCAC GTGTTGGCTCA-30 , reverse 50 -CTCCAGCCTACTCATTG GGATCA-30 ). Quantitative real-time PCR assays were carried out with SYBR Primescript RT-PCR kit (TaKaRa). The reactions were run in 20mL mixture containing 10mL SYBR Premix EX Taq (2), 0.4mL of each primer (10mmol/mL), 1.6mL cDNA, and 7.6mL ddH2O. The PCR was performed in a Rotor-Gene 3000 (Corbett Research, Sydney, Australia), and the thermocycling parameters were as follows: 10 seconds at 95 C, 1 cycle; and 5 seconds at 95 C, 20 seconds at 60 C, 45 cycles. Fluorescence level was detected at the annealing stage of each cycle. A melting curve was generated during the reactions to check for the possibility of primer-dimer formation. The 2DDCt method was used to calculate the relative mRNA level of each gene (11). 1449
TABLE 1 Dynamic changes in lesions after endometriosis (EM) induction. Day 1 2 3 4 5 6 9 12 15 18 21
Recipient mice (n)
Mice with induced EM (n)
Lesions (n)
Weight (mean ± SD, mg)
Color
Adhesion
10 10 10 10 10 10 10 10 10 10 10
0 3 6 9 8 10 9 8 10 9 9
0 7 15 20 16 25 23 19 22 23 22
0 26.5 2.8 32.3 3.3 51.5 4.7 61.3 4.5 66.5 5.5 71.2 6.3 67.8 5.2 65.7 5.8 72.3 6.6 68.9 7.2
— red red red red dark red dark red or blue reddish or blue transparent transparent transparent
— N N N N N Y Y Y Y Y
Note: N ¼ no adhesion found in the pelvic cavity; Y ¼ adhesion developed in the pelvic cavity. Chen. Cytokine expression in endometriosis. Fertil Steril 2010.
Measurement of Cytokines Concentrations of IL-1b, TNF-a, and VEGF in peritoneal fluids were measured by using respective mouse-specific ELISA according to the manufacturer’s instructions. The intra-assay and interassay coefficients of variation were <5% in these assays. Statistical Analysis Statistical analysis was performed on a personal computer by using SPSS (version 14.0). Lilliefors-adjusted KolmogorovSmirnov test was used to test whether the variables used in the study were normally distributed. Data were expressed as mean SD for weight (normally distributed data). Bonferroni-adjusted Mann-Whitney t test was used for the analysis of the data. A P value of < .05 was considered to be statistically significant. RESULTS Animal Model In each of the 11 observation days after the induction of endometriosis, ten BALB/c mice were killed. No lesion was found on the 1st day, and the endometriotic lesions began to appear on the 2nd day, imparting bright-red color during the first 6 days after the induction. The adhesion developed on the 9th day, with the lesions presenting as dark-red, blue, brown, pink, or transparent blotches, small cysts, or scars on the surface of organs or peritoneum of the abdominopelvic cavity. All of the lesions turned white or transparent on the 15th day. The endometriotic lesions were collected by excision. The total number and weight of endometriotic lesions in each group were determined and the results compared. The total weight of endometriotic lesions from the 4th day (51.5 4.7 mg) to the 21st day (68.8 5.9 mg) was not statistically different. The results are given in Table 1 and Figure 1. 1450
Chen et al.
Cytokine expression in endometriosis
mRNA of Cytokines at Different Days The mRNA expressions of IL-1b, TNF-a,VEGF and MCP-1 from the peritoneal cavity cells in the 120 mice (110 model mice and ten control mice) were determined by quantitative real-time PCR, and the results are shown in Figure 2. The level of IL-1b mRNA reached the first peak on the 1st day, the second peak emerged on the 12th day, the level of the 2nd day to the 9th day fluctuated around a certain level, and the level of the 15th day to the 21st day fluctuated around another level. The first peak (48.00 0.40) was significantly higher than the second one (20.83 1.15; P<.01), and the average of the 2nd to 9th days (11.27 1.64) was significantly higher than that of the 15th to 21st days (7.31 1.26; P<.01), both values being higher than that of control group (1.00 0.38; P<.05). The level of TNF-a mRNA reached the first peak on the 1st day, the second peak on the 4th day, and the third peak on the 12th day, and during other time periods, the level fluctuated around a certain level. The first peak (18.02 1.28) was significantly higher than the second one (9.56 0.57) and the third one (9.30 0.26; P<0.01), and no significant difference was noted between the second and the third peaks (P>.05), with both of them being higher than the average level (3.59 0.92; P<0.01), which was higher than that of control group (1.00 0.21; P<.05). The first peak of VEGF mRNA appeared on the first day, the second peak on the 3rd day, the third peak on the 12th day. During other time periods, the VEGF mRNA level fluctuated around a certain level from the 2nd day to the 12th day, and it fluctuated around a lower level from the 15th day to the 21st day. The first peak (19.19 0.61) was significantly higher than the second (8.91 1.13) and the third (8.31 0.62) peaks (P<.01), and the second peak was not significantly different from the third one in terms of magnitude (P>.05). The levels of the 2nd day to the 12th day (6.12 1.21) were Vol. 93, No. 5, March 15, 2010
FIGURE 1 Morphologic changes of the endometriotic lesions at different days: (A) 2nd day; (B) 4th day; (C) 6th day; (D) 9th day; (E) 15th day; (F) 21st day.
Chen. Cytokine expression in endometriosis. Fertil Steril 2010.
significantly higher than those of the 15th day to the 21st day (2.81 0.55; P<.05), and the latter was still higher than that of the control group (1 0.25; P<.05). The level of MCP-1 mRNA reached a peak on the 1st day, and during other time periods the level fluctuated around a certain level. The first peak (57.00 0.40) was significantly higher than the second (16.03 1.14) and the third (11.47 1.78) peaks (P<.01), and the second peak was not significantly different from the third in terms of magnitude (P>.05). However, the magnitudes of both were higher than the average level (6.88 1.63; P<.01), and the average value was higher than that of control group (1 0.40; P<.05). Concentrations of Cytokines at Different Days The total proteins of IL-1b, TNF-a, and VEGF were determined by ELISA, and the results are given in Figure 3. Fertility and Sterility
The protein level of IL-1b showed the first peak lasting from the 1st to 3rd days, the second peak appeared on the 15th day, and during other time periods it fluctuated around a certain level. The magnitude of the first peak (0.160 0.003 ng/mL) was not significantly different from that of the second peak (0.178 0.011 ng/mL; P>.05), both of them being higher than the average level (0.105 0.010 ng/mL; P<.05), and the average level being significantly higher than that of the control group (0.053 0.005 ng/mL; P<.05). The protein level of TNF-a reached the first peak on the 1st to 2nd days and the second peak on the 6th day, and during other time periods it fluctuated around certain level. There was no obvious difference in terms of magnitude between the first peak (0.588 0.027 ng/mL) and the second peak (0.650 0.040 ng/mL; P>.05), with both being significantly higher than the average level (0.379 0.066 ng/mL; P<.05), 1451
FIGURE 2 Change in mRNA level of cytokines over time. All of them reached the first peak on the 1st day, and the second and the third peaks appeared during the time periods from the 3rd day to the 6th day and from the 12th day to the 15th day. IL-1b ¼ interleukin-1b; MCP-1 ¼ monocyte chemoattractant protein 1; TNF-a ¼ tumor necrosis factor a; VEGF ¼ vascular endothelial growth factor.
Chen. Cytokine expression in endometriosis. Fertil Steril 2010.
and the average values being significantly higher than that of the control group (0.180 0.016; P<.05). The protein level of VEGF reached at a peak which lasted from the 1st day to the 12th day, and during other time periods it fluctuated around a certain level. No significant difference was observed among the first peak (0.173 0.020 ng/mL), the second peak (0.216 0.023 ng/mL), and the average level (0.157 0.022 ng/mL; P>.05), and all of them were significantly higher than that of the control group (0.056 0.005; P<.05). DISCUSSION Although endometriosis is one of the most-investigated gynecologic disorders, a full understanding of its etiology and pathogenesis remains elusive. The current consensus is that endometriosis is associated with an abnormal local pelvic inflammatory process plus accompanying high level of proinflammatory cytokines in the peritoneum (12). The cytokines and chemokines, such as IL-1b, TNF-a, VEGF, and MCP-1, are fundamental factors involved in the endometriosis-associated intraperitoneal inflammation. Interleukin-1b is well known to play a critical role in the pathogenesis of this disor1452
Chen et al.
Cytokine expression in endometriosis
der by inducing the growth, adhesion, invasiveness, and angiogenesis of endometrial fragments outside the uterus (13). Tumor necrosis factor a can directly or indirectly promote proliferation, adhesion, and invasion of endometrial cells and the angiogenesis seen in endometriosis (14). Vascular endothelial growth factor is an important vasoactive growth factor which is involved in both the development and progression of peritoneal endometriosis (15). Our previous study demonstrated that VEGF was expressed in epithelial and stromal cells (16). A substantial body of evidence has shown that VEGF gene expression was higher in ectopic endometrium than in its eutopic counterpart (17). Monocyte chemoattractant protein 1 is a chemotactic cytokine that can influence both innate immunity, through its effects on monocytes, and adaptive immunity, via T helper cell polarization (18). An earlier study (19) demonstrated that MCP-1 could substantially increase the production of Fas ligand in cultured endometrial and stromal cells, and, interestingly, this increased production did not up-regulate the apoptosis of endometrial cells but increased the apoptosis of T lymphocytes. This might result in the development of immunotolerance by increasing the apoptosis of leukocytes and thereby helping the survival of ectopic endometrial cells (19). Vol. 93, No. 5, March 15, 2010
FIGURE 3
Therefore, animal model is a useful tool for the investigation of the pathogenesis and treatment of endometriosis.
Change in the protein level of cytokines over time. All of them arrived at the first peak on the 1st day, and the second and the third peaks appeared during the time period from the 3rd day to the 6th day and from the 12th day to the 15th day. Abbreviations as in Figure 2.
The present study revealed that the cytokines increased substantially on the 1st day although no lesions developed. This finding is of great importance, because, for a long time it has been controversial whether the elevated cytokine levels are causes or results of endometriosis. Some researchers have shown that the secretion of these proinflammatory proteins in the peritoneal microenvironment might contribute to the establishment and further progression of endometriosis (21), whereas others have suggested that these molecular alterations might be more a consequence of the disease than a cause (22). The dispute seems to fall into the ‘‘chicken-and-egg’’ dilemma. We found that the rise of the cytokines preceded the development of ectopic lesions, suggesting that the high-level cytokines might be more a cause than a result of endometriosis. This issue warrants further investigation. A question may arise concerning the origin of these cytokines. Research has shown that macrophages play key roles in endometriosis by producing and secreting cytokines, chemokines, and growth factors, which are believed to contribute significantly to the pathogenesis of the disease (23). In a previous study (8), we found that the peritoneal macrophages in the murine endometriosis model reached the first peak on the 1st day after the induction of endometriosis. Furthermore, according to Dmowski and Braun (24), the endometrial fragments, when misplaced into the pelvic cavity, might secrete a large number of cytokines to induce a local inflammatory response which further facilitates the cell invasion and promotes progression of the disease. The four cytokines arriving at peak on the 1st day supports our hypothesis that the endometrial cells that have entered the abdominal cavity can secrete cytokines by themselves or by recruiting macrophages.
Chen. Cytokine expression in endometriosis. Fertil Steril 2010.
Although the levels of IL-1b, TNF-a, VEGF, and MCP-1 have been reported to be abnormally high in the peritoneal fluid of women with endometriosis, and the role of cytokines has been intensively studied, it is unclear whether the rise of the cytokines takes place before or after the development of ectopic lesion. Moreover, it is not known if the change of cytokine levels follows any pattern. Answers to these questions are critical for the elucidation of the pathogenesis of endometriosis. One obstacle to the investigation of the disease is our inability to track the changes in cytokine levels during the initial stage of endometriosis in human beings, and animal models offer us a possibility to dynamically follow these changes. Umezawa et al, (20) demonstrated that the expressions of cytokines and chemokines in rats with endometriosis were similar to those in human endometriosis patients. Fertility and Sterility
As we know it, endometriotic lesions can be classified according to their morphology and their behaviors. The red lesions are more vascularized and have a higher epithelial mitotic index than the typical puckered black or bluish peritoneal lesions, and the vascularization and the mitotic index are still lower in the white lesions (25). Interestingly, in the present study, we found that the lesions were red during the initial 6 days. The adhesion developed on the 9th day and the lesions presented as dark red, blue, brown, or transparent blotches, small cysts, or scars on the surface of organs or on peritoneum of the abdominopelvic cavity. All of the lesions developed into white or transparent lesions on the 15th day. Thus, red lesions are thought to correspond to the first active stage of implantation of endometriotic lesions and evolve into the typical black or bluish lesions. The white or transparent lesions, which are believed to correspond to fibrotic quiescent lesions, showed less vascularization and were less active (26). It is noteworthy that, corresponding to the process of the endometriotic lesions transforming from red to white, the cytokines presented three peaks: the first peak took place on the 1st day, the second and the third peaks appeared during the time periods from the 3rd day to the 6th day and from the 12th day to the 15th day, and during other time periods the 1453
cytokines fluctuated around a certain level higher than the level of the control group. Resolution is an active process, and acute inflammatory response lasts for approximately 3–4 days (27). In the present study, the gene and protein levels of the cytokines dropped on the 2nd day but did not decline on the 3rd–4th day as natural resolution of inflammation proceeds, and instead on the 3rd-6th day it rose. The elevated cytokines might be related to the secretion of the newly developed endometriotic lesions. The occurrence of third peak might be due to the endometriotic lesions appearing on the 12th day to the 15th day, which contained more macrophages, myofibroblasts, and vascular endothelial cells (28). The pattern of change in the cytokines is similar to that of phagocytic function of macrophages, as we had previously reported (8), suggesting, from one perspective, that the two functions (secretion and phagocytosis) of macrophages complement each other to play an important role in the development and progression of endometriosis. Therefore, we support the notion that endometriosis per se might create a condition that further promotes its progression (29). Further research needs to be conducted on whether the estrous cycle of mice is related to the change in cytokines (30). In conclusion, this study, for the first time, demonstrated that cytokines (IL-1b, TNF-a, VEGF, and MCP-1) followed certain patterns from the 1st day to the 21st day, suggesting that endometriosis might be a positive feedback process. This finding is helpful in the understanding of the pathogenesis of endometriosis.
REFERENCES 1. Giudice LC, Kao LC. Endometriosis. Lancet 2004;364:1789–99. 2. Rier SE. Environmental immune disruption: a comorbidity factor for reproduction? Fertil Steril 2008;89:103–8. 3. Zhang XM, Wen J, Deng L, Lin J. Decreased levels of peritoneal interleukin-1 receptor antagonist in patients with endometriosis and disease-related dysmenorrheal. Fertil Steril 2007;88:594–9. 4. Bedaiwy MA, Falcone T, Sharma RK, Goldberg JM, Attaran M, Nelson DR, et al. Prediction of endometriosis with serum and peritoneal fluid markers: a prospective controlled trial. Hum Reprod 2002;17: 426–31. 5. Machado DE, Abrao MS, Berardo PT, Takiya CM, Nasciutti LE. Vascular density and distribution of vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 (Flk-1) are significantly higher in patients with deeply infiltrating endometriosis affecting the rectum. Fertil Steril 2008;90:148–55. 6. Ulukus M, Ulukus EC, Goker ENT, Tavmergen E, Zheng W, Arici A. Expression of interleukin-8 and monocyte chemotactic protein 1 in women with endometriosis. Fertil Steril. In Press. 7. Gazvani R, Templeton A. Peritoneal environment, cytokines and angiogenesis in the pathophysiology of endometriosis. Reproduction 2002;123:217–26. 8. Chen QH, Pu DM, Li T, Zhou WD, Wang Q, Chen QX. The changing pattern of phagocytotic function of peritoneal macrophages in the mouse endometriosis model. Chin J Histochem Cytochem 2008;17:362–8. 9. Dabrosin C, Gyorffy S, Margetts P, Ross C, Gauldie J. Therapeutic effect of angiostatin gene transfer in a murine model of endometriosis. Am J Pathol 2002;161:909–18.
1454
Chen et al.
Cytokine expression in endometriosis
10. Cao ZY. Chinese obstetrics and gynecology. In: Gao Y, ed. The pathology of endometriosis. Vol. 2. Beijing: People’s Health Publishing House, 2004:1486–8. 11. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2DDCt method. Methods 2001;25:402–8. 12. Dionyssopoulou E, Vassiliadis S, Evangeliou A, Koumantakis EE, Athanassakis I. Constitutive or induced elevated levels of l-carnitine correlate with the cytokine and cellular profile of endometriosis. J Reprod Immunol 2005;65:159–70. 13. Kyama CM, Overbergh L, Mihalyi A, Meuleman C, Mwenda JM, Mathieu C, et al. Endometrial and peritoneal expression of aromatase, cytokines, and adhesion factors in women with endometriosis. Fertil Steril 2008;89:301–10. 14. Grund EM, Kagan D, Tran CA, Zeitvogel A, Palmer S, Nataraja S. TNF-a regulates the expression of inflammatory and invasive genes via differential kinase signaling in human endometriotic epithelial cells. Fertil Steril 2006;86:S32. 15. McLaren J. Vascular endothelial growth factor and endometriotic angiogenesis. Hum Reprod Update 2000;6:45–55. 16. Chen QH, Qu JY, Zhong S, Fang QQ, Ma YH. Expression of the vascular endothelial growth factor in ectopic and eutopic endometrium in patients with endometriosis. Prog Obstet Gynecol 2004;13:279–83. 17. Bourlev V, Volkov N, Pavlovitch S, Lets N, Larsson A, Olovsson M. The relationship between microvessel density, proliferative activity and expression of vascular endothelial growth factor-A and its receptors in eutopic endometrium and endometriotic lesions. Reproduction 2006;132:501–9. 18. Gu L, Tseng S, Horner RM, Tam C, Loda M, Rollins BJ. Control of TH2 polarization by the chemokine monocyte chemoattractant protein-1. Nature 2000;404:407–11. 19. Selam B, Kayisli UA, Akbas GE, Basar M, Arici A. Regulation of FAS ligand expression by chemokine ligand 2 in human endometrial cells. Biol Reprod 2006;75:203–9. 20. Umezawa M, Sakata C, Tanaka N, Kudo S, Tabata M, Takeda K, et al. Cytokine and chemokine expression in a rat endometriosis is similar to that in human endometriosis. Cytokine 2008;43:105–9. 21. Gazvani R, Templeton A. Peritoneal environment, cytokines and angiogenesis in the pathophysiology of endometriosis. Reproduction 2002;123:217–26. 22. Seery JP. Endometriosis associated with defective handling of apoptotic cells in the female genital tract is a major cause of autoimmune disease in women. Med Hypotheses 2006;66:945–9. 23. Wu RJ, Van der Hoek KH, Ryan NK, Norman RJ, Robker RL. Macrophage contributions to ovarian function. Hum Reprod Update 2004;10: 119–33. 24. Dmowski WP, Braun DP. Immunology of endometriosis. Best Pract Res Clin Obstet Gyn 2004;18:245–63. 25. Donnez J, Squifflet J, Casanas-Roux F, Pirard C, Jadoul P, Langendonckt AV. Typical and subtle atypical presentations of endometriosis. Obstet Gyn Clin North Am 2003;30:83–93. 26. Kats R, Metz CN, Akoum A. Macrophage migration inhibitory factor is markedly expressed in active and early-stage endometriotic lesions. J Clin Endocrinol Metabol 2002;87:883–9. 27. Levy BD, Clish CB, Schmidt B, Gronert K, Serhan CN. Lipid mediator class switching during acute inflammation: signals in resolution. Nat Immunol 2001;2:612–9. 28. Hull ML, Escareno CR, Godsland JM, Doig JR, Johnson CM, Phillips SC, et al. Endometrial-peritoneal interactions during endometriotic lesion establishment. Am J Pathol 2008;173:700–15. 29. Lebovic DI, Mueller, Taylor RN. Immunobiology of endometriosis. Fertil Steril 2001;75:1–10. 30. Li YG, Pu DM, Li YL. The expression of cyclooxygenase-2 in cervical cancers and hela cells was regulated by estrogen/progestogen. J Huazhong Univ Sci Technol 2007;27:457–60.
Vol. 93, No. 5, March 15, 2010