1 Department of Obstetrics and Gynecology, 2 Laboratory of Host Defense, Institute for Advanced Medical Sciences and 3 Department of Surgical Pathology, Hyogo College of Medicine, Nishinomiya, Hyogo 663-8501, Japan
4 To whom correspondence should be addressed. e-mail: yoshi416{at}hyo-med.ac.jp
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Abstract |
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Key words:
COX-II/cytokine/endometriosis/IL-18/IL-1
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Introduction |
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IL-18 was initially conceived to be a Th1 cytokine which, together with IL-12, releases interferon- (IFN-
) and TNF-
from T cells and natural killer cells (Okamura et al., 1995
; Tsutsui et al., 1996
). However, IL-18 was later found to play a role in stimulating the Th2 status, releasing IL-4, IL-5 and IL-13 (Hoshino et al., 1999
; Nakanishi et al., 2001
). IL-18 also stimulates a subset of macrophages to induce cyclooxygenase (COX)-II and, subsequently, prostaglandins (PGs) (Kashiwamura et al., 2002
), which mediate various biological responses, including pain (Olee et al., 1999
; Futani et al., 2002
). It has been reported recently that the peritoneal fluid of endometriosis patients contains elevated levels of PGs (Wu et al., 2002
). This, together with the complex immune status in endometriosis patients, prompted us to examine the role of IL-18 in the pathogenesis of endometriosis.
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Materials and methods |
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Preparation of sera, peritoneal fluids and tissue specimens
The peritoneal fluid was collected using laparoscopy, mixed with heparin and centrifuged, and the supernatant was stored at 80°C in 500 µl aliquots until use. Blood samples were processed and stored in a similar manner. Tissue specimens were obtained from peritoneal lesions and endometriomas. They were fixed in 10% buffered formalin and embedded in paraffin.
Measurement of cytokines
IL-18 in peritoneal fluids and sera was measured by enzyme-linked immunosorbent assay (ELISA) using the Human IL-18 Immuno Assay Kit (MBL Co. Ltd, Nagoya, Aichi, Japan). IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, TNF-
, granulocytemacrophage colony-stimulating factor (GM-CSF) and IFN-
were determined by Bio-Plex Protein Array System (Bio-Rad Laboratories, Hercules, CA) using Human Cytokine Assay reagents (Bio-Rad).
Cytokines
Recombinant human IL-18 was purchased from MBL Co. Ltd and recombinant human IL-1 from R&D systems (Minneapolis, MN).
Preparation of CD14-positive cells from the peritoneal fluid and treatment with IL-18
Cells in peritoneal fluids were collected by centrifugation, treated with ACK solution to deplete red blood cells, and then incubated with micro bead-conjugated mouse anti-human CD14 monoclonal antibody (mAb) (Miltenyi Biotec, Gladbach, Germany). CD14-positive cells were separated using a magnetic cell sorter and a biomolecules (MACS) column (Miltenyi Biotec). The purity of cells was analysed by flow cytometry using fluorescein isothiocyanate (FITC)-labelled mouse anti-human CD14 mAb (BD Biosciences, San Jose, CA). CD14-positive cells (2 x 105 cells/ml) were cultured with IL-18 (20 ng/ml) or IL-1 (20 ng/ml) in phenol red-free RPMI-1640 (Invitrogen Corp., Carlsbad, CA) supplemented with 10% heat-inactivated fetal calf serum at 37°C for 16 h under 5% CO2.
Preparation of endometriotic cells and treatment with IL-18
Endometriotic tissues were incubated in RPMI-1640 (Invitrogen) containing 15 µg/ml DNase (Sigma Chemical Co., St Louis, MO) and 400 U/ml collagenase (Wako Pure Chemical Industries, Ltd, Osaka, Japan) at 37°C for 1 h. Cells released were collected by centrifugation at 150 g for 10 min and used for experiments without separation of inner and interstitial cells. Treatment of these cells with IL-18 or IL-1 was carried out in the same way as described above for CD14-positive cells.
RTPCR analysis
Total RNA was extracted using Isogen (NIPPON GENE Co. Ltd, Toyama, Japan) and reverse transcribed by MuLV reverse transcriptase. The resultant cDNA was used as the template for PCR using primers specific to human COX-I, COX-II, IL-18, IL-18R and IL-18R
as follows: COX-I sense, 5'-GAGTCTTTCTCCAAC GTGAGC-3'; antisense, 5'-ACCTGGTACTTGAGTTTCCCA-3'; COX-II sense, 5'-TGAAACCCACTCCAAACACAG-3'; antisense, 5'-TCATCAGGCACAGGAGGAAG-3'; IL-18 sense, 5'-GCTTGAA TCTAAATTATCAGTC-3'; antisense, 5'-GAAGATTCAAATTGC ATCTTAT-3'; IL-18R
sense, 5'-GTTGAGTTGAATGACACAGG-3'; antisense, 5'-TCCACTGCAACATGGTTAAG-3'; and IL-18R
sense, 5'-ATGCTCTGTTTGGGCTGGATA-3'; antisense, 5'-CATC TTGACACAACAGGCTAC-3'. PCRs were denaturation at 94°C for 5 min, then 32 cycles at 94°C for 30 s, at 58°C for 30 s, and at 72°C for 1 min, and extension at 72°C for 7 min.
PCR of -actin cDNA was conducted using a sense primer, 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3', and an antisense primer, 5'-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3', at 94°C for 5 min, then 35 cycles at 94°C for 30 s, at 66°C for 30 s, and at 72°C for 1 min, and then at 72°C for 7 min.
PCR products were electrophoresed in agarose gels, stained with ethidium bromide and visualized under UV illumination.
Immunohistochemical staining
Paraffin-embedded tissue specimens were cut into 4 µm sections, deparaffinized and incubated with mouse anti-human IL-18 mAb (a kind gift from Dr Torigoe, Institute of Bio-Research, Hayashibara, Okayama, Japan), mouse anti-human IL-18R mAb (a kind gift from Dr Torigoe), mouse anti-human COX-II mb (IBL, Fujioka, Gunma, Japan) or mouse anti-human CD68 mAb (DAKO, Glostrup, Denmark), and then with peroxidase-conjugated rabbit anti-mouse IgG polyclonal antibody (Vector, CA). Specific bindings of antibodies were detected using a DAB reagent kit (DAKO).
Western blot analysis
Endometriotic cells were lysed in ice-cold NP-40 lysis buffer consisting of 20 mM TrisHCl pH 8.8, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 10% glycerol and 50 mM phenylmethysulphonyl fluoride, supplemented with proteinase inhibitor cocktail (one tablet/10 ml of lysis buffer) (Roche Molecular Biochemicals, Mannheim, Germany). Each proteinase inhibitor cocktail tablet contains 3.0 mg of antipapain, 0.5 mg of bestatin, 1.0 mg of chymostatin, 3.0 mg of E-64, 0.5 mg of leupeptin, 0.5 mg of pepstatin, 3.0 mg of phosphramidon, 20.0 mg of pefabloc SC and 0.5 mg of aprotinin. The lysate (20 µg of protein) was subjected to SDSPAGE under reduced conditions. Separated proteins were transferred onto PDFV membranes (Hybond-P, Amersham Biosciences Co., Piscataway, NJ). The membrane was soaked in phosphate-buffered saline (PBS) containing 2% non-fat milk, and incubated with 1 µg/ml of mouse anti-human COX-II mAb (IBL Co. Ltd, Fujioka, Gunma, Japan) and affinity-purified rabbit anti-human -actin antiboby (Sigma Chemical Co.). Horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibody (1:1000 diluted) and the F(ab')2 fragment of HRP-conjugated donkey anti-rabbit IgG antibody were used as the secondary antibodies (Amersham Biosciences). Specific bindings of antibodies were detected by the luminescent image analyser, LAS-1000 plus (Fuji Photo Film Co. Ltd, Tokyo, Japan), using ECL plus western blotting detection reagents (Amersham Biosciences).
Statistical analysis
Data were analysed by MannWhitney U-test for multiple comparisons. A P-value <0.05 was considered to be statistically significant. Results were expressed as the means ± SEs of repeated experiments. The Pearson correlation coefficient (r) was determined using Stat Mate III software (Atms K.K., Tokyo, Japan).
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Results |
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RTPCR analysis of expression of IL-18, IL-18R and COX-II
RT-PCR analysis showed that endometriotic tissues of endometriosis patients expressed mRNAs of IL-18, IL-18 receptors (IL-18R, IL-18R
) and COX-II (Figure 5).
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Discussion |
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Endometriosis patients generally accumulate peritoneal fluids rich in macrophages in the pelvic cavity (Chacho et al., 1986; Harada et al., 1997
). Our fluorescence-activated cell sorting (FACS) analysis using FITC-labelled anti-human-CD14 antibody confirmed that the majority of cells in the peritoneal fluid of endometriosis patients were macrophages. We found that these macrophages express IL-18R
and produce COX-II in response to treatment with IL-18. We also showed the expression of IL-18 and IL-18R
in endometriotic tissue and found that COX-II was induced by IL-18 in endometriotic cells. In addition, COX-II was found to be expressed in CD68-positive cells, probably macrophages, infiltrating into endometriotic tissues. Finally, we detected caspase-1, which converts inactive IL-18 precursors to an active form (Gu et al., 1997
), in the stroma of endometriotic tissues (data not shown). These results suggest that functional IL-18 is generated in situ and induces COX-II, which would lead to the production of PGs, eventually causing pain.
Elevation of IL-18 levels in endometriosis is of interest from the standpoint of the production of autoantibodies in endometriosis, since IL-18 has been shown to enhance both Th1 and Th2 immune responses (Hoshino et al., 1999; Nakanishi et al., 2001
; Ogura et al., 2001
). With IL-12, IL-18 induces IFN-
as a Th1 cytokine, but without IL-12, it induces IL-10 as a Th2 cytokine (Nakanishi et al., 2001
; Ogura et al., 2001
). It has been shown that simultaneous treatment of systemic lupus erythematosis (SLE) model mice MRL/1pr with IL-18 and IL-12 exacerbates bufferfly facial rash but reduces autoantibody levels (Esfandiari et al., 2001
). On the other hand, treatment of these mice with IL-18 alone results in remission of facial rash and increases in autoantibody levels. It is possible that, in endometriosis, IL-18 may induce PGs in ectopic endometriotic cells, converting their environment into a Th2 dominant status to favour local autoimmunity leading to autoantibody production. In this way, IL-18 may play a crucial role in the pathogenic network in endometriosis.
In conclusion, our study shows for the first time that IL-18 might be a key cytokine in developing the pathogenesis of endometriosis. This raises the possibility that certain chemical agents which block IL-18 functions may be of use for the treatment of endometriosis patients.
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Acknowledgements |
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References |
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Submitted on October 22, 2002; resubmitted on September 22, 2003; accepted on November 12, 2003.