| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Department of Obstetrics and Gynecology (H.K., J.K., M.T., Y.K., H.I., H.Ho.), Graduate School of Medicine, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan; Institute of Bio-Response Informatics (H.O.), Kyoto 602-0845, Japan; Department of Clinical Chemistry, Kobe Pharmaceutical University (M.O.), Kobe 658-8558, Japan; and Laboratory of Clinical Pharmaceutics, Gifu Pharmaceutical University (H.Ha., T.A.), Gifu 502-8585, Japan
Address all correspondence and requests for reprints to: Dr. Jo Kitawaki, Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kamigyo-ku, Kyoto 602-8566, Japan. E-mail: kitawaki{at}koto.kpu-m.ac.jp.
 |
Abstract |
|---|
 Top Abstract IntroductionPatients and MethodsResultsDiscussionReferences |
|---|
(P < 0.05). These results demonstrate for the first time that AIF-1 is expressed in eutopic endometrium and endometriotic tissue, suggesting that AIF-1 is one cytokine in the local network involved in the onset of menstruation. AIF-1 derived from peritoneal macrophages may also possibly play a significant role in the pathophysiology and progression of endometriosis. |
Introduction |
|---|
|
TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
|---|
Endometriosis is defined as the presence of endometrial glands and stroma outside the uterine cavity, and it is common in women of reproductive age. Its exact pathogenesis is unclear; various factors are implicated, including estrogen dependency and inflammatory and immune responses (10, 11). Patients with endometriosis show increased levels of activated macrophages, lymphocytes, and various kinds of cytokines in the peritoneal fluid (12, 13).
In the present study we investigated the involvement of AIF-1 in menstruation and the pathophysiology of endometriosis through examination of AIF-1 expression in human eutopic endometrium and endometriosis. We also determined AIF-1 concentrations in peritoneal fluids from endometriosis patients and women without endometriosis. To investigate whether AIF-1 is one of the cytokines in a network involved in the pathophysiology of endometriosis, we also examined the relationship between AIF-1 secretion and other cytokines known to be elevated in the peritoneal fluid of women with endometriosis.
|
Patients and Methods |
|---|
|
TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
|---|
Eutopic endometrial tissue, ovarian endometriotic tissue, peritoneal fluid, and peripheral blood were obtained from women who had undergone laparotomy or laparoscopy at the Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine. This study protocol was approved by the Kyoto Prefectural University of Medicine institutional review board, and informed consent was obtained from each patient. All patients were of reproductive age, with normal menstrual cycles (mean ± SD, 28.7 ± 2.6 d; range, 25–34 d), and were not receiving any form of endocrine therapy, such as GnRH analog, danazol, or steroids. Participants were classified by disease type. Endometriosis, adenomyosis, and/or leiomyomas were diagnosed by histological examination using excised tissues or by laparoscopy findings. Women with cervical cancer in situ without other gynecological disease were defined as disease-free. However, women with malignant neoplasms other than cervical carcinoma in situ or with benign ovarian neoplasms, pelvic inflammation, or pregnancy were excluded from this study. A total of 60 women met the criteria for enrollment: 40 patients with endometriosis, 10 patients with adenomyosis and/or leiomyomas, and 10 disease-free patients. The mean age was 35.9 ± 5.5 yr (mean ± SD; range, 24–44 yr). There was no significant difference in mean age among the endometriosis, adenomyosis, leiomyoma, and disease-free groups.
Eutopic endometrial specimens were obtained from all patients according to chronological dating and were histologically adjusted according to the criteria of Noyes et al. (14). The menstrual cycle was divided into six phases: menstrual (d 1 to the last day of menstruation), midproliferative (the first day after menstruation to d 10), late proliferative (d 11–14), early secretory (d 15–19), midsecretory (d 20–24), and late secretory (d 25–28). An ovarian endometrioma, also known as a chocolate cyst, was resected for analysis from 30 patients. The severity of endometriosis was assessed according to the revised American Society for Reproductive Medicine (r-ASRM) scoring system (15). The endometriosis group was classified into r-ASRM stages of I–IV. Each stage consisted of 10 patients. Ectopic endometrial tissue samples were obtained from the inner walls of chocolate cysts. Peritoneal fluid was obtained from all patients and centrifuged at 800 x g for 15 min. The supernatant was stored at –80 C until use for ELISA, and the resulting cellular pellet was centrifuged through a Ficoll-Hypaque solution (Organon Teknika, Inc., Durham, NC) to isolate macrophages according to the manufacturer’s instructions. Peripheral blood was drawn before surgery, and serum was separated by centrifugation (1700 x g for 10 min) and kept at –80 C until used in the ELISA.
RNA isolation and RT-PCR
Total RNA was extracted from eutopic and ectopic endometrial tissue samples using TRIzol (Invitrogen Life Technologies, Inc., Carlsbad, CA), and the first strand cDNA synthesis from total RNA was catalyzed by the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen Life Technologies, Inc.) using oligo(deoxythymidine). The resulting first strand cDNA was used for PCR amplification with forward primer 5'-AGACGATCCCAAATATAGCAG-3' and reverse primer 5'-TAGCTCTAGGTGAGTCTTGG-3' for AIF-1 (4) and with the human glyceraldehyde-3-phosphate dehydrogenase (G3PDH) amplimer set for G3PDH (BD Clontech, Palo Alto, CA). The PCR mixture comprised 1 µl first strand cDNA, 0.8 µM of each of the primers mentioned above, and 1.25 U Taq polymerase (Takara Premix Ex Taq; Takara Biochemicals, Inc., Tokyo, Japan) in a total volume of 50 µl PCR buffer provided by the manufacturer. After an initial denaturation at 94 C for 5 min, PCR was carried out at 94 C for 45 sec, at 58 C (for AIF-1) or 50 C (for G3PDH) for 45 sec, and at 72 C for 1 min, followed by a final extension step at 72 C for 5 min. PCR products were electrophoresed in 2% agarose gel and stained with ethidium bromide. The intensity of each band was measured with a densitometer. Relative changes in mRNA expression were assessed by a semiquantitative RT-PCR method as described previously (16). The intensity of the transcript increased exponentially with up to 26 cycles for AIF-1 and 22 cycles for G3PDH, with each eventually reaching a plateau. We calculated the intensity of AIF-1 transcript at 26 cycles relative to that of G3PDH at 22 cycles to compare initial mRNA levels. Peritoneal macrophages were analyzed in the same manner. Negative controls were carried out containing no reverse transcriptase. No bands were detected. DNA bands were excised from the agarose gel and extracted using NucleoTrap Gel Extraction Kit (BD Clontech). Aliquots of PCR products were sequenced using a DNA analyzer (Applied Biosystems Japan, Inc., Tokyo, Japan).
Peptide synthesis and preparation for antihuman AIF-153–71 and AIF-1113–129 antibodies
Two synthetic peptides, which corresponded to residues 53–71 and 113–129 of human AIF-1 (AIF-153–71 and AIF-1113–129, respectively) as deduced from the nucleotide sequence of the human AIF-1 gene, were obtained with an additional cysteine residue at the N terminal (Biologica Co., Nagoya, Japan). After purification by reverse phase HPLC, the synthetic peptide (purity, >90%) was coupled to keyhole limpet hemocyanin with N-(
-maleimidocaproyloxy)succinimide (Sigma-Aldrich Corp., St. Louis, MO). The carrier-conjugated peptide was then emulsified with Freund’s complete adjuvant (Difco Laboratories, Inc., Detroit, MI) and injected sc (0.5 mg/injection) into rabbits six times at 10-d intervals. Blood samples were collected 10 d after the last injection. The specific antibody in the sera was purified using an AIF-1 peptide-coupled, cyanogen bromide-activated, Sepharose affinity column. The antibodies reacted with protein from abdominal adipose tissue and peripheral blood mononuclear cells that was identical in molecular size to purified recombinant human AIF-1.
Expression of recombinant AIF-1 and preparation of antirecombinant AIF-1 antibody
Human AIF-1 cDNA was amplified from human peripheral lymphocyte cDNA (BD Clontech) using PCR. The forward and reverse primers were 5'-GTGGATCCATGAGCCAAACCAGGGATTT-3' (containing the BamHI site) and 5'-CACTCGAGTCAGATAGGGCTTTCTTGGCT-3' (containing the XhoI site), respectively. To express AIF-1 as a glutathione-S-transferase fusion protein, the DNA fragment obtained was inserted into BamHI/XhoI sites of pGEX-4 (Amersham Biosciences, Piscataway, NJ) in-frame. The fusion protein was purified by a glutathione-S-transferase purification system (Amersham Biosciences) and affinity chromatography with anti-AIF-1113–129 antibody. Antirecombinant AIF-1 antiserum was raised in a similar manner as antisynthetic peptide antibody by injecting 50 mg recombinant human AIF-1 into a rabbit. The human AIF-1 antibody IgG fraction was prepared by chromatography on a recombinant human AIF-1-coupled cyanogen bromide-activated Sepharose affinity column and biotinylated with 5-(N-succinimidyloxycarbonyl)pentyl D-biotinamide (Dojindo Laboratories, Inc., Kumamoto, Japan).
Western blot analysis
Western blot analysis was performed as previously described (16) with modifications. Eutopic and ectopic endometrial specimens were homogenized, solubilized in lysis buffer (PBS, pH 7.4, containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and 0.57 mM phenylmethylsulfonylfluoride), and centrifuged. Thirty micrograms of each protein extract were electrophoresed in SDS-PAGE, electrotransferred to a polyvinylidene difluoride membrane, and incubated with antihuman AIF-153–71 antibody (1:2,000), followed by incubation with a second antibody conjugated with horseradish peroxidase (1:20,000; Amersham Biosciences). Immunoreactions were detected with enhanced chemiluminescence using ECL Plus Western blotting detection reagents (Amersham Biosciences). The intensity of each band was measured with a densitometer and expressed in arbitrary units. AIF-1 protein expression in peritoneal macrophages was analyzed in the same manner. Negative controls were carried out by incubating the membrane with either nonimmunized serum or only the second antibodies. No bands were detected.
Immunohistochemistry
Immunohistochemical staining was performed as described previously (17) with modifications. Paraformaldehyde-fixed eutopic and ectopic endometrial tissue samples were embedded in paraffin, cut into 4-µm sections, deparaffinized, autoclaved at 121 C for 20 min in 0.01 M citrate buffer (pH 6.0), and treated with 3% H2O2. After washing with 0.05 M Tris-HCl buffer (pH 7.6) containing 0.05% Tween 20, the sections were incubated with Dako Protein Block Serum-Free (DakoCytomation, Inc., Carpinteria, CA), then incubated for 24 h at 4 C with antihuman AIF-153–71 antibody diluted 1:200, washed, incubated with biotinylated antirabbit Ig, washed, and incubated with streptavidin conjugated to horseradish peroxidase (Amersham Biosciences). After washing, sections were colored with 3,3-diaminobenzidine hydrochloride solution and counterstained with hematoxylin. The intensity of immunostaining was evaluated by two independent observers using a semiquantitative index, H score = 
Pi, where i is the intensity of staining with a value of 0, 1, 2, or 3 (negative, weak, moderate, or strong, respectively), and P is the percentage of stained cells for each given i (range, 0–100%). The H score was determined by observing 20 glands or 200 stromal cells. Negative controls were incubated with the same dilution of nonimmunized rabbit serum. No staining was detected.
ELISA
The AIF-1 concentrations in peritoneal fluid and serum were measured by ELISA. Microtiter plates were coated with affinity-purified anti-AIF-1113–129 IgG (1.5 mg/ml) diluted with 10 mM carbonate buffer (pH 9.3) for 1 h at room temperature. After washing, nonspecific binding sites in each well were blocked with 10 mM carbonate buffer containing 0.5% BSA. Standard solution (0–2,500 pg/ml recombinant human AIF-1) and samples diluted 1:2 with sample buffer (50 mM Tris-HCl buffer, pH 7.0, containing 200 mM NaCl, 10 mM CaCl2, 0.1% Triton X-100, and 1% BSA) were added to the wells, and the plate was incubated for 1 h at room temperature. After washing with BSA-free sample buffer, biotinylated antirecombinant human AIF-1 IgG was added to each well. The plate was incubated for 1 h at room temperature, washed, and incubated for an additional 1 h at room temperature with streptavidin-horseradish peroxidase diluted 1:10,000. After final washing, the plate was treated for 20 min with a substrate solution of 3,3',5,5'-tetramethylbenzidine, and H2O2 was added to each well and allowed to react for 20 min at room temperature. The reaction was stopped by the addition of 1 M phosphoric acid, after which OD values at 450 nm were read with an ELISA plate reader. The detection limit was 25 pg/ml, and intra- and interassay coefficients of variation were 6.9–9.7% at 170 pg/ml and 4.9–7.1% at 600 pg/ml, respectively. IL-1ß, IL-6, and IL-8 concentrations were measured using the corresponding ELISA kits (Genzyme TECHNE Co., Minneapolis, MN) according to the manufacturer’s instructions.
Culture of peritoneal macrophages and endometriotic cells
Macrophages in peritoneal fluid were isolated through a Ficoll-Hypaque solution (Organon Teknika, Inc.) and grown in RPMI 1640 medium (Invitrogen Life Technologies, Inc.) containing 10% dextran-coated charcoal-treated fetal bovine serum (FBS) in a 24-well microplate at 37 C in a humidified atmosphere of 95% air-5% CO2. The purity of the macrophages was confirmed to be greater than 95% by ingestion of latex (18). After reaching confluence and incubation with FBS-free medium overnight, the macrophages were also incubated with or without 0.1–1.0 µg/ml lipopolysaccharide (Escherichia coli LPS; Sigma-Aldrich Corp.), 100-1000 U/ml interferon- (IFN; PeproTech EC, Inc., London, UK), or 0.01–10 ng/ml IL-1ß (Genzyme TECHNE Co.) in serum-free RPMI 1640 medium for 24 h.
Separation and culture of endometriotic cells were performed as described previously (19). Endometriotic tissue samples were digested with 0.25% collagenase (type 1; Sigma-Aldrich Corp.) in serum-free DMEM/Ham’s F-12 medium (Invitrogen Life Technologies, Inc.) at 37 C for 2 h. No attempt was made to separate glandular epithelial cells and stromal cells. Cells were cultured in DMEM/Ham’s F-12 medium containing 10% charcoal-treated FBS in 35-mm petri dishes in a humidified atmosphere of 95% air-5% CO2. After reaching confluence, the cells were also incubated with or without 0.1–1.0 ng/ml recombinant human AIF-1 in serum-free DMEM/Ham’s F-12 medium for 24 h. Each culture was performed in triplicate, and supernatants were separated by centrifugation (800 x g for 15 min) and kept at –80 C until assay.
For the proliferation assay, endometriotic cells were cultured in 96-well microplates at a concentration of 104 cells/well. After incubation with or without recombinant human AIF-1 for 48 h, the number of cells was estimated spectrophotometrically by the incorporation of tetrazolium dye using Cell Count Reagent SF (Nacalai Tesque, Inc., Kyoto, Japan). The reagent was added, and the cells were incubated for an additional 4 h, after which an OD value at 450 nm was determined using a microplate reader. All assays were performed in triplicate.
Statistics
Differences in mRNA and protein levels of AIF-1; peritoneal fluid concentrations of AIF-1, IL-1ß, and IL-6; and serum concentrations of AIF-1 among the patient groups were analyzed by nonparametric Mann-Whitney or Kruskal-Wallis test, followed by multiple comparisons using Dunn’s procedure. Correlations between the AIF-1 concentration in peritoneal fluid/serum and the r-ASRM score as well as between levels of AIF-1 mRNA and protein in ectopic endometrial specimens and the r-ASRM score were examined by Spearman’s correlation coefficient by rank test. Differences in age among the groups; in AIF-1 concentrations in the supernatants of cultured peritoneal macrophages; in IL-1ß, IL-6, and IL-8 concentrations in the supernatants of cultured endometriotic cells; and in the number of cultured endometriotic cells were analyzed by one-factor ANOVA, followed by multiple comparisons using Dunnett’s procedure. P < 0.05 was considered significant. Data are expressed as the median with the interquartile range or as the mean ± SEM.
|
Results |
|---|
|
TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
|---|
In RT-PCR analysis, AIF-1 transcript was detected in all 60 eutopic endometrial specimens. The sequences of these PCR products were confirmed by direct sequencing to be the same as that for human AIF-1 mRNA reported in the GenBank databank (U19713). With the use of semiquantitative RT-PCR, AIF-1 mRNA expression was significantly greater in the menstrual phase (P < 0.05) and the late secretory phase (P < 0.01) than in other phases of the menstrual cycle (Fig. 1A
). However, AIF-1 mRNA expression showed no significant difference among the three patient groups based on disease type: endometriosis, adenomyosis/leiomyomas, and disease-free (Fig. 1B).
|
). As with AIF-1 mRNA expression, protein expression in eutopic endometrial samples showed no difference among the various patient groups (Fig. 2B).
|
). The immunostaining intensity was greater in glandular cells than in stromal cells (P < 0.01). In glandular cells, H scores in the menstrual phase were significantly greater than those in the late proliferative phase, those in the early secretory phase, and those in the midsecretory phase (P < 0.05; Fig. 4A).
|
|
In RT-PCR analysis, AIF-1 transcript was detected in all 30 endometrioma specimens. With the use of semiquantitative RT-PCR, the level of AIF-1 mRNA expression showed no significant difference during the menstrual cycle (Fig. 1A
), nor was there any significant relationship between the level of AIF-1 mRNA expression and the r-ASRM score. With Western blot analysis, AIF-1 protein was detected in protein extracts from all endometrioma specimens. Protein expression showed no significant difference during the menstrual cycle (Fig. 2A), nor was there any significant relationship between the level of protein expression and the r-ASRM score. Immunostaining for AIF-1 was detected in the cytoplasm and nuclei of glandular cells and in the nuclei of stromal cells of all endometrioma specimens (Fig. 3). Immunostaining intensity was greater in glandular cells than in stromal cells (P < 0.01). However, there was no significant difference in immunostaining intensity during the phases of the menstrual cycle (Fig. 4B).
AIF-1 concentration in peritoneal fluid
The AIF-1 concentration in peritoneal fluid was elevated in endometriosis patients (median, 222 pg/ml; interquartile range, 118–325 pg/ml) compared with that in disease-free patients (113 pg/ml; 105–120 pg/ml; P < 0.01; Fig. 5A). Within the endometriosis group, peritoneal fluid AIF-1 level rose significantly with increase in r-ASRM stage (P < 0.001; Fig. 5A). The AIF-1 concentration in peritoneal fluid correlated with the progression of r-ASRM score (rs = 0.693; P < 0.0001; Fig. 6). IL-1ß (20, 21) and IL-6 (22, 23) levels are known to be elevated in patients with endometriosis. In the current study, peritoneal fluid IL-1ß (Fig. 5B) and IL-6 (Fig. 5C) concentrations were increased in patients with endometriosis (P < 0.01) and with progression of r-ASRM stage (P < 0.05 for IL-1ß and P < 0.01 for IL-6) in a manner parallel that seen for AIF-1 concentrations. However, there was no significant difference in the peritoneal fluid AIF-1 concentration during the phases of the menstrual cycle. By contrast, the serum concentration of AIF-1 showed no significant difference among the three patient groups (disease-free: 16.0 pg/ml, 12.0–19.0 pg/ml; adenomyosis and/or leiomyomas, 20.5 pg/ml, 12.7–23.0 pg/ml; endometriosis, 15.0 pg/ml, 8.2–22.7 pg/ml) or during the different phases of the menstrual cycle.
|
|
In peritoneal macrophages, intense expression of AIF-1 mRNA (Fig. 1A) and protein (Fig. 2A) was observed. When peritoneal macrophages were isolated and cultured, basal secretion of AIF-1 was greater in women with endometriosis than in those without the disease (P < 0.05). The AIF-1 concentration in the culture supernatant increased approximately 8-fold from the basal level after stimulation for 24 h by IL-1ß in a dose-dependent manner (P < 0.01). To a lesser extent (but still significant), stimulation of AIF-1 release was produced by addition of IFN (P < 0.05). However, AIF-1 secretion was not stimulated by the addition of LPS (Fig. 7). The degree of IL-1ß stimulation was similar for peritoneal macrophages obtained from disease-free patients (Fig. 7A) and patients with endometriosis (Fig. 7B).
|
) or the culture supernatant. The addition of IL-1ß and IFN failed to induce AIF-1 to a detectable level. Similarly, the secretion of IL-1ß, IL-6, and IL-8 from cultured endometriotic cells did not change with the addition of recombinant human AIF-1 (Fig. 8). The addition of AIF-1 did not significantly change the number of cultured endometriotic cells.
|
|
Discussion |
|---|
|
TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
|---|
Cytokines are one of the key factors involved in the pathogenesis, pathophysiology, and progression of endometriosis. Peritoneal fluid obtained from women with endometriosis contains leukocytes, of which approximately 85% are macrophages (30). Furthermore, cell number and activity to secrete cytokines are increased in endometriosis. In the peritoneal fluid of women with endometriosis, concentrations of cytokines, including IL-1ß (20, 21), IL-6 (22, 23), IL-8 (31, 32), IL-10 (33, 34), TNF
(21, 35), and VEGF (36), are increased, and at least some of them are correlated with the clinical severity of endometriosis. Activated macrophages are considered to be the dominant source of cytokines in peritoneal fluid. These cytokines are considered to be involved in macrophage activation, inflammatory change, and enhanced angiogenesis. Peritoneal macrophages obtained from women with endometriosis secrete more IL-1ß, IL-6, IL-8, IL-10, and TNF- than macrophages from women without endometriosis (32, 37, 38), and the secretion of IL-6, IL-8, IL-10, IL-12, and TNF is stimulated by LPS (32, 39). The present study shows that the concentration of AIF-1 in peritoneal fluid is elevated in patients with endometriosis, and the AIF-1 concentration correlates with the severity of endometriosis, a pattern parallel to those observed for peritoneal concentrations of IL-1ß and IL-6. The present study demonstrates that AIF-1 is released from peritoneal macrophages and that release is higher from macrophages obtained from patients with endometriosis than those obtained from patients without endometriosis. The release of AIF-1 is stimulated by IL-1ß and IFN. Taken together, IL-1ß may stimulate AIF-1 secretion from peritoneal macrophages. The primary increase in IL-1ß secretion may result in the elevated concentrations of AIF-1 and IL-6 in the peritoneal fluid of women with endometriosis. Because there was no significant difference in peritoneal fluid AIF-1 concentration during the phases of the menstrual cycle, it is unlikely that the major source of peritoneal fluid AIF-1 is endometrial fragments that reach the peritoneal cavity through retrograde menstruation.
Endometriotic tissues also secrete various cytokines. Ectopic and eutopic endometrial cells from women with endometriosis produce more IL-6 than endometrial cells from women without endometriosis (24). In cultured endometriotic cells, IL-1ß stimulates the production of IL-6 (24, 40), VEGF (41), and IL-8 (42). TNF- promotes the proliferation of endometriotic stromal cells by inducing IL-8 gene and protein expression (43). These cytokines are thought to be involved in the neovascularization that surrounds endometriotic lesions (41). The present study demonstrates that AIF-1 transcript and protein are expressed constitutively in both the glandular and stromal cells of endometriotic tissue. In contrast to the pattern seen in eutopic endometrium for other cytokines and steroid-related markers, the level of AIF-1 expression in endometriotic tissue showed no significant difference during the menstrual cycle, nor was there any significant relationship between the level of AIF-1 mRNA expression and clinical severity (e.g. r-ASRM score). AIF-1 did not affect the proliferation of cultured endometriotic cells. However, unlike the pattern for other cytokines, we failed to detect AIF-1 release from cultured endometriotic cells even after stimulation by other cytokines. One reason for this finding may be that AIF-1 acts at a transcriptional level and is not secreted outside cells (1). However, because we failed to detect AIF-1 transcript in cultured endometriotic cells despite documented secretion of other cytokines, the other possibility is that the ability to secrete AIF-1 was lost during the culture process.
Interestingly, Brauner et al. (44) reported an elevated concentration of AIF-1 in the peritoneal fluid of subjects with peritonitis developed during continuous ambulatory peritoneal dialysis. This may imply that AIF-1 is secreted from peritoneal macrophages in nonspecific inflammatory conditions, including peritonitis and endometriosis. However, peritoneal macrophages from women with endometriosis have been shown to share specific characteristics with those from women without endometriosis, as we stated above. In addition, peritoneal macrophages are different from those in peripheral blood. Although the present study showed that AIF-1 release from cultured peritoneal macrophages was stimulated by IL-1ß and IFN, secretion from a macrophage cell line derived from peripheral blood was induced by live bacteria, but not by IL-1ß or IFN (44). AIF-1 was induced by IL-1ß in human vascular smooth muscle cells at the transcriptional level (3) and was induced by IFN within cells of mouse macrophage cell lines and rat bone marrow-derived macrophages (1). Nevertheless, AIF-1 was not detected in extracts from the supernatant of IFN-stimulated cells (1). The reasons for these differences remain to be solved.
In conclusion, we have demonstrated AIF-1 expression in human eutopic endometrium with an increase in expression around the time of menstruation, indicating that AIF-1 is involved in its onset. AIF-1 is expressed in human endometriotic tissue, and the AIF-1 concentration in the peritoneal fluid of patients with endometriosis is elevated and correlated with the clinical severity of endometriosis. Peritoneal macrophages obtained from patients with endometriosis secrete more AIF-1 than those obtained from patients without endometriosis. AIF-1 release is stimulated by IL-1ß and IFN. Our results suggest that AIF-1 is a novel member of the cytokine network involved in the pathophysiology and progression of endometriosis. However, the membrane receptor for AIF-1 has not yet been identified. Additional studies are needed, because little is currently known about the molecular mechanism of AIF-1 activity.
|
Footnotes |
|---|
First Published Online October 26, 2004
Abbreviations: AIF-1, Allograft inflammatory factor-1; FBS, fetal bovine serum; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; IFN, interferon-; LPS, lipopolysaccharide; VEGF, vascular endothelial growth factor.
Received May 10, 2004.
Accepted October 20, 2004.
|
References |
|---|
|
TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
|---|
by human peritoneal macrophages in vivo and in vitro. Am J Obstet Gynecol 161:1718–1725[Medline]
(IFN-) and interleukin-6 (IL-6) in peritoneal fluid and macrophage-conditioned media of women with endometriosis. Am J Reprod Immunol 32:180–183
, and IL-2 in peritoneal fluid and macrophage-conditioned media of women with endometriosis. Am J Reprod Immunol 34:381–385
promotes proliferation of endometriotic stromal cells by inducing interleukin-8 gene and protein expression. J Clin Endocrinol Metab 85:824–829This article has been cited by other articles:
 |
|
 |
|
  S. H. Kim, H. W. Lee, Y. H. Kim, Y. H. Koo, H. D. Chae, C. H. Kim, P. R. Lee, and B. M. Kang Down-regulation of p21-activated kinase 1 by progestin and its increased expression in the eutopic endometrium of women with endometriosis Hum. Reprod., May 1, 2009; 24(5): 1133 - 1141. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Kimura, Y. Kawahito, H. Obayashi, M. Ohta, H. Hara, T. Adachi, D. Tokunaga, T. Hojo, M. Hamaguchi, A. Omoto, et al. A Critical Role for Allograft Inflammatory Factor-1 in the Pathogenesis of Rheumatoid Arthritis J. Immunol., March 1, 2007; 178(5): 3316 - 3322. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
, Glandular cells; 
, stromal cells. Mens, Menstrual phase; MP and LP, midproliferative and late proliferative phases; ES, MS, and LS, early, mid, and late secretory phases, respectively. Each phase represents data for five to 13 cases. In both eutopic endometrial tissue and endometriotic tissue, the intensity was greater in glandular cells than in stromal cells (P < 0.01, as analyzed by Mann-Whitney test). a vs. b, P < 0.05 (as analyzed by Kruskal-Wallis test, followed by multiple comparisons using Dunn’s procedure). The box plot includes the median (horizontal line) and interquartile range (box), whereas the whiskers represent the 10–90th percentiles.
), and IL-8 (
) by cultured endometriotic cells in the presence or absence (Control) of 0.1–1.0 ng/ml human recombinant AIF-1. Each bar represents the mean ± SEM of six experiments. AIF-1 did not affect the production of ILs.