Hum. Reprod. Advance Access originally published online on July 1, 2008
Human Reproduction 2008 23(10):2210-2219; doi:10.1093/humrep/den195
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Toll-like receptor 4-mediated growth of endometriosis by human heat-shock protein 70
1 Department of Obstetrics and Gynecology, Graduate School of Biomedical Sciences, Nagasaki University, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan 2 Nagasaki Municipal Hospital, Nagasaki, Japan 3 Department of Molecular Pathology, Atomic Bomb Disease Institute, Nagasaki University, Nagasaki, Japan 4 Sasebo Chuo Hospital, Nagasaki, Japan
5 Correspondence address. Tel: +81-95-819-7363; Fax: +81-95-819-7365; E-mail: nemokhan{at}nagasaki-u.ac.jp
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Abstract |
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BACKGROUND: We investigated the role of human heat-shock protein 70 (Hsp70) in Toll-like receptor 4 (TLR4)-mediated growth of endometriosis.
METHODS: TLR4 expression was examined in macrophages (M
) isolated in primary culture from the peritoneal fluid of women with and without endometriosis. The production of a number of macromolecules by non-treated M, Hsp70-treated M and after treatment with anti-TLR4 antibody was examined by enzyme linked immunosorbent assay (ELISA). The single and combined effects of Hsp70 and lipopolysaccharide (LPS) on the growth of endometrial stromal cells were analyzed by 5-bromo-2-deoxyuridine (BrdU) incorporation study. Hsp70 levels in eutopic and ectopic endometria were measured by ELISA.
RESULTS: TLR4 was detected in isolated M at protein and gene level. Hsp70 (10 µg/ml) significantly stimulated the production of hepatocyte growth factor, vascular endothelial cell growth factor, interleukin-6 and tumor necrosis factor alpha by M derived from women with endometriosis compared with M derived from women with no endometriosis (P < 0.05 for each). This effect of Hsp70 was abrogated after pretreatment of M with anti-TLR4 antibody. BrdU incorporation indicated that Hsp70 significantly enhanced the growth of endometrial stromal cells (
50% increase) from women with endometriosis compared to non-treated cells. A synergistic effect on cell proliferation was observed between exogenous Hsp70 and LPS and this was significantly suppressed by pretreatment of cells with anti-TLR4 antibody (P < 0.05). Tissue levels of Hsp70 were significantly higher in the eutopic endometria (P < 0.05) and opaque red lesions (P < 0.01) derived from women with endometriosis than from other peritoneal lesions or from women with no endometriosis.
CONCLUSIONS: A prominent stress reaction was observed in blood-filled opaque red peritoneal lesions. Human Hsp70 induces pelvic inflammation and may be involved in TLR4-mediated growth of endometrial cells derived from women with endometriosis.
Key words: cell growth/endometriosis/TLR4/Hsp70/LPS/macrophages
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Introduction |
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Endometriosis induces a variable amount of inflammatory reaction in the pelvic environment depending on the stage and morphology of the disease (Halme et al., 1987
; Harada et al., 2001; Lebovic et al., 2001; Khan et al., 2003; Wira et al., 2005). The inflammatory reaction associated with endometriosis was demonstrated both in vitro and in vivo by the infiltration of immune cells and the presence of a number of primary and secondary inflammatory mediators in tissue and body fluids (Halme et al., 1988, 1989; Keenan et al., 1994, 1995; Osuga et al., 1999; Mahnke et al., 2000; Khan et al., 2002a,b, 2004a, 2005a, 2007). The primary inflammatory mediators derived from Gram-negative and -positive microbes can elicit an immune response in the pelvic environment through pattern-recognition receptors, which belong to the family of Toll-like receptors (TLRs) (Akira and Takeda, 2004; Takeda and Akira, 2005; Khan et al., 2007). Other secondary inflammatory mediators that are produced in response to primary inflammatory mediators interact with their respective receptors in immune cells or endometrial cells and may be involved in the growth of endometriosis either alone or in combination (Halme et al., 1987; McLaren et al., 1996; Harada et al., 2001; Khan et al., 2005a,b,c). There is a possibility that, in addition to the pelvic inflammation, endometriosis may also produce a stress reaction and release endogenous heat-shock proteins (Hsps) in the pelvic environment as a result of tissue damage, tissue invasion and by inflammatory reaction itself. However, studies are limited regarding immune cell-mediated regulation of pelvic endometriosis by human Hsp, their mechanism of action and levels in the pelvic environment.
A wide variety of stressful stimuli, such as heat shock, ultraviolet radiation, viral or bacterial infections, internal physical stress, chemical stress and pelvic inflammation, induce an increase in the intracellular synthesis of Hsps (Zugel and Kaufmann, 1999; Asea et al., 2000, 2002). Mammalian Hsp60, Hsp70 and Hsp90 have been implicated in a variety of autoimmune and inflammatory conditions (Wallin et al., 2002). The so-called ‘danger theory’ states that antigen presenting cells can be activated by endogenous substances released by damaged or stressful tissues (Matzinger, 1998). Members of the Hsp family are candidate molecules that potentially signal tissue damage or cellular stress to the immune system.
Human Hsps (Hsp60, Hsp70 and Hsp90) are reported to be produced by macrophages (M), vascular endothelial cells, smooth muscle cells, endometrial cells and other dendritic cells (Wallin et al., 2002). In a manner similar to the recognition of lipopolysaccharide (LPS), recognition of Hsp60 and Hsp70 seems to be mediated by a complex of TLR4 and MD-2 (Kol et al., 1999; Wallin et al., 2002). Since the biological potentiality of human Hsp70 is stronger than that of either Hsp60 or Hsp90 (Wallin et al., 2002), we report here TLR4 expression in M derived from the peritoneal fluid (PF) of women with and without endometriosis and TLR4-mediated growth of pelvic endometriosis in response to human Hsp70. We also examined the pattern of stress reaction by measuring endogenous Hsp70 concentration in the eutopic endometria and different peritoneal lesions of women with pelvic endometriosis.
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Subjects
A total of 25 women between 20 and 38 years of age undergoing laparoscopy for pelvic pain, dysmenorrhea and/or infertility were recruited in this study. Among them, 12 women had endometriosis of Stages I–II and the remaining 13 women had endometriosis of Stages III–IV at the time of diagnostic laparoscopy. The control group consisted of 12 fertile women between 21 and 36 years of age without any evidence of pelvic or ovarian endometriosis and operated for dermoid cysts by laparoscopy. The staging and the morphological distribution of peritoneal lesions were based on the revised classification of The American Society of Reproductive Medicine (r-ASRM) (ASRM, 1997
). Neither the study group nor the endometriosis free group had been on hormonal medication in 3 months prior to the surgical procedure. All control women and women with endometriosis had regular menstrual cycles (28–32 days). The phase of the menstrual cycle was determined by histological dating of eutopic endometrium samples taken simultaneously with the PF samples. Menstrual dating was carried out by an independent pathologist. All induced menstrual cycles were excluded from the current study. The distribution of patients in different menstrual cycles is as follows: control women, proliferative phase, n = 4; secretory phase, n = 8; endometriosis women, proliferative phase, n = 10; secretory phase, n = 15.
Peritoneal lesions of endometriosis were diagnosed by their macroscopic appearances according to the published criteria (Jansen and Russel, 1996) and categorized as red, black and white lesions as proposed in the latest revision of the ASRM classification (ASRM, 1997). As we described recently (Khan et al., 2004b), the distribution of peritoneal lesions according to color appearance for our current study was done as follows: total red lesions (n = 20) [blood-filled opaque red lesions (n = 8) and non-opaque transparent and/or translucent red lesions (n = 12)]; black lesions (n = 21), and white lesions (n = 11). Biopsy specimens from each of these peritoneal lesions were collected for subsequent experimental analysis. The details of physical collection of tissue biopsies and the PF were reported elsewhere (Khan et al., 2002b, 2004a,b, 2006).
All biopsy specimens and the PF were collected in accordance with the guidelines of the Declaration of Helsinki and with the approval by the Nagasaki University Institutional Review Board. An informed consent was obtained from all women.
Isolation of the M from the PF
The PF was obtained from all women with or without endometriosis with the use of laparoscopy. The M were isolated in primary culture from the PF of six women with and without endometriosis. The detailed procedure of M isolation in primary culture was described previously (Khan et al., 2005a,b). The M were allowed to adhere to the culture plate for 2 h, after which the non-adherent cells were removed by washing the plates three times with RPMI medium. The adherent cells remaining on the plates were more than 95% M as estimated by their morphology and by immunocytochemical staining using CD68 (KP1), a mouse monoclonal antibody from Dako, Denmark. An aliquot of M was plated in four-well chamber slides (Nunc, Naperville, IL, USA) for immunostaining and the rest were used for culture. The detailed procedure of immunocytochemical staining is described elsewhere (Rana et al., 1996; Khan et al., 2003, 2004a). Non-immune mouse immunoglobulin (Ig) G1 antibody in 1:50 dilution was used as a negative control. A counter staining of M with hematoxylin–eosin was also performed and we did not find any contaminating cells, such as gland cells or stromal cells in isolated M (data not shown).
Isolation of stromal cells in primary culture
Stromal cells were collected from the biopsy specimens of the eutopic endometria derived from six women with and without endometriosis. The detailed procedure of the isolation of stroma is described previously (Osteen et al., 1989; Sugawara et al., 1997; Khan et al., 2005c).
The characteristics of the cultured stromal cells were determined by morphological and immunocytochemical studies. An aliquot of stromal cells was placed in a four-chamber slide (Nunc) for immunostaining and the rest was used for culture. After 24 h, the slides were washed in PBS, fixed with 4% paraformaldehyde for 10 min and rinsed with PBS. The slides then were incubated in 0.1% Triton X-100 for 5 min and were incubated for 3 h at 37°C as follows: against human cytokeratin monoclonal antibodies (mAb) (epithelial cell specific) at a dilution of 1:50 (MNF 116;Dako, Denmark), against human vimentin mAb (stromal cell specific) at a dilution of 1:20 (V9; Dako), against human von Willebrand factor mAb (endothelial cell specific) at a dilution of 1:50 (Dako) and against CD45 mAb (other leukocytes) at a 1:50 (Dako) dilution. The specificity of the immunocytochemical staining was confirmed by the deletion of the first antibody. Immunocytochemical staining was performed on at least three different isolated cells with similar results. The purity of stromal preparation was more than 95%, as judged by positive cellular staining for vimentin.
Treatment of M and stromal cells
The isolated peritoneal M were cultured in triplicate (105cells/well) for 24 h to assess basal (constitutive) production of cytokines. To evaluate the stimulated (induced) secretion of cytokines, after initial culture with serum containing RPMI medium, M were serum starved for 24 h and then serum free M were cultured for another 24 h with different concentrations of highly purified recombinant human Hsp70 (1, 5, 10, 15 and 20 µg/ml) (low endotoxin, ESP-555, Stressgen, Victoria, Canada). A blocking experiment was performed with anti-TLR4 antibody (10 µg/ml) (HTA-125, HyCult Biotechnology) 20 min prior to the treatment with recombinant human Hsp70 (10 µg/ml) in order to examine any change in the secretion of cytokines and growth factors in culture media without washing the pre-incubated antibodies. After 24 h, the cultured media were collected in triplicate, pooled and frozen at –70°C until testing. Possible contamination of endotoxin with Hsp70 was examined by measuring endotoxin levels in the culture media by the limulus amoebocyte lysate test (Endotoxin-Single Test; Wako-Jun-Yaku Co. Ltd, Tokyo, Japan), pre-treatment of cells with polymyxin B (1 µg/ml, Sigma), an LPS antagonist and by heat treatment (65°C) of Hsp70-treated cells.
Next, to examine the direct effects of recombinant Hsp70 (1, 5 and 10 µg/ml) and LPS (10 ng/ml) derived from Escherichia coli (serotype 0111:b4; Sigma, St Louis, MO, USA) on the proliferation of endometrial stromal cells, 104cells/ml were plated in 96-well microtitre plate and treated with various doses of recombinant Hsp70 either alone or in combination with LPS (10 ng/ml) in serum free RPMI medium and incubated for another 24 h. The neutralizing effects of polymyxin B (1 µg/ml) and anti-TLR4 antibody (10 µg/ml) on stromal cell growth were examined.
The production of macromolecules in the culture media of basal and stimulated M and cell proliferation assay were studied in six women with and without endometriosis (three each in proliferative phase and three in secretory phase).
Cytokine assays in the culture media of M
The culture media of basal (non-treated) and stimulated (treated with Hsp70) M were prospectively collected in triplicate and assays were performed retrospectively. The concentrations of hepatocyte growth factor (HGF), vascular endothelial cell growth factor (VEGF), interleukin (IL)-6 and tumor necrosis factor alpha (TNF
) in the culture media were measured in duplicate using a commercially available sandwich enzyme linked immunosorbent assay (ELISA) developed by R&D system in a blind fashion (Quantikine, R&D system, Minneapolis, MN, USA). The antibodies used in HGF, VEGF, IL-6 and TNF determinations do not cross-react with other cytokines. The limits of detection were 40.0 pg/ml for HGF, 9.0 pg/ml for VEGF, 0.70 pg/ml for IL-6 and 4.4 pg/ml for TNF. Both the intra- and inter-assay coefficients of variation were <10% for all these assays.
Immunolocalization of TLR4 in M
In order to immunolocalize TLR4 in the CD68-immunoreactive isolated M, we performed immunocytochemical staining of TLR4 using corresponding antibody (HTA-125, 1:50, Santa Cruz). The immunoreaction of TLR4 was examined in M derived from women with or without endometriosis. The detailed procedure of immunocytochemistry was described previously (Fujishita et al., 1997; Nisolle et al., 1997; Khan et al., 2005a,b). Non-immune mouse Ig G1 antibody (1:50) was used as a negative control.
Western blotting
Cultured cells in six-well plates were homogenized in the lysis buffer containing 50 mM Tris–HCl (pH 6.8), 2% sodium dodecyl sulfate, 10% glycerol, 50 mM dithiothreitol, and 0.1% bromophenol blue and were diluted to 1 mg total protein per milliliter. Plasma lysates and total cell lysates of M and Ramos, a B-lymphoma cell line were resolved in 8% SDS–PAGE. The procedure in the preparation of plasma lysates and total cell lysates was described elsewhere (Crawford et al., 1982). Proteins were blotted onto a nitrocellulose membrane and incubated with a rabbit antibody to TLR4 (1:300) as a primary antibody and an anti-rabbit antibody (1:1000; Amersham Pharmacia Biotech, UK) as a secondary antibody. Immune complexes were visualized by the use of an enhanced chemiluminescence western blotting system (Amershan Pharmacia Biotech).
Gene expression of TLR4 and HGF in M
Ribonucleic acid (RNA) was extracted from the cultured M in 60 mm petridish (Greiner) using the monophasic solution of 40% phenol and ISOGEN method (Molecular Research Center, Tokyo), according to the manufacturer's protocol.
The expression of TLR4 was analyzed in the M derived from women with or without endometriosis. The presence of mRNA encoding TLR4 in the basal M was determined using forward and reverse primers synthesized to anneal with cDNA for TLR4. Amplification of cDNA reaction mixture for TLR4 was done. The mRNA expressions of TLR4 were analyzed by RT-PCR and using the sense and anti-sense primers of TLR4 as described previously (Hirata et al., 2005). PCR generated bands were cloned and found to match the published sequences for the expected products.
The mRNA expression of HGF in response to Hsp70 and anti-TLR4 antibody was also examined by standard RT-PCR and using sense and anti-sense primers as described previously (Khan et al., 2005a,b,c). A scanner densitometer was used to determine the ratio of intensity of each band relative to β-actin that was used as an internal control. Autoradiographs were analyzed to quantify the differences in levels of transcripts between Hsp70-treated and non-treated samples derived from the control women and women with endometriosis. Values of each transcript after the treatment with Hsp70 were normalized to 1. Densitometric analysis of gel bands was performed using the National Institutes of Health image analysis program.
Real-time quantitative PCR was performed as reported previously (Koga et al., 2000). To assess TLR4 and HGF mRNA expression, real-time quantitative PCR and data analysis were performed using Light Cycler (Roche Diagnostics GmbH, Mannheim, Germany). Expression of TLR4 and HGF mRNA was normalized to RNA loading for each sample using β-actin mRNA as an internal control. The primers for TLR4, HGF and β-actin were the same as those used for standard PCR. PCR conditions were as follows: for TLR4, 40 cycles at 95°C for 10 s, 64°C for 10 s and 72°C for 12 s; for HGF, 40 cycles at 95°C for 10 s, 64°C for 10 s and 72°C for 12 s; for β-actin, 30 cycles at 95°C for 10 s, 60°C for 10 s and 72°C for 10 s. All PCR conditions were followed by the melting curve analysis.
Each PCR product was purified with a QIAEX II gel extraction kit (QIAGEN), and their identities were confirmed using an ABI PRISMTM 310 genetic analyzer (Applied Biosystems, Foster city, CA, USA).
Cell proliferation assays
5-Bromo-2-deoxyuridine (BrdU) can be used to measure cell proliferation by quantifying BrdU incorporated into the newly synthesized DNA of replicating cells (Takagi, 1993; Khan et al., 2005a,b). The incorporated BrdU can be detected by a quantitative cellular enzyme immunoassay (Biotrak, Amersham Pharmacia Biotech Ltd, UK) using monoclonal antibodies directed against BrdU. It offers a non-radioactive alternative to the [3H]-thymidine-based cell proliferation and carries equal sensitivity and specificity (Takagi, 1993). The detailed procedure of BrdU incorporation assay was described previously (Khan et al., 2005a,b,c). We examined the proliferation of endometrial stromal cells in response to Hsp70, LPS, polymyxin B and anti-TLR4 antibody and the differences in cell proliferation were expressed as the percentage of controls. The absorbance values correlated directly with the amount of DNA synthesis and thereby with the number of proliferating cells in culture.
Endogenous Hsp70 assays in tissue extracts
A fraction of biopsy specimen from eutopic endometria of women with and without endometriosis and from different peritoneal lesions of women with endometriosis was homogenized in homogenizing buffer using a Polytron homogenizer (Kinematics, Luzern, Switzerland) and according to the procedure described previously (Miura et al., 2006). The respective tissue suspension was centrifuged at 1500 rpm for 5 min to obtain the supernatant and stored at –80°C for the subsequent measurement of endogenous human Hsp70.
The tissue concentrations of human Hsp70 in the homogenized supernatant were measured in duplicate using a commercially available sandwich enzyme-linked immunosorbent assay (StressXpressTM, EKS-700, Stressgen, Victoria, Canada) according to the manufacturer's instructions. The protein concentration of samples was measured by the method of Bradford (1976) to standardize Hsp70 levels.
The antibodies used in Hsp70 determination do not cross-react with other cytokines. The sensitivity of this assay kit has been determined to be 200 pg/ml. Both the intra- and inter-assay coefficients of variation were <10% for this assay. The tissue concentration of Hsp70 was expressed as nanogram per microgram protein.
Statistical analysis
The clinical characteristics of the subjects were evaluated by one-way analysis of variance. The data are expressed as either mean ± SEM or mean ± SD. The concentrations of the studied cytokines were not distributed normally and the data were analyzed using non-parametric test. The differences between endometriosis and non-endometriosis, red lesions and other peritoneal lesions, Hsp70- or LPS-treated and non-treated groups were compared using Mann–Whitney U-test or Student's t-test. For comparison among three or more groups, the Kruskal–Wallis test was used to determine the difference among the groups. P < 0.05 was considered statistically significant.
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There were no significant differences in clinical characteristics between women with and without endometriosis (data not shown). As an initial study, we also examined five women with endometriosis but without infertility. We did not find any differences in cytokine profile or cell growth in response to Hsp70 in these five women compared with the women with endometriosis and infertility. Therefore, we represented our data only in women with infertility.
Expression of TLR4 in M
We detected both protein and gene expression of TLR4 in M, endometrial stromal cells and epithelial cells and also in eutopic and ectopic endometria derived from women with and without endometriosis. TLR4 was immunolocalized in CD68-positive M (Fig. 1A), vimentin-positive stromal cells and cytokeratin-positive epithelial cells. A parallel expression of TLR4 was also found in the glandular epithelial cells and stromal cells derived from both eutopic and ectopic endometria of women with and without endometriosis (data not shown). A 78 kDa molecular size of TLR4 was also visualized by western blot analysis in M and this was prominent in total cell lysates (Lane 3 for Ramos cells and Lane 4 for M) (Fig. 1B). This was confirmed at the mRNA levels (406 bp) in basal M derived from the PF of women with and without endometriosis (Fig. 1C). The amount of TLR4 mRNA was dose-dependently increased in basal M with a maximum amount found at 24–48 h of incubated cells. Although an apparent increase in the amount of TLR4 mRNA was found in basal M derived from women with endometriosis, there was no significant difference in TLR4 expression between women with and without endometriosis (Table I). The protein and gene expression of TLR4 in endometrial cells were reported elsewhere (Young et al., 2004; Hirata et al., 2005; Khan et al., 2005d,e, 2007).
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TLR4-mediated production of different cytokines by human Hsp70-treated M
In our initial time-dependent and dose-dependent study, we found a maximum increase in the levels of different macromolecules and cell growth at 24–48 h and in response to 10–15 µg/ml of Hsp70. Therefore, we obtained all current experimental data in response to 10 µg/ml of Hsp70 with a treatment duration of 24 h. We found that the concentrations of HGF, VEGF, IL-6 and TNF
were significantly higher in the culture media of Hsp70-treated M than that in non-treated M (P < 0.01 or < 0.05 for each, Fig. 2). The levels of these cytokines and growth factors were also markedly higher in M derived from women with endometriosis when compared with control women without endometriosis (P < 0.05 for each, Fig. 2). When we pre-treated M with antibody against TLR4, then again treated them with Hsp70, the levels of all these cytokines and growth factors were significantly decreased in comparison with cells without antibody (P < 0.05, for each of HGF, VEGF, IL-6 and TNF) (Fig. 3). This effect was observed in M derived from women with endometriosis but not from control women. No difference in the production of these macromolecules was observed between M collected during the proliferative phase and the secretory phase (data not shown).
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TLR4-mediated gene expression of HGF by human Hsp70
We found that gene expression of HGF in response to Hsp70 is stronger in M
derived from women with endometriosis than from women without endometriosis (Fig. 4A). Although a dose-dependent increase in gene expression of HGF (505 bp) was observed in both these two groups of women, a significant and a 2-fold increase in mRNA expression of HGF was found in women with endometriosis (P < 0.05) compared with women without endometriosis in response to Hsp70 (Fig. 4B). When we pre-treated M with antibody against TLR4, then again treated them with Hsp70, HGF mRNA expression level was significantly decreased in comparison with cells without antibody (P < 0.05, Fig. 4B). When we examined the difference in the amount of HGF mRNA in response to Hsp70 and anti-TLR4 antibody by RT-PCR, results in M were almost parallel to those obtained by standard RT-PCR (Table II). This indicates that the stimulating effect of Hsp70 on the production of HGF and other macromolecules is mediated by TLR4.
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Exclusion of endotoxin contamination of Hsp70-treated cells
In order to exclude the possible contamination by endotoxin of Hsp70-treated cells, both M
and endometrial stroma, we repeatedly measured endotoxin level in the culture media. We could not detect any endotoxin in the culture media of Hsp70-treated M or Hsp70-treated stromal cells. Pre-treatment of M with polymyxin B (1 µg/ml) failed to decrease the levels of any of these macromolecules in the culture media of Hsp70-treated cells (data not shown). Since LPS is heat stable and Hsp70 is heat labile (Wallin et al., 2002), we further excluded endotoxin contamination by heat treatment (65°C) of Hsp70-treated cells for 20 min. We could not detect any cytokine or growth factor in the culture media of Hsp70-treated cells, possibly due to degradation of Hsp70 after heat treatment.
Proliferation of stromal cells by Hsp70 and LPS
A BrdU incorporation study indicated that stromal cells derived from eutopic endometria of women with endometriosis proliferated dose-dependently and significantly in response to Hsp70 (P < 0.05 at 5 and 10 µg/ml versus non-treated cells, Fig. 5A). No significant difference was observed between treated and non-treated cells derived from women without endometriosis.
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We also found that the individual treatment with Hsp70 (10 µg/ml) or LPS (10 ng/ml) was able to significantly stimulate proliferation of stromal cells derived from eutopic endometria of women with endometriosis (1.5- to 1.7-fold increase) compared with non-treated cells (P < 0.05 for each, Fig. 5B). A synergistic effect in cell proliferation was observed between Hsp70 and LPS. In fact, combined treatment of stromal cells with Hsp70 and LPS further increased BrdU incorporation when compared with cells treated with Hsp70 alone (P < 0.05, Fig. 5B). In order to confirm the cellular specificity of LPS, we treated cells with polymyxin B (1 µg/ml) and an LPS antagonist. We found that polymyxin B significantly abrogated LPS-promoted cell proliferation (P < 0.05) but failed to decrease combined Hsp70- and LPS-promoted proliferation of stromal cells (Fig. 5B). However, pre-treatment of stromal cells with anti-TLR4 antibody (10 µg/ml) was able to significantly decrease the combined Hsp70- and LPS-promoted proliferation of stromal cells (P < 0.05, Fig. 5B). These results further indicate that both Hsp70 and LPS have the capacity to directly stimulate stromal cell proliferation and this growth promoting effect is mediated by TLR4.
Tissue levels of endogenous Hsp70 in eutopic and ectopic endometria
In order to examine the in vivo variation of stress reaction at the tissue level, we measured levels of endogenous Hsp70 in the eutopic endometria of women with and without endometriosis and in different peritoneal lesions as shown in Fig. 6. The tissue concentrations of Hsp70 were significantly higher in the homogenized samples of eutopic endometria derived from women with endometriosis than in similar tissues derived from control women (P < 0.05, Fig. 6A). No significant difference was observed in tissue levels of Hsp70 between endometriosis of Stages I–II and III–IV (data not shown). Although an apparent increase in tissue levels of Hsp70 was found in the samples derived from women in the secretory phase, we did not find any significant difference in Hsp70 levels in endometrial samples between the secretory phase and the proliferative phase (data not shown).
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When we analyzed endogenous Hsp70 levels in samples according to color appearance of peritoneal lesions in women with pelvic endometriosis, we found that the tissue levels were highest in red lesions, intermediate in black lesions and lowest in white peritoneal lesions (P < 0.05 by Kruskal–Wallis test for red lesions, Fig. 6B). No difference in tissue levels of Hsp70 was found between the samples of black lesions and white lesions. When total red lesions were subdivided into blood-filled opaque red lesions and non-opaque transparent or translucent red lesions, as we described previously (Khan et al., 2004b
), opaque red lesions showed significantly higher levels of Hsp70 at the tissue level when compared with either non-opaque red lesions, black lesions or white peritoneal lesions (P < 0.01 for each, Fig. 6B). No difference in tissue levels of Hsp70 was found among less active peritoneal lesions, such as non-opaque red lesions, black lesions and white peritoneal lesions. |
Discussion |
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In our current study, we demonstrated that pelvic endometriosis induces stress reaction in the pelvic environment in addition to inducing pelvic inflammation. This was confirmed by the release of a variable amount of endogenous Hsp70 by the different peritoneal lesions and eutopic endometria of women with endometriosis. We also demonstrated that locally produced Hsp70 might be responsible for TLR4-mediated induction of inflammatory reaction and direct promotion in the growth of endometriosis. Although polymyxin B, a potent LPS antagonist, is able to suppress LPS-mediated growth of endometrial cells derived from women with endometriosis as reported previously (Hirata et al., 2005
; Khan et al., 2007), in our current study, polymyxin B was unable to suppress combined LPS- and Hsp70-mediated growth of endometriosis. In contrast, the growth promoting effect of combined LPS and Hsp70 was significantly suppressed when the biological function of TLR4 was blocked with anti-TLR4 antibody. Our current findings indicated that LPS- and Hsp70-mediated inflammatory reaction and growth of endometriosis may be mediated by TLR4 in the pelvic environment.
TLR4-mediated production of different cytokines and growth factors and endometrial cell proliferation in response to LPS has been demonstrated previously (Hirata et al., 2005; Khan et al., 2005d,e, 2007). All of these studies were done in in vitro culture system. When we consider the internal pelvic environment, besides LPS, there are a number of other exogenous and endogenous ligands for TLR4 (Kiechl et al., 2002; Akira and Takeda, 2004). TLR4 is an essential receptor for bacterial endotoxin or LPS recognition. In addition to LPS, as a potential endogenous ligand, Hsp70 can also transmit signal through TLR4 (Wallin et al., 2002; Triantafilou and Triantafilou, 2004). Therefore, we presumed that growth of endometriosis may be regulated by endogenous Hsp70 or LPS either alone or in combination after their binding with TLR4. The expression level of TLR4 was reported to be higher in the samples derived from the secretory phase of menstrual cycle (Fazeli et al., 2005; Aflatoonian et al., 2007), however, we did not find any significant difference in TLR4 expression levels between proliferative phase and secretory phase samples or between women with and without endometriosis in our current study. This could be due to the small number of samples we used in the current study.
As a component of the innate immune system, we found that M derived from the PF of women with and without endometriosis equally expressed TLR4 both at the protein and gene level. When we measured the secretion levels of HGF, VEGF, IL-6 and TNF in the culture media of Hsp70-treated M, we found that the production of all these macromolecules was significantly higher in treated cells when compared with non-treated cells or in M derived from women with endometriosis when compared with women without endometriosis. Again, direct stimulation with Hsp70 was able to significantly enhance the proliferation of endometrial stromal cells derived from women with endometriosis compared with similar cells derived from control women. All these pro-inflammatory response and cell promoting effects of Hsp70 were mediated by TLR4. In fact, we found a similar increase in the amount of HGF mRNA in M in response to Hsp70 and abrogation of cytokine secretion, HGF mRNA levels and cell proliferation after pretreatment of cells with anti-TLR4 antibody. Our current findings provide further evidence that an internal stress reaction in the pelvic environment could be responsible for pelvic inflammation and growth of endometriosis in addition to estrogen and other primary or secondary inflammatory mediators.
We learned from our present study that blocking of TLR4 could be more effective in reducing pro-inflammatory response and growth of pelvic endometriosis, because there are other endogenous and exogenous ligands for TLR4 in addition to LPS (Kiechl et al., 2002). The possible contamination by endotoxin in the study of Hsp70-treated cells during bio-culture procedure was a matter of concern in different reports (Byrd et al., 1999; Triantafilou et al., 2001; Wallin et al., 2002; Triantafilou and Triantafilou, 2004). However, we carefully excluded the possible contamination of Hsp70-treated cells with endotoxin by our serial exclusion experiments.
Endogenous Hsps including Hsp70 can be produced in response to environmental stimuli (heat shock, ultraviolet radiation and heavy metals), pathological stimuli (viral, bacterial, parasitic infection, inflammation, malignancy or autoimmunity) and physiological stimuli (different physical or chemical stress) (Asea et al., 2000, 2002). Besides inflammation, pelvic endometriosis may induce a variable degree of physical stress (cell to cell contact, cell proliferation, cell differentiation or tissue invasion) or chemical stress (receptor–ligand interaction) in the pelvic environment. In order to examine the degree of stress reaction in the pelvic environment, we measured tissue levels of endogenous Hsp70 in the eutopic endometria and different peritoneal lesions of women with endometriosis. We found that endogenous stress reaction in eutopic endometria as measured by tissue levels of Hsp70 was significantly higher in women with endometriosis when compared with control women. Although a tendency of higher tissue levels of Hsp70 was observed in early endometriosis (Stages I–II) and in the secretory phase, there was no significant difference in Hsp70 levels between r-ASRM Stages I–II and III–IV or between proliferative and secretory phases of the menstrual cycle.
When we analyzed tissue levels of Hsp70 according to color appearance of endometriotic lesions, we found the highest levels in blood-filled opaque red lesions compared with other peritoneal lesions. This could be due to a higher stress reaction caused by increased mitogenic, angiogenic and tissue invasion properties of opaque red lesions as we reported previously (Khan et al., 2003, 2004b). In fact, both opaque and non-opaque red lesions are included in the same groups of red lesions according to the morphological classification of r-ASRM (ASRM, 1997). These results further indicated that this subgroup of red lesions displayed strong stress reaction when compared with other peritoneal lesions in the pelvic environment.
Finally, we conclude that women with endometriosis harboring peritoneal lesions of different color appearances suffer different in vivo stress reaction. Among them, a prominent stress reaction was observed in blood-filled opaque red peritoneal lesions. We suggest that human Hsp70 also induces pelvic inflammation and may regulate TLR4-mediated growth of endometriosis. A variation in pelvic inflammatory reaction and stress reaction may function together to regulate the growth of pelvic endometriosis. Our current findings may provide a clue in targeting TLR4 as a new therapeutic strategy in women with endometriosis. Current ongoing studies from our laboratory regarding relationships between stress reaction and inflammation or between stress reaction and angiogenic response in the pelvic environment may provide further evidence for the importance of stress protein in women with different endocrine diseases.
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This work was supported by Grants-in-Aid for Scientific Research (Grant No. 16591671 and 18591837) from the Ministry of Education, Sports, Culture, Science and Technology of Japan (to KN Khan).
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
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We thank Miss Kazumi Hayashida and Miss Kyoko Ishida, Department of Obstetrics and Gynecology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan, for their excellent technical assistance.
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Aflatoonian R, Tuckerman E, Elliot SL, Bruce C, Aflatoonian A, Li TC, Fazeli A. Menstrual cycle-dependent changes of toll-like receptors in endometrium. Hum Reprod (2007) 22:586–593.
Akira S, Takeda K. Toll-like receptor signaling. Nat Rev Immunol (2004) 4:499–511.[CrossRef][Web of Science][Medline]
Asea A, Krafft SK, Kurt-Jones EA, Stevenson MA, Chen LB, Fingerg RW, Koo GC, Calderwood SK. HSP70 stimulates cytokine production through a CD14-dependent pathway, demonstrating its dual role as a chaperone and cytokine. Nat Med (2000) 6:435–442.[CrossRef][Web of Science][Medline]
Asea A, Rehli M, Kabingu E, Boch JA, Bare O, Auron PE, Stevenson MA, Calderwood SK. Novel signal transduction pathway utilized by extracellular HSP70: role of toll-like receptor (TLR) 2 and TLR4. J Biol Chem (2002) 277:15028–15034.
Bradford M. A Rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem (1976) 72:315–323.[CrossRef][Web of Science][Medline]
Byrd CA, Bornmann W, Erdjument-Bromage H, Tempst P, Pavletich N, Rosen N, Nathan CF, Ding A. Heat-shock protein 90 mediates macrophage activation by Taxol and bacterial lipopolysacccharide. Proc Natl Acad Sci USA (1999) 96:5645–5650.
Crawford L, Leppard K, Lane D, Harlow E. Cellular proteins reactive with monoclonal antibodies directed against simian virus 40 T-antigen. J Virol (1982) 42:612–620.
Fazeli A, Bruce C, Anumba DO. Characterization of Toll-like receptors in the female reproductive tract in humans. Hum Reprod (2005) 20:1372–1378.
Fujishita A, Nakane PK, Koji T, Masuzaki H, Chavez RQ, Yamabe T, Ishimaru T. Expression of estrogen and progesterone in endometrium and peritoneal endometriosis: an immunohistochemical and in situ hybridization study. Fertil Steril (1997) 67:856–864.[CrossRef][Web of Science][Medline]
Halme J, Becker S, Haskill S. Altered maturation and function of peritoneal macrophages: possible role in pathogenesis of endometriosis. Am J Obstet Gynecol (1987) 156:783–789.[Web of Science][Medline]
Halme J, White C, Kauma S, Estes J, Haskill S. Peritoneal macrophages from patients with endometriosis release growth factor activity in vitro. J Clin Endocrinol Metab (1988) 66:1044–1049.
Halme J. Release of tumor necrosis factor-alpha by human peritoneal macrophages in vivo and in vitro. Am J Obstet Gynecol (1989) 161:1718–1725.[Web of Science][Medline]
Harada T, Iwabe T, Terakawa N. Role of cytokines in endometriosis. Fertil Steril (2001) 76:1–10.[CrossRef][Web of Science][Medline]
Hirata T, Osuga Y, Hirota Y, Koga K, Yoshino O, Harada M, Morimoto C, Yano T, Nishii O, Tsutsumi O, et al. Evidence for the presence of Toll-like receptor 4 system in the human endometrium. J Clin Endocrinol Metab (2005) 90:548–556.
Jansen RPS, Russel P. Non-pigmented endometriosis: clinical, laparoscopic and pathologic definition. Am J Obstet Gynecol (1996) 155:1154–1159.
Keenan JA, Chen TT, Chadwell NL, Torry DS, Caudle MR. Interferon-gamma (IFN-gamma) and interleukin-6 (IL-6) in peritoneal fluid and macrophage- conditioned media of women with endometriosis. Am J Reprod Immunol (1994) 32:180–183.[Web of Science][Medline]
Keenan JA, Chen TT, Chadwell NL, Torry DS, Caudle MR. IL-1 beta, TNF-alpha, and IL-2 in peritoneal fluid and macrophage-conditioned media of women with endometriosis. Am J Reprod Immunol (1995) 34:381–385.[Medline]
Khan KN, Fujishita A, Kitajima M, Masuzaki H, Sekine I, Ishimaru T. Infiltrated macrophage activity in intact tissue of endometriosis. Proc. Endometriosis (2002) a 23:137–142. (in Japanese).
Khan KN, Masuzaki H, Fujishita A, Hamasaki T, Kitajima M, Hasuo A, Ishimaru T. Association of interleukin-6 and estradiol with hepatocyte growth factor in peritoneal fluid of women with endometriosis. Acta Obstet Gynecol Scand (2002) b 81:764–771.[CrossRef][Web of Science][Medline]
Khan KN, Masuzaki H, Fujishita A, Kitajima M, Sekine I, Ishimaru T. Immunoexpression of hepatocyte growth factor and c-Met receptor in eutopic endometrium predicts the activity of ectopic endometrium. Fertil Steril (2003) 79:173–181.[CrossRef][Web of Science][Medline]
Khan KN, Masuzaki H, Fujishita A, Kitajima M, Sekine I, Ishimaru T. Differential macrophage infiltration in early and advanced endometriosis and adjacent peritoneum. Fertil Steril (2004) a 81:652–661.[CrossRef][Web of Science][Medline]
Khan KN, Masuzaki H, Fujishita A, Kitajima M, Sekine I, Ishimaru T. Higher activity by opaque endometriotic lesions than non-opaque lesions in women with endometriosis. Acta Obstet Gynecol Scand (2004) b 83:375–382.[CrossRef][Web of Science][Medline]
Khan KN, Masuzaki H, Fujishita A, Kitajima M, Kohno T, Sekine I, Matsuyama T, Ishimaru T. Regulation of hepatocyte growth factor by basal and stimulated macrophages in women with endometriosis. Hum Reprod (2005) a 20:49–60.
Khan KN, Masuzaki H, Fujishita A, Kitajima M, Sekine I, Matsuyama T, Ishimaru T. Estrogen and progesterone receptor expression in macrophages and regulation of hepatocyte growth factor by ovarian steroids in women with endometriosis. Hum Reprod (2005) b 20:2004–2013.
Khan KN, Masuzaki H, Fujishita A, Kitajima M, Hiraki K, Sekine I, Matsuyama T, Ishimaru T. Interleukin-6- and tumor necrosis factor -mediated expression of hepatocyte growth factor by stromal cells and its involvement in the growth of endometriosis. Hum Reprod (2005) c 20:2715–2723.
Khan KN, Fujishita A, Kitajima M, Hiraki K, Masuzaki H, Sekine I, Matsuyama T, Ishimaru T. Increased endotoxin concentration in peritoneal fluid promotes toll-like receptor 4 (TLR4)-mediated growth of endometriosis. Am J Reprod Immunol (2005) d 53:292–293.
Khan KN, Fujishita A, Kitajima M, Hiraki K, Masuzaki H, Sekine I, Matsuyama T, Ishimaru T. Detection of Escherichia coli in menstrual blood and endotoxin in peritoneal fluid: an implication in pelvic inflammation and toll-like receptor 4 (TLR4)-mediated growth of endometriosis. Eur J Obstet Gynecol Reprod Biol (2005) e 123:S15–S16.
Khan KN, Masuzaki H, Fujishita A, Kitajima M, Hiraki K, Miura S, Sekine I, Ishimaru T. Peritoneal fluid and serum levels of hepatocyte growth factor may predict the activity of endometriosis. Acta Obstet Gynecol Scand (2006) 85:458–466.[Web of Science][Medline]
Khan KN, Kitajima M, Hiraki K, Fujishita A, Ishimaru T, Masuzaki H. Toll-like receptors in innate immunity: role of bacterial endotoxin and toll-like receptor 4 (TLR4) in endometrium, endometriosis and placenta. Inflamm Immun (2007) 15:56–68. (review article in Japanese).
Kiechl S, Lorenz E, Reindl M, Wiedermann CJ, Oberhollenzer F, Bonora E, Willeit J, Schwartz DA. Toll-like receptor 4 polymorphisms and atherogenesis. N Engl J Med (2002) 347:185–192.
Koga K, Osuga Y, Tsutsumi O, Momoeda M, Suenaga A, Kugu K, Fujiwara T, Takai Y, Yano T, Taketani Y. Evidence for the presence of angiogenin in human follicular fluid and the up-regulation of its production by human chorionic gonadotropin and hypoxia. J Clin Endocrinol Metab (2000) 85:3352–3355.
Kol A, Bourcier T, Lichtman AH, Libby P. Chlamydial and human heat-shock proteins activate human vascular endothelium, smooth muscle cells and macrophages. J Clin Invest (1999) 103:571–577.[Web of Science][Medline]
Lebovic DI, Mueller MD, Taylor RN. Immunobiology of endometriosis. Fertil Steril (2001) 75:1–10.[CrossRef][Web of Science][Medline]
Mahnke JL, Dawood Y, Huang JH. Vascular endothelial growth factor and interleukin-6 in peritoneal fluid of women with endometriosis. Fertil Steril (2000) 73:166–170.[CrossRef][Web of Science][Medline]
Matzinger P. An innate sense of danger. Semin Immunol (1998) 10:399–415.[CrossRef][Web of Science][Medline]
McLaren J, Prentice A, Charnock-Jones DS, Millican SA, Muller KH, Sharkey AM, Smith SK. Vascular endothelial growth factor is produced by peritoneal fluid macrophages in endometriosis and is regulated by ovarian steroids. J Clin Invest (1996) 98:482–489.[Web of Science][Medline]
Miura S, Khan KN, Kitajima M, Hiraki M, Shingo M, Masuzaki H, Samejima T, Fujishita A, Ishimaru T. Differential infiltration of macrophages and prostaglandin production by different uterine leiomyomas. Hum Reprod (2006) 21:2545–2554.
Nisolle M, Casanas-Roux F, Donnez J. Immunohistochemical analysis of proliferative activity and steroid receptor expression in peritoneal and ovarian endometriosis. Fertil Steril (1997) 68:912–919.[CrossRef][Web of Science][Medline]
Osuga Y, Tsutsumi O, Okagaki R, Takai Y, Fujimoto A, Suenaga A, Maruyama M, Momoeda M, Yano T, Taketani Y. Hepatocyte growth factor concentrations are elevated in peritoneal fluid of women with endometriosis. Hum Reprod (1999) 14:1611–1613.
Osteen KG, Hill GA, Hargrove JT, Gorstein F. Development of a method to isolate and culture highly purified populations of stromal and epithelial cells from human endometrial biopsy specimens. Fertil Steril (1989) 52:965–972.[Web of Science][Medline]
Rana N, Gebel H, Braun DP, Rotman C, House R, Dmowski WP. Basal and stimulated secretion of cytokines by peritoneal macrophages in women with endometriosis. Fertil Steril (1996) 65:925–930.[Web of Science][Medline]
Sugawara J, Fukaya T, Murakami T, Yoshida H, Yajima A. Increased secretion of hepatocyte growth factor by eutopic endometrial stromal cells in women with endometriosis. Fertil Steril (1997) 68:468–472.[CrossRef][Web of Science][Medline]
Takagi S. Detection of 5-bromo-2-deoxyuridine (BrdU) incorporation with monoclonal anti-BrdU antibody after deoxyribonuclease treatment. Cytometry (1993) 14:640–649.[CrossRef][Web of Science][Medline]
Takeda K, Akira S. Toll-like receptors in innate immunity. Intl Immunol (2005) 17:1–14.
The American Society for Reproductive Medicine. Revised American Society for Reproductive Medicine classification of endometriosis: 1996. Fertil Steril (1997) 67:817–821.[CrossRef][Web of Science][Medline]
Triantafilou M, Triantafilou K. Heat-shock protein 70 and heat-shock protein 90 associate with Toll-like receptor 4 in response to bacterial lipopolysaccharide. Biochem Soc Trans (2004) 32:636–639.[CrossRef][Web of Science][Medline]
Triantafilou K, Triantafilou M, Dedrick RL. A CD14-independent LPS receptor cluster. Nat Immunol (2001) 2:338–345.[CrossRef][Web of Science][Medline]
Wira CR, Fahey JV, Sentman CL, Pioli PA, Shen L. Innate and adaptive immunity in female genital tract: cellular responses and interactions. Immunol Rev (2005) 206:306–335.[CrossRef][Web of Science][Medline]
Wallin RPA, Lundqvist A, More SH, von Bonin A, Kiessling R, Ljunggren H-G. Heat-shock proteins as activators of the innate immune system. TRENDS Immunol (2002) 23:130–135.[CrossRef][Web of Science][Medline]
Young SL, Lyddon TD, Jorgenson RL, Misfeldt ML. Expression of Toll-like receptors in human endometrial epithelial cells and cell lines. Am J Reprod Immunol (2004) 52:67–73.[Medline]
Zugel U, Kaufmann SH. Immune response against heat-shock proteins in infectious diseases. Immunobiology (1999) 201:22–35.[Web of Science][Medline]
Submitted on December 24, 2007; resubmitted on April 16, 2008; accepted on April 25, 2008.
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