1 Department of Obstetrics and Gynecology, 2 Division of Cytokine Signaling, Department of Molecular Microbiology and Immunology, Graduate School of Biomedical Sciences and 3 Department of Molecular Pathology, Atomic Bomb Disease Institute, Nagasaki University, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan
4 To whom correspondence should be addressed. Email: nemokhan{at}net.nagasaki-u.ac.jp
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Abstract |
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Key words: cell growth/endometriosis/hepatocyte growth factor/lipopolysaccharide/macrophage
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Introduction |
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We and others have already demonstrated that hepatocyte growth factor (HGF) and vascular endothelial cell growth factor (VEGF) are also higher in the PF of women with endometriosis than that of healthy controls (Donnez et al., 1998; Fujishita et al., 1999
; Osuga et al., 1999
; Mahnke et al., 2000
; Khan et al., 2002a
). HGF was discovered as a mitogen for adult hepatocytes and is identical to scatter factor (Miyazawa et al., 1989
; Nakamura et al., 1989
; Weidner et al., 1991
). Several lines of evidence have implied that HGF, produced by mesenchymal cells, exerts mitogenic, motogenic (migration), morphogenic and angiogenic activity after binding with its receptor, c-Met, on various epithelial cells derived from rodents and humans (Nakamura et al., 1986
; Tajima et al., 1992
). The role of HGF in the proliferation, migration and metaplastic transformation of endometrial epithelial cells has been demonstrated in vitro and in vivo (Sugawara et al., 1997
; Ishimaru et al., 2004
).
Although production of HGF by hepatic kupffer cells and alveolar macrophages has been reported (Skrtic et al., 1999; Morimoto et al., 2001
; Crestani et al., 2002
), information regarding production of HGF by peritoneal M
is unknown. Therefore, we report for the first time the production of HGF by the PF M
derived from women with or without endometriosis and examined the ability of PF M
to synthesize HGF in basal conditions and after treatment with lipopolysaccharide (LPS).
Since our initial study (Khan et al., 2003a) demonstrated that PF of women with endometriosis contains a higher concentration of LPS (endotoxin) than that of those without endometriosis, we speculated that LPS could be an inflammatory mediator of M
stimulation in the pelvic microenvironment. In addition, we also demonstrated the metabolic activity of peritoneal M
in different stages and morphological appearances of endometriosis. Finally, we examined the effect of HGF on the growth of bovine endometrial epithelial cells and isolated stroma or M
derived from women with or without endometriosis.
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Materials and methods |
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Isolation of M from the PF
PF was obtained from all women with or without endometriosis with the use of laparoscopy. M were isolated in primary culture. PF samples were centrifuged at 400 g for 10 min and the cellular pellet was under-layered with lymphocyte separation medium (ICN, Aurora, OH) and centrifuged at 400 g for 10 min. M
were collected from the interface and cultured in RPMI-1640 medium (GIBCO, Grand Island, NY) supplemented with 100 IU/ml of penicillin G, 50 mg/ml of streptromycin, 2.5 µg/ml of amphotericin B and 10% fetal bovine serum at 37°C in 5% CO2 in air.
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 >95% M
as estimated by their morphology and by immunocytochemical staining using CD68 (KP1), a mouse monoclonal antibody from Dako, Denmark. The cells used for immunocytochemical staining were plated in 4-well chamber slides (Nunc, Naperville, IL) and grown to near confluence. The detailed procedure of immunocytochemical staining is described elsewhere (Rana et al., 1996
). Non-immune mouse immunoglobulin (Ig) G1 antibody at 1:50 dilution was used as a negative control. A counter-staining of M
was also performed and we did not find any contaminating cells in isolated M
(data not shown).
The isolated peritoneal M were cultured in triplicate (105 per 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 medium, M
were serum starved for 24 h and then serum-free M
were cultured for another 24 h with LPS derived from Escherichia coli (serotype 0111:B4; Sigma, St Louis, MO). After 24 h, the cultured media were collected in triplicate, pooled, and frozen at 70°C until testing.
Proliferation of M by MTT assay
The proliferative status of M can be assessed by the metabolic activity of viable cells using MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolioum bromide] assay. The principle of this assay is that the tetrazolium salt MTT is cleaved to formazan by the succinate-tetrazolium reductase which belongs to the mitochondrial respiratory chain and is active only in viable cells. A microtitre plate assay which uses the tetrazolium salt MTT is now widely used to quantitate cell proliferation and cytotoxicity (Mosmann, 1983
; Sugawara et al., 1997
).
Isolated M from women with or without endometriosis were first placed in a 96-well microtitre plate (Griener labortechnik, Germany) at a density of 104 cells per well and the time-dependent (6, 12, 24, 48 and 72 h) proliferation of M
examined. For the dose-dependent study, after a 24 h pre-incubation period without serum, culture media were replaced for another 24 h with serum-free media, containing 0, 1, 5, 10 and 15 ng/ml of LPS (Sigma). The dose-dependent study of LPS was applied in isolated M
derived from women with or without endometriosis and according to the dominant distribution of either blood-filled red lesions or black lesions of pelvic endometriosis as we reported previously (Khan et al., 2002a
, 2004a
).
The MTT assay was then performed in triplicate samples by adding 10 µl of 5 mg/ml MTT solution per well and incubated for 4 h. After that, 100 µl of dimethyl sulphoxide (DMSO) was added, kept at room temperature for a few minutes to dissolve the dark blue crystals (formazan), and finally their absorbance was measured at 570 nm with a microplate reader. We found a direct relationship between cell number and the amount of MTT formazan generated. This indicates that the absorbance at MTT assay was directly proportional to the number of viable cells.
Cytokine assays in the culture media of treated and non-treated M
The culture media of basal (non-treated) and stimulated (5 ng/ml of LPS) M were collected in triplicate and assays were performed retrospectively. The concentrations of HGF and VEGF in the culture media were measured in duplicate in a blind fashion using a commercially available sandwich enzyme-linked immunosorbent assay (ELISA) developed by R&D Systems (Quantikine, R&D Systems, Minneapolis, MN). The antibodies used in VEGF and HGF determination do not cross-react with other cytokines. The limits of detection were 9.0 pg/ml for VEGF and 40.0 pg/ml for HGF. Both the intra- and inter-assay coefficients of variation were <10% for all these assays.
Immunolocalization of HGF in M
In order to immunolocalize HGF in CD68-immunoreactive M, we performed immunohistochemistry using the respective antibody and using serial sections of eutopic endometrium derived from women with endometriosis. A 1:50 dilution of a rabbit polyclonal antibody against a recombinant protein of HGF
(H-145) (sc-7949; Santa Cruz Biotechnology, Santa Cruz, CA) of human origin was used.
Immunohistochemistry was performed in the 5 µm thick serial sections of paraffin-embedded tissues as described previously (Khan et al., 2003b). Non-immune mouse IgG1 antibody (1:50) was used as a negative control. Placental tissue, which is known to exhibit high levels of HGF, was used as a positive control.
Determination of HGF and c-Met mRNA by RTRCR in M
We have used the RTPCR technique to determine the mRNA levels of HGF and its receptor, c-Met, in basal (non-treated) and stimulated M (treated by 1, 5 and 10 ng/ml of LPS) derived from women with or without endometriosis. RNA was isolated from each of 106 M
cultured in a 60 mm Petri dish (Greiner) using a monophasic solution of 40% phenol and the ISOGEN method (Molecular Research Center, Tokyo), according to the manufacturer's protocol.
RNA was treated with RNase-free DNase I in 10 mmol/l TrisHCl, pH 8.3, 50 mmol/l KCl and 1.5 mmol/l MgCl2 in the presence of RNasin ribonuclease inhibitor and incubated at 37°C for 30 min to remove DNA contamination. After extraction with phenolcholoform and ethanol precipitation, the RNA was re-dissolved in autoclaved ultrapure water (Milli-Q, MILLIPORE Inc. Corp., Yonezawa, Japan).
cDNA synthesis. The first-strand cDNA was synthesized using an RTPCR kit (Stratagene, La Jolla, CA) with oligo(dT) primers. A 5 µg aliquot of total RNA was heated to 65°C for 5 min in the presence of 3 µl of oligo(dT) primer and then rapidly chilled on ice. A master mix contained 5 µl of 10x first-strand buffer, 2 µl of 10 mmol/l dNTP mix, 1 µl of Moloney murine leukaemia virus (MMLV) reverse transcriptase (50 U/µl) and 1 µl of RNase inhibitor (40 U/µl), and each reaction was incubated at 37°C for 1 h with further incubation at 90°C for 5 min.
PCR amplification. Aliquots from the cDNA reaction were PCR amplified in 20 µl reactions as follows: 10x PCR buffer [100 mmol/l TrisHCl (pH9.0), 400 mmol/l KCl, 15 mmol/l MgCl], 10 mmol/l of each of the deoxynucleotide triphosphates (dNTP mixture), 10 µmol/l of each primer and 1 U of Taq DNA polymerase (Bioneer Corporation, Seoul). A 1 µl aliquot of cDNA was added for each PCR. The reaction was initiated by heat denaturation at 94°C for 1 min, annealing of the primers for I min at 59°C, and then extension for 1 min at 72°C. This was repeated for 32 cycles for HGF and c-Met using the PCR apparatus (Takara Biomedicals, Tokyo). The amplification protocol for -actin used as an internal control was the same as above except for the annealing condition of the primer (62°C for 1 min), and the reaction was repeated for 23 cycles. After the final cycle, the temperature was maintained at 72°C for 10 min to allow completion of synthesis of the amplification products. A control with no reverse transcription was run with each sample to confirm that PCR products were free of DNA contamination.
Analysis of PCR-amplified products was performed by fractionation over a 1.5% agarose gel followed by ethidium bromide staining of DNA bands. A scanner densitometer was used to determine the ratio of intensity of each band relative to -actin and is represented as the relative expression of the target gene. Autoradiographs were analysed to quantitate differences in levels of transcripts between non-treated samples and LPS-treated samples derived from control women and women with endometriosis. The values of each transcript after treatment with LPS were nomalized to 1. Densitometric analysis of gel bands was performed using the National Institutes of Health image analysis program.
In order to confirm the PCR products, the corresponding PCR product was size fractionated and subcloned into plasmid pCRII (Invitrogen, San Diego, CA). The PCR products were sequenced by the dideoxy chain termination method and the product of each primer pair was confirmed in both directions (sense and antisense). There was no difference between the sequence products after subcloning and the sequence of the target gene used for the study.
Primer design and controls. Optimal oligonucleotide primer pairs for RTPCR amplification of oligo(dT)-primed reverse-transcribed cDNA were selected with the aid of the computer program Oligo, version 4.0 (National Biosciences, Inc., Plymouth, MN). Human oligonucleotide primers of HGF, c-Met and -actin which we used for our current study, their location on cDNA and corresponding GenBank accession numbers are shown in Table I.
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Isolation of stroma in primary culture
Stroma was collected from the biopsy specimens of the eutopic and ectopic endometrium derived from the women with or without endometriosis. The detailed procedure for the isolation of stroma has been described previously (Osteen et al., 1989; Sugawara et al., 1997
).
The characteristics of the cultured stromal cells were determined by morphological and immunocytochemical studies. The isolated cells were placed in a four-chamber slide (Nunc, Naperville, IL). After 24 h, the slides were washed in phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 10 min, and rinsed with PBS. Slides were then incubated in 0.1% Triton X-100 for 5 min and incubated for 3 h at 37°C as follows: against human cytokeratin monoclonal antibodies (mAbs) (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 dilution (Dako). 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. A counter-staining of stroma was also performed to exclude the contamination by epithelial or endothelial cells in isolated stromal cell culture (data not shown).
DNA synthesis assay by bromodeoxyuridine (BrdU) incorporation
Unlike the MTT assay, the BrdU labelling and detection kit measures cell proliferation by quantitating BrdU incorporated into the newly synthesized DNA of replicating cells. The incorporated BrdU can be detected by a quantitative cellular enzyme immunoassay (Biotrak, Amersham Pharmacia Biotech Ltd, UK) using mAbs directed against BrdU. It offers a non-radioactive alternative to [3H]thymidine-based cell proliferation and carries equal sensitivity and specificity (Takagi, 1993).
Besides isolated stroma and M, we also used a bovine endometrial epithelial cell line (BEND cells, ATCC, USA, CRL-2398) as a source of non-cancerous epithelial cells derived from bovine uterus. These bovine endometrial cells retain functional characteristics similar to those of human endometrial cells, as described previously (Austin et al., 1999
; Parent et al., 2003
). These normal endometrial epithelial cells were grown in a medium containing a 1:1 mixture of Ham's F12 medium and Eagle's minimal essential medium (EMEM) with Earle's salt and 1.5 mmol/l L-glutamine adjusted to contain 2.2 g/l sodium bicarbonate and supplemented with 0.126 g/l D-valine, 10% fetal bovine serum and 10% horse serum.
Briefly, the desired cells (bovine endometrial epithelial cells, human stroma and M) were cultured in 96-well microtitre plates (104 cell/well). An average of 48 h were required to reach confluence for these various cells.. After a 24 h pre-incubation period without serum, the respective cells were treated with or without HGF (recombinant HGF, R&D Systems) in a serum-free medium and incubated for an additional 24 h. After that, the cells were labelled with 10 µmol/l BrdU (100 µl/well) and incubated for 4 h at 37°C. The cells were fixed and genomic DNA was denatured by adding 200 µl/well of blocking reagent (1:10) for 30 min at room temperature. Peroxidase-labelled anti-BrdU antibody (1:100) was added (100 µl/well) and incubated for 90 min at room temperature. After washing three times, TMB (3,3'5,5'-tetramethylbenzidine) substrate solution was added (100 µl/well) and incubated for 15 min at room temperature for colour appearance, and finally optical density was measured using a microplate reader at an absorbance of 450 nm. The absorbance values correlated directly to the amount of DNA synthesis and thereby to the number of proliferating cells in culture.
In order to confirm that the growth-promoting factor in the LPS-treated culture medium is HGF, we used antibody to deplete HGF in the conditioned medium. M were pre-treated with anti-HGF antibody (10 µg/ml, R&D Systems), incubated for 4 h and then treated with 5 ng/ml of LPS for another 24 h and examined for the change in cell growth.
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 analysed using a non-parametric test. The differences between endometriosis and non-endometriosis, red lesions and black lesions, LPS- or HGF-treated and non-treated groups were compared using the MannWhitney U-test or Student's t-test. The Tukey test of significant differences was used post hoc for multiple comparisons. Differences were considered as statistically significant for P<0.05.
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Results |
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Proliferation of basal and LPS-stimulated M
The isolated PF M placed into primary culture which were immunoreactive to CD68 and their negative control are shown in Figure 1A and B, respectively. The co-existing contamination by other leukocytes was excluded by their negative immunostaining for CD45 (data not shown).
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HGF production by the basal and LPS-stimulated M
The production of HGF in culture media by the LPS-treated (5 ng/ml) and non-treated M derived from the PF of control women, women with stage III endometriosis and women with stage IIIIV endometriosis is shown in Figure 3.
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As shown in Figure 3, a 4-fold (243.6±45.8 versus 66±23.7 pg/ml, P<0.001) and 3-fold (148±34.6 versus 49.6±17.3 pg/ml, P<0.001) increase in the production of HGF was observed in the LPS-treated M derived from women with stage III and stage IIIIV endometriosis, respectively, when compared with non-LPS-treated M
. However, only a 1.5-fold increase in the production of HGF was observed in the control women after LPS treatment compared with no treatment (74.2±20.6 versus 51.6±19.6 pg/ml, P<0.05). In contrast to HGF, the production of VEGF was increased by both basal and LPS-stimulated M
in all three groups of women (data not shown). Although the production of VEGF in response to LPS was higher than that of HGF, we did not find any significant difference between the production of HGF and VEGF in these women (data not shown).
Production of HGF by M in intact tissue
As shown in Figure 4, tissue localization of HGF (Figure 4C) was demonstrated in the same position as CD68-immunoreactive M (Figure 4B) (shown by the corresponding arrowheads) in the serial section of intact tissues derived from the eutopic endometrium of a woman with endometriosis. This indicates that HGF is also being synthesized and secreted by the infiltrated M
of intact tissue in addition to in vitro production of HGF by the basal or LPS-stimulated M
.
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A 3-, 5- and 4-fold increase in the expression of HGF mRNA in M was found in women with endometriosis at 1 (P<0.01), 5 (P<0.001) and 10 ng/ml (P < 0.01) of LPS treatment, respectively (Figure 5B). Although a very low expression of HGF mRNA was observed in the gel band, a 1.5- to 2-fold increase in the relative expression of HGF mRNA was found in women without endometriosis (Figure 5B).
Similar to the dose-dependent increase in c-Met mRNA expression by gel band, a 2-to 4-fold increase in the relative expression of c-Met was observed in the M of women with or without endometriosis and after LPS treatment (Figure 5C). This indicates that M
derived from women with or without endometriosis carry the receptor for HGF but the HGF mRNA expression varies, resulting in the differential production of HGF at the protein level between these two groups of women.
Effect of culture media from the basal and LPS-treated M on cell growth
According to the above results, the culture media of the basal and LPS-treated M contained a variable concentration of cytokines and growth factors including HGF. Therefore, we tried to examine the effect of 10% culture medium derived from the basal and LPS (5 ng/ml)-treated M
of women with endometriosis on the growth of normal epithelial cells derived from bovine endometrium, and stroma which was isolated from the eutopic endometrium of women with endometriosis.
We confirmed the purity of isolated stroma by their positive immunoreaction to vimentin (Figure 1C and D) and negative immunoreaction to cytokerartin, von Willebrand factor and CD45 (data not shown). This indicates that our isolated stroma were free of contamination with epithelial cells, endothelial cells or other leukocytes.
The cell growth, as shown in Figure 6, was analysed by counting the total cell number (initial plating 105 per well). We found that the application of 10% culture medium from the treated M (LPS, 5 and 10 ng/ml) on bovine endometrial epithelial cells and isolated human endometrial stroma for an incubation period of 24 h significantly increased the growth of these cells (>50% of control) when we compared them with basal M
or low dose treatment (LPS 1 ng/ml) of M
(P<0.05 for both epithelial cell and stroma) (Figure 6A and B).
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In order to prove that the growth-promoting factor in the LPS-treated conditioned medium is HGF, we extended our experiment by using antibody to deplete HGF in the conditioned medium. We found that although not significant, the blocking effect of HGF tended to reverse the growth of both epithelial cells and stroma towards the growth by non-treated conditioned medium (Figure 6A and B, hatched bars). This further indicates that besides other growth factors, LPS-treated culture medium also contains HGF and this may promote the growth of endometrial cells.
Effect of HGF on cell proliferation by study of BrdU incorporation
The enhanced cell growth after application of culture medium derived from the LPS-treated M is the concerted effect of different cytokines and growth factors including HGF. We tried to investigate the direct effect of different concentrations of recombinant HGF on the proliferation of bovine endometrial cells, epithelial cells and stroma or M
of women with or without endometriosis by BrdU incorporation.
The application of recombinant HGF, at a concentration of 10100 ng/ml and with an incubation period of 24 h, was able to significantly increase the proliferation of epithelial cells compared with non-treated cells or other low dose treatment with HGF (P<0.05 for 10, 50 and 100 ng/ml) (Figure 7A). HGF was unable to induce the proliferation of stroma isolated from women without endometriosis. However, HGF at a concentration of 10, 50 and 100 ng/ml stimulated the proliferation of stroma (>50% of control) derived from women with endometriosis (P<0.05 versus non-treated stroma) (Figure 7B). We also studied the effect of exogenous HGF on stromal cells of ectopic endometrium and we did not find any difference in cell proliferation between similar cells of eutopic and ectopic endometrium (data not shown).
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Since the BrdU incorporation study represents the simple incorporation of BrdU into the proliferated DNA of these cells and does not reflect the actual cell growth as accounted for by increased cell number, we also tried to examine the cell growth of epithelial cells, stroma and M by determining the cell number (initial plating 105 cells/well) under HGF stimulation. We found a parallel and significantly increased cell growth under a stimulation dose of 10, 50 and 100 ng/ml of HGF for epithelial cells and stroma and at a dose of 50 and 100 ng/ml of HGF for M
(data not shown).
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Discussion |
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The increased infiltration of M in intact tissue and PF of women with endometriosis has been reported (Halme et al., 1987
; Khan et al., 2002b
, 2004b
). However, the metabolic activity of these M
in women with or without endometriosis and based on their morphological appearance by laparoscopy is not well described. We found that proliferation of M
as a measure of their metabolic activity was significantly higher in women with endometriosis and also in those harbouring dominant red peritoneal lesions rather than other pigments. This indicates that M
in the PF of women with active endometriosis equally retain higher metabolic activity for their consequent production of different macromolecules.
A number of growth factors, cytokines or chemokines are reported to be produced by epithelial cells, endothelial cells, mesothelial cells or the mesenchymal cells (Tabibzadeh et al., 1989; Betjes et al., 1993
). In addition, the production of cytokines by LPS-stimulated M
from women with endometriosis has been reported (Wu et al., 1999
). The growth or persistence of endometriosis has been considered as an inflammatory response as manifested by increased concentrations of IL-6, IL-8 and TNF-
in the PF of women with endometriosis (Harada et al., 2001
; Khan et al, 2004a
). As we reported recently (Khan et al., 2003a
), concentration of LPS (endotoxin) in the PF of women with endometriosis appeared to be higher when compared with that in those without endometriosis. Therefore, we presume that the peritoneal macrophages in women with endometriosis are at an activation state to produce more HGF after stimulation with LPS.
We demonstrated for the first time that beside alveolar M and hepatic kupffer cells, a substantial amount of HGF is also produced by the peritoneal M
derived from women with endometriosis. This is demonstrated both at the protein level and at the transcriptional level in response to LPS. The production of HGF and other macromolecules by the peritoneal M
in the pelvic microenvironment may be antigen primed because peritoneal M
retain Toll-like receptor 4 (TLR4) for LPS as demonstrated by a recent study (Triantafilou and Triantafilou, 2002
). This is documented here by the increased mRNA expression of HGF, the secretion of HGF and other macromolecules in the culture medium in response to LPS treatment compared with that by the basal M
. Comparing the transcriptional level, the decreased levels of HGF production at the protein level can be explained by the post-transcriptional or post-translational degradation of HGF. This dissociation in the regulation of HGF between the transcriptional level and the protein level requires further investigation.
Besides in vitro production of HGF by basal and LPS-stimulated M, the immunoreaction of HGF was also found to be co-localized in the same position of tissue M
as demonstrated in the serial section of intact endometrium. This result has shown for the first time that in addition to mesenchymal origin, HGF could also be synthesized and secreted by the infiltrated M
of the PF or the intact tissue derived from women with endometriosis. We have already established that highly active blood-filled ectopic endometrium and corresponding eutopic endometrium equally harbour abundant M
(Khan et al., 2004b
). Our current findings further strengthened the notion that the metabolic and biological activity of infiltrated M
in the eutopic endometrium of stage III endometriosis is higher than that of stage IIIIV endometriosis or in control women.
We demonstrated production of a small amount of HGF by the M of control women in our current study and it is also reported that HGF can be produced by hepatic kupffer cells and alveolar macrophages (Skrtic et al., 1999
; Morimoto et al., 2001
; Crestani et al., 2002
) in response to any stress, injury, apoptosis or reactive oxygen species. Therefore, it is quite reasonable to speculate that besides endometriosis, HGF may also stimulate cells from normal subjects. Further studies are required to prove this finding in normal subjects.
We demonstrated a 3- to 5-fold increase in the expression of HGF mRNA in M derived from women with endometriosis. However, M
of women without endometriosis displayed a 1.5- to 2-fold increase in HGF mRNA. However, it was interesting to observe that these inflammatory cells of women with or without endometriosis displayed a dose-dependent and an almost equal expression of c-Met. In fact, a 2- to 4-fold increase in the expression of c-Met mRNA was observed in M
of these two groups of women.
The common belief until now is that the synthesis of HGF by mesenchymal cells and its interaction with c-Met as located on human endometrial epithelial and endothelial cells confer a paracrine mode of action (Sonnenberg et al., 1993). We recently reported a homogenous immunoexpressions of HGF and c-Met in the glandular epithelium and a heterogenous expression of this ligandreceptor in the stroma of women with endometriosis (Khan et al., 2003b
).
If we consider that a typical lesion of pelvic endometriosis has three major cellular components, i.e. glandular epithelial cells, stroma and infiltrated M, then we can postulate a possible inter-relationship between the co-expressions of HGF and its receptor, c-Met, among these three types of cells. We believe that besides a paracrine mode of action, the ligandreceptor interaction of HGF in epithelial cells, stroma and activated M
may also exert an autocrine or intracrine mode of action in the regulation of the growth of endometriosis.
The autocrine and paracrine modes of action of HGF among epithelial cells, stroma and M were documented further by the stimulated effect of HGF on the proliferation of endometrial epithelial cells, stroma and M
. The proliferation of epithelial and mesothelial cells in response to HGF is well accepted (Yashiro et al., 1996
; Sugawara et al., 1997
). However, the proliferation of stroma and M
in response to HGF treatment is completely new in our current study. The increased proliferation of stroma and M
by HGF was only evident for the cells which were obtained from women with endometriosis and not from those without endometriosis. This can be explained by the increased co-expression of HGF and its receptor, c-Met, in these cells compared with those from non-endometriosis.
Our current findings of increased production of HGF and its receptor c-Met by LPS-treated M and their exogenous response to cell proliferation by endometriotc cells are in agreement with some recently published reports (Sugawara et al. 1997
; Khan et al., 2003b
). The report by Khan et al. (2003b)
demonstrated that the biological activity of eutopic endometrium, as measured by the immunoreaction of HGF and c-Met in the glandular epithelium and stroma, was similar to that of highly active red peritoneal lesions and was significantly higher than that of controls and other lesions. The most recent publication by Khan et al. (2004b)
further demonstrated that peritoneal lesions of stage III endometriosis, their adjacent peritoneum and corresponding eutopic endometrium harbour abundant M
that could be involved in the growth of endometriosis. This indicates that a persistent inflammatory response in the PF as well as in intact tissue may be responsible for the growth and progression of endometriosis.
Finally we conclude that peritoneal M of women with endometriosis can also produce a significant amount of HGF in response to LPS. These results further evaluated the crucial role of HGF in the histogenesis of endometriosis and its possible involvement in the growth or persistence of endometriosis. In fact, our current findings of HGF at both the transcriptional and protein levels indicate that besides other cytokines and growth factors, HGF may also play an important role in the pathogenesis of endometriosis.
Besides ovarian steroid hormones, the role of the innate immune system in the regulation of endometriosis is also important. Since peritoneal M retain the receptor (TLR4) for LPS derived from Gram-negative bacteria (Triantafilou and Triantafilou, 2002
), we can speculate that a subclinical concentration of endotoxin could stimulate M
, produce different cytokines and growth factors and interact with its neighbouring cells in the pathology of endometriosis. Further studies are needed to find out the exact source of stimulation in regulating the activity of M
and their possible association with infertility or the growth of endometriosis.
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Acknowledgements |
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Submitted on April 8, 2004; resubmitted on July 28, 2004; accepted on August 20, 2004.