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
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
Submitted on December 24, 2007; resubmitted on April 16, 2008; accepted on April 25, 2008.