Additive effects of inflammation and stress reaction on Toll-like receptor 4-mediated growth of endometriotic stromal cells.

STUDY QUESTION: Is there any combined effect between inflammation and stress reaction on Toll-like receptor 4 (TLR4)-mediated growth of endometriotic cells? SUMMARY ANSWER: A combined effect of local inflammation and stress reaction in the pelvic environment may be involved in TLR4-mediated growth of endometriotic stromal cells. WHAT IS KNOWN ALREADY: In endometriosis, higher endotoxin levels in the menstrual fluid (MF) and peritoneal fluid (PF) and higher tissue concentrations of human heat shock protein 70 (HSP70) in the eutopic and ectopic endometria promote TLR4-mediated growth of endometriotic cells. STUDY DESIGN, SIZE AND DURATION: This is a case-controlled research study with prospective collection and retrospective evaluation of sera, MF, PF and endometrial tissues from 43 women with and 20 women without endometriosis. PARTICIPANTS/MATERIALS, SETTING,
METHODS: PF was collected from 43 women with endometriosis and 20 control women during laparoscopy. Sera and endometrial biopsy specimens were collected from a proportion of these women. MF was collected from a separate population of 20 women with endometriosis and 15 control women. HSP70 concentrations in sera, MF, PF and in culture media were measured by ELISA. Gene expression of HSP70 by endometrial cells in response to lipopolysaccharide (LPS) was examined by qRT-PCR. The individual and combined effects of LPS and HSP70 on the secretion of interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) by PF-derived macrophages (M[Symbol: see text]) were examined by ELISA, while their effects on endometrial cell proliferation were examined by bromodeoxyuridine and [(3)H]-thymidine incorporation assay. MAIN RESULTS AND THE ROLE OF CHANCE: Concentrations of HSP70 were maximal in MF, intermediate in PF and the lowest in sera. In MF and PF, HSP70 levels were higher in women with endometriosis than in controls. LPS stimulated gene expression and secretion of HSP70 by eutopic endometrial stromal cells (ESCs) and this effect was abrogated after pretreatment of cells with an anti-TLR4 antibody. This effect was significantly higher for ESCs derived from women with endometriosis than for ESCs from control women. Exogenous treatment with either HSP70 or LPS significantly stimulated the production of IL-6 and TNFα by M[Symbol: see text] and promoted the proliferation of ESCs, and a significant additive effect between LPS and HSP70 was observed. While individual treatment with either polymyxin B, an LPS antagonist, or anti-HSP70 antibody was unable to suppress the combined effects of LPS and HSP70 on cytokine secretion or ESC proliferation, pretreatment of cells with the anti-TLR4 antibody was able to significantly suppress their combined effects. LIMITATIONS, REASONS FOR CAUTIONS: Further studies are needed to examine the mutual role between other secondary inflammatory mediators and endogenous stress proteins in promoting pelvic inflammation and growth of endometriotic stromal cells. WIDER IMPLICATIONS OF THE
FINDINGS: Our findings suggest that endotoxin and HSP70 are mutually involved in a stress reaction and in inflammation. A combined effect between local inflammation and a stress reaction in pelvic environment may be involved in TLR4-mediated growth of endometriotic cells. Since endometriosis is a multi-factorial disease, it is difficult to explain uniformly its growth regulation by a single factor. Our findings may provide some new insights in understanding the physiopathology or pathogenesis of endometriosis and may hold new therapeutic potential. STUDY FUNDING/COMPETING INTEREST(S): 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 K.N.K.). There is no conflict of interest related to this study. TRIAL REGISTRATION NUMBER: Not applicable.