17β-estradiol and lipopolysaccharide additively promote pelvic inflammation and growth of endometriosis

Abstract

Endometriosis is a multifactorial disease mostly affecting women of reproductive age. An additive effect between inflammation and stress reaction on the growth of endometriosis has been demonstrated. Here we investigated the combined effect between 17β-estradiol (E2) and lipopolysaccharide (LPS) on pelvic inflammation and growth of endometriotic cells. Peritoneal fluid was collected from 46 women with endometriosis and 30 control women during laparoscopy. Peritoneal macrophages (Mφ) and stromal cells from eutopic/ectopic endometrial stromal cells (ESCs) were isolated from 10 women each with and without endometriosis in primary culture. Changes in cytokine secretion (interleukin 6 [IL-6] and tumor necrosis factor α [TNF-α]) by Mφ and proliferation of ESCs in response to single and combined treatment with E2 and LPS were measured by enzyme-linked immunosorbent assay and by bromodeoxyuridine incorporation assay, respectively. A significantly increased secretion of IL-6 and TNF-α in Mφ culture media was found in response to E2 (10(-8) mol/L) compared to nontreated Mφ. This effect of E2 was abrogated after pretreatment of cells with ICI 182720 (10(-6) mol/L; an estrogen receptor [ER] antagonist). Combined treatment with E2 and LPS (10 ng/mL) additively promoted IL-6 and TNF-α secretion by peritoneal Mφ and growth of eutopic/ectopic ESCs. The additive effects of E2 + LPS on cytokine secretion and growth of ESCs were effectively suppressed after combined blocking of ER and Toll-like receptor 4. An additive effect was observed between E2 and LPS on promoting proinflammatory response in pelvis and growth of endometriosis.

Keywords: LPS; endometriosis; estradiol; macrophages; stromal cells.

Figure 1.

Concentrations of 17β-estradiol (E₂; A) and endotoxin (lipopolysaccharide [LPS]; B) in sera, menstrual fluid (MF), and peritoneal fluid (PF) derived from control women (white box) and women with endometriosis (hatched box). B, *P < .05 versus controls for either MF or PF. Boxes represent the distance (interquartile range) between the first (25%) and the third (75%) quartiles, and horizontal lines in the boxes represent median values.

Figure 2.

Time-dependent changes in the secretion of interleukin (IL) 6 (A) and tumor necrosis factor α (TNF-α; B) in the culture media of macrophages (Mφ) derived from the peritoneal fluid of 10 women with endometriosis (black bar) and 10 control women (white bar) and in response to 17β-estradiol (E₂; 10⁻⁸ mol/L). *P < .05 versus controls for each indicated incubation time. The results are expressed as mean ± standard error of the mean (SEM) of triplicate experiments with Mφ derived from each woman.

Figure 3.

Production of interleukin (IL) 6 (A) and tumor necrosis factor α (TNF-α; B) in the culture media of peritoneal fluid macrophages (Mφ) derived from 6 women each with endometriosis and control women and in response to 17β-estradiol (E₂), progesterone (P), a combination of E₂ and P (E₂ + P), and ICI, an estrogen receptor (ER) antagonist + E₂, and ICI + E₂ + P. *P < .05 versus E₂-untreated Mφ (A and B). The results are expressed as mean ± standard error of the mean (SEM) of triplicate experiments for each treatment.

Figure 4.

Production of interleukin (IL) 6 (A) and tumor necrosis factor α (TNF-α; B) in the culture media by lipopolysaccharide (LPS)-activated (10 ng/mL) and/or 17β-estradiol (E₂; 10⁻⁸ mol/L)- and progesterone (P; 10⁻⁶ mol/L)-treated macrophages (Mφ) that were derived from the peritoneal fluid of 10 women without endometriosis. The results are expressed as mean ± standard error of the mean (SEM) of triplicate experiments with Mφ.

Figure 5.

The exogenous single or combined effect of 17β-estradiol (E₂) and LPS on the production of interleukin (IL) 6 (A) and tumor necrosis factor α (TNF-α; B) in the culture media of Mφ derived from the peritoneal fluid of 10 women each with endometriosis (black bar) and control women (white bar). *P < .05 versus control; ^† P < .05 versus individual LPS- or E₂-treated Mφ; ^‡ P < .05 versus ICI-untreated Mφ; ^¶ P < .05 versus single treatment with ICI or anti-Toll-like receptor 4 (anti-TLR4) antibody. The results are expressed as mean ± standard error of the mean (SEM) of triplicate experiments for each treatment. LPS indicates lipopolysaccharide.

Figure 6.

The exogenous single or combined effect of LPS and 17β-estradiol (E₂) on the bromodeoxyuridine (BrdU) incorporation into eutopic (A) and ectopic (B) endometrial stromal cells (ESCs) derived from 10 women each with endometriosis (black bar) and control women (white bar). Results of BrdU incorporation are expressed as a percentage of control. The BrdU incorporation of nontreated cells equals 100%. Like cytokine secretion as shown in Figure 5, a similar pattern of BrdU incorporation in eutopic/ectopic ESCs was observed in response to treatment with E₂, LPS, LPS + E₂, ICI (an estrogen receptor [ER] antagonist), and anti-TLR4 antibody (A and B). *P < .05 versus control ESCs (A); *P < .05 versus nontreated ectopic ESCs (B); ^† P < .05 versus single LPS or E₂ treatment (A and B); ^‡ P < .05 in (A) and ^# P < .05 in (B) versus LPS-treated ESCs; ^‡ P < .05 versus LPS + E₂-treated cells (B); ^¶ P < .05 versus single treatment with ICI or anti-TLR4 antibody (A and B). The results are expressed as mean ± standard error of the mean (SEM) of triplicate experiments for each treatment. LPS indicates lipopolysaccharide; TLR4, Toll-like receptor 4.

Figure 7.

Concentrations of interleukin (IL) 6 (white box) and tumor necrosis factor α (TNF-α; hatched box) in the peritoneal fluid derived from 30 control women, 46 women with endometriosis and 20 gonadotropin-releasing hormone agonist (GnRHa)-treated women. Boxes represent the distance (interquartile range) between the first (25%) and third (75%) quartiles, and horizontal lines in the boxes represent median values.