Inflammatory cytokine profile of co‑cultivated primary cells from the endometrium of women with and without endometriosis

Abstract

Endometriosis is a chronic gynecological disorder defined as the presence of endometrial tissue within extra-uterine sites. The primary symptoms are infertility and chronic pain. The inflammatory environment and aberrant immune responses in women with endometriosis may be directly associated with the initiation and progression of endometriotic lesions. In the present study, the secretion of inflammatory cytokines was evaluated in cultures of primary endometrial cells (ECs) isolated from the endometrium of women with and without endometriosis. The presence of endometriotic cells leads to alterations in the secretory profile of healthy ECs. The expression of the inflammatory cytokines interleukin (IL)‑6 and IL‑8 was significantly increased in endometriotic and co‑cultured cells compared with healthy ECs. IL‑6 expression was strongly correlated with IL‑8 expression in endometriotic cells. IL‑1β expression was increased on day 10 of co‑culture to 48.30 pg/ml and may be associated with the long‑term co‑culture, rather than IL‑6 and IL‑8 expression. IL‑6 expression was strongly correlated with cell number, whereas IL‑8 expression was moderately correlated with cell number. Additionally, it was observed that co‑cultured cells exhibited a different population of cells, with expression of the mesenchymal stem cell marker cell surface glycoprotein MUC18, indicating a putative role of endometrial mesenchymal stem cells in the secretion of cytokines and disease development. These results indicate a predominant role of primary endometriotic cells in the secretion of cytokines, which contributes to the disrupted peritoneal and endometrial environment observed in the women with endometriosis.

Figure 1.

Representative fluorescent images of co-cultured ECs. Endometriotic ECs were unstained and healthy ECs were stained with Calcein AM 24 h after cell seeding. Calcein AM-stained cells are visualized in green. (A) Blank channel. (B) Green channel. Scale bar, 100 µm. ECs, endometrial cells.

Figure 2.

IL-6 and IL-8 culture medium secretion over time. (A) IL-6 concentration curves of each group. (B) IL-8 concentration curves of each group mean. (C) Statistical analysis of IL-6 and (D) IL-8 concentrations between groups. Data are presented as the mean ± standard error of the endometriosis, healthy and co-cultured endometrial cells. *Repeated measures analysis of variance between the three groups. ^#Significance within subjects. IL, interleukin; EIV, endometriotic endometrial cells; H, healthy cells, Co, co-cultured cells.

Figure 3.

Flow cytometry analysis of the healthy, endometriotic and co-cultured ECs. (A) Endometriotic ECs cultivated alone prior to co-culture and (B) healthy ECs cultured alone prior to co-culture. (C) Co-cultured ECs at day 1, (D) day 3, (E) day 7 and (F) day 10. (G) Frequency of P1 and P2 during co-culture. (H) Statistical analysis of each population frequency between each day of co-culture and cells cultured alone. The elliptical delimitations 1 and 2 indicate the two populations delimited by differences in size and complexity in each sample. The images are representative for all samples. Data are presented as the mean of each sample group ± standard error. ECs, endometrial cells; EIV, endometriotic ECs; D1, day 1 of co-culture; D3, day 3 of co-culture, D7, day 7 of co-culture; D10, day 10 of co-culture; FSC, forward scatter; H, healthy ECs; P1, population one; P2, population two; SSC, side scatter.

Figure 4.

CD13 expression in each cell population. In the histograms, light gray dotted lines indicate background fluorescence obtained with the isotype control immunoglobulin G1. CD13 expression is represented by the light gray filled areas. The × axis represents fluorescence intensity and the y axis represents cell count. (A) Histogram for CD13 expression in P1 of healthy ECs and (B) EIV. (C) Histogram for CD13 expression in P2 of healthy ECs and (D) EIV. (E) Histogram for CD13 expression on P1 at day 1, (F) day 3, (G) day 7 and (H) day 10 of EC co-culture. (I) Histogram for CD13 expression on P2 at day 1, (J) day 3, (K) day 7 and (L) day 10 of EC co-culture. (M) CD13 expression in each population. (N) Statistical analysis of CD13 expression between each day of co-culture for P1 and P2. Data are presented as the mean of each sample group ± standard error. ECs, endometrial cells; CD13, aminopeptidase N; P1, population one; P2, population two; H, healthy ECs; EIV, endometriotic ECs; D1, day 1 of co-culture; D3, day 3 of co-culture; D7, day 7 of co-culture; D10, day 10 of co-culture.

Figure 5.

CD146 expression in cell each population. In the histograms, light gray dotted lines indicate background fluorescence obtained with the isotype control immunoglobulin G1. CD146 expression is represented by the light gray filled areas. The × axis represents fluorescence intensity and the y axis represents cell count. (A) Histogram for CD146 expression in P1 of healthy ECs and (B) EIV. (C) Histogram for CD146 expression in P2 of healthy ECs and (D) EIV. (E) Histogram for CD146 expression on P1 at day 1, (F) day 3, (G) day 7 and (H) day 10 of EC co-culture. (I) Histogram for CD146 expression on P2 at day 1, (J) day 3, (K) day 7 and (L) day 10 of EC co-culture. (M) CD146 expression in each population. (N) Statistical analysis of CD146 expression between each day of co-culture for P1 and P2. Data are presented as the mean of each sample group ± standard error. ECs, endometrial cells; CD146, cell surface glycoprotein MUC18; P1, population one; P2, population two; H, healthy; ECs, EIV, endometriosis ECs; D1, day 1 of co-culture; D3, day 3 of co-culture; D7, day 7 of co-culture; D10, day 10 of co-culture.