Effects of Endocrine Disruptor Compounds, Alone or in Combination, on Human Macrophage-Like THP-1 Cell Response

International audienceThe aim of the present study was to evaluate the immunological effects on human macrophages of four endocrine disruptor compounds (EDCs) using the differentiated human THP-1 cell line as a model. We studied first the effects of these EDCs, including Bisphenol A (BPA), di-ethylhexyl-phthalate (DEHP), dibutyl phthalate (DBP) and 4-tert-octylphenol (4-OP), either alone or in combination, on cytokine secretion, and phagocytosis. We then determined whether or not these effects were mediated by estrogen receptors via MAPK pathways. It was found that all four EDCs studied reduced strongly the phagocytosis of the differentiated THP-1 cells and that several of these EDCs disturbed also TNF-alpha, IL-1 beta and IL-8 cytokine secretions. Furthermore, relative to control treatment, decreased ERK 1/2 phosphorylation was always associated with EDCs treatments-either alone or in certain combinations (at 0.1 mu M for each condition). Lastly, as treatments by an estrogen receptor antagonist suppressed the negative effects on ERK 1/2 phosphorylation observed in cells treated either alone with BPA, DEHP, 4-OP or with the combined treatment of BPA and DEHP, we suggested that estrogen receptor-dependent pathway is involved in mediating the effects of EDCs on human immune system. Altogether, these results advocate that EDCs can disturb human immune response at very low concentrations

Effect of EDCs alone and in combination on the secretion of IL-1β by differentiated THP-1 cells.

<p>Differentiated THP-1 cells were exposed to different concentrations of BPA or DEHP or DBP or 4-OP or a combination of both for 24 hours. Culture medium was replaced by fresh medium containing 10 ng/mL of LPS for a subsequent 24 hours of incubation. IL-1β concentrations were determined using a sandwich ELISA test. Values are means ± SD (N = 3). Each experiment has been performed three times and in triplicate. a: <i>vs</i> control (Tukey’s test, p<0.05); b: <i>vs</i> combination (One-way ANOVA, Tukey post test, p<0.05).</p

Effect of EDCs alone and in combination on the secretion of TNF-α by differentiated THP-1 cells.

<p>Differentiated THP-1 cells were exposed to BPA, DEHP, DBP 4-OP or a combination of both for 24 hours. Culture medium was replaced by fresh medium containing 10 ng/ml of LPS for a subsequent 24 hours of incubation. TNF-α concentrations were determined using a sandwich ELISA test. Values are means ± SD (N = 3). Each experiment has been performed three times and in triplicate. a: <i>vs</i> control (Tukey’s test, p<0.05); b: <i>vs</i> combination (One-way ANOVA, Tukey post test, p<0.05).</p

Effect of EDCs alone and in combination on estrogen receptor protein expression and signaling.

<p>(A) Differentiated THP-1 cells (PMA 5 ng/ml for 48 hours) were exposed to ICI-182780 (1 μM), BPA, DEHP, BPA + DEHP, DBP, BPA + DBP, 4-OP or 4-OP + DEHP (each EDC at 0.1 μM) for 24 hours. Culture medium was replaced by fresh medium containing 10 ng/mL of LPS for a subsequent 24 hours of incubation. Whole cell lysates (20 μg) were separated by SDS-PAGE (10%) and immunoblotted with the anti-estrogen receptor antibody as described in Materials and Methods. The same blot was stripped and immunoblotted with control antibody (anti-β-actin). Histograms represent the relative quantifications of ERα which were performed in comparison with β-actin. Values in non-treated cells are taken as 100%. Each experiment has been performed four times. Values are means ±SD (N = 4). a: <i>vs</i> control (One-way ANOVA, Tukey post test, p<0.05). (B) Differentiated THP-1 cells (PMA 5 ng/ml for 48 hours) were exposed to ICI-182780 alone and in combination with BPA, DEHP, BPA + DEHP, DBP, BPA + DBP, 4-OP, 4-OP + DEHP for 24 hours. Culture medium was replaced by fresh medium containing 10 ng/mL of LPS for a subsequent 24 hours of incubation. Each EDC was also tested without ICI. EDCs and ICI were used at 0.1 μM and 1 μM respectively throughout this experiment. Whole cell lysates (20 μg) were separated by SDS-PAGE (10%) and immunoblotted with the anti-phospho-ERK antibody as described in Materials and Methods. The same blot was stripped and immunoblotted with anti-ERK antibody. Histograms represent the relative quantifications of phospho-ERK which were performed in comparison with ERK. Values in non-treated cells are taken as 100%. Each experiment has been performed three times. Values are means ±SD (N = 3). a: <i>vs</i> control (One-way ANOVA, Tukey post test, p<0.05); b: <i>vs</i> exposure to corresponding EDCs after treatment by ICI (One-way ANOVA, Tukey post test, p<0.05).</p

Effect of EDCs alone and in combination on phagocytosis by differentiated THP-1 cells.

<p>Differentiated THP-1 cells (PMA 5 ng/ml for 48 hours) were exposed to different concentrations (0.001, 0.1, 1 and 10 μM) of BPA, DEHP, DBP, 4-OP or a combination of both for 24 hours. Culture medium was replaced by fresh medium containing 10 ng/ml of LPS for a subsequent 24 hours of incubation. Panels A and B depict confocal laser microscopy analysis of phagocytosis of FITC-latex beads by untreated differentiated THP-1 cells (A) or by differentiated THP-1 cells exposed to BPA (B). Arrows show examples of phagocytized FITC-beads. Panel C represents the effects of BPA, DEHP, DBP, 4-OP and a combination of both (BPA + DEHP, BPA + DBP, 4-OP + DEHP) on phagocytosis of latex beads by differentiated THP-1 cells. Values are means ± SD (N = 3). Each experiment has been performed three times and in triplicate. a: <i>vs</i> control (One-way ANOVA, Tukey post test, p<0.05).</p