Influence of phthalates on in vitro innate and adaptive immune responses

Abstract

Phthalates are a group of endocrine disrupting chemicals, suspected to influence the immune system. The aim of this study was to investigate the influence of phthalates on cytokine secretion from human peripheral blood mononuclear cells. Escherichia coli lipopolysaccharide and phytohemagglutinin-P were used for stimulation of monocytes/macrophages and T cells, respectively. Cells were exposed for 20 to 22 hours to either di-ethyl, di-n-butyl or mono-n-butyl phthalate at two different concentrations. Both diesters were metabolised to their respective monoester and influenced cytokine secretion from both monocytes/macrophages and T cells in a similar pattern: the secretion of interleukin (IL)-6, IL-10 and the chemokine CXCL8 by monocytes/macrophages was enhanced, while tumour necrosis factor (TNF)-α secretion by monocytes/macrophages was impaired, as was the secretion of IL-2 and IL-4, TNF-α and interferon-γ by T cells. The investigated phthalate monoester also influenced cytokine secretion from monocytes/macrophages similar to that of the diesters. In T cells, however, the effect of the monoester was different compared to the diesters. The influence of the phthalates on the cytokine secretion did not seem to be a result of cell death. Thus, results indicate that both human innate and adaptive immunity is influenced in vitro by phthalates, and that phthalates therefore may affect cell differentiation and regenerative and inflammatory processes in vivo.

Fig 1. Influence of phthalates on the cytokine response of innate immune cells.

MNC cultures were stimulated with 100 pg/ml E. coli LPS and exposed to di-ethyl phthalate (DEP), di-n-butyl phthalate (DnBP), or mono-n-butyl phthalate (MnBP), at two different concentrations, for 20–22 hours. The resulting production of IL-1β, IL-6, CXCL8, IL-10 and TNF-α are shown as ratio to the respective ethanol control. The red dashed line indicates the level of the ethanol controls (ratio = 1). * = p<0.05 compared to ethanol control, # = p<0.05 compared to low phthalate exposure (0.1 μM).

Fig 2. Influence of phthalates on T-cell responses.

MNC cultures were stimulated with phytohemagglutinin-P and exposed to di-ethyl phthalate (DEP), di-n-butyl phthalate (DnBP) or mono-n-butyl phthalate (MnBP), at two different concentrations, for 20–22 hours. The resulting production of IL-2, IL-4, IL-6, IL-10, TNF-α and IFN-γ are shown as ratios to the respective ethanol control. The red dashed line indicates the level of the ethanol controls (ratio = 1). * = p<0.05 compared to ethanol control, # = p<0.05 compared to low phthalate exposure (0.1 μM).

Fig 3. LDH contents (y-axis) in supernatants from LPS- and PHA-P-stimulated MNC exposed to phthalates.

The LDH content was proportional to the produced fluorescence (given in relative fluorescence units (RFUs)).