Phthalates Potentiate the Response of Allergic Effector Cells

Abstract

Several studies strongly indicate that there is a positive correlation between the level of urbanization and industrial development and the incidence of allergic diseases. Increasingly more time is being spend in an indoor environment and the existence of allergy-promoting factors in private homes and public buildings has been suggested (Aas et al. 1997; Poulsen et al. 2000). New chemicals such as plasticizer-containing plastics have been introduced to the indoor environment. The plasticizers are not covalently bound to the plastic matrix and can therefore be released from products and become airborne or absorbed to small dust particles leading to human exposure by inhalation (Oie et al. 1997; Jaakkola et al. 1999; Clausen et al. 2003). Recently, a study in a murine model suggested phthalates and monophthalates to stimulate the IgE and IgG1 production (Larsen et al. 2002). Yet another study shows an increase in intracellular Ca2+ concentration and degranulation in the rat basophillic leukemia cell line RBL-2H3 when stimulated with diphthalates (Nakamura et al. 2002). To test for a possible effect in man we have used the basophil histamine release assay, which models the inflammatory part of allergic disease. We examined five commonly used diphthalates and their corresponding five monophthalates, which are the primary metabolites after hydrolysis. Peripheral blood mononuclear cells containing 0.1–1% basophils obtained from freshly drawn blood or blood bank buffy coats were preincubated with mono- or di-phthalate (5–500 μM) for 15 to 60 min. (mono-n-butyl phthalate (MBuP), mono-n-octyl phthalate (MOP), mono-iso-nonyl phthalate (MINP) and mono-iso-decyl phthalate (MIDP) were all synthesised at the National Institute of Occupational Health, Copenhagen, Denmark. The rest of the mono- and di- phthalates were obtained from Tokyo Kasei Organic Chemicals (TCI), Tokyo, Japan (Larsen et al. 2001 & 2002). Subsequently, phthalate was removed by washing and the cells stimulated with anti-IgE (A0094, DAKO A/S, Denmark), fMLP (N-formyl-methionyl-leucyl-phenylalanine, Sigma, Denmark), Calcium Ionophore A23187 (Sigma, Denmark) or allergen (cat hair extract, ALK-Abelló, Denmark) for 60 min. Cells were washed and the histamine release was determined by using the o-phthaldialdehyde method and expressed in percentages of the total content of histamine (Skov et al. 1985). The spontaneous histamine release from the basophils in this assay was less than 10% of the total histamine content of the cells. In the experiments stimulating with cat hair extract blood samples were obtained from three atopic patients. All patients had a positive (mean wheal diameter >=3 mm) duplicate skin prick test to cat hair extract (Soluprick, ALK-Abelló, Denmark) and specific IgE to Cat (>=CAP class 2, Pharmacia, Sweden). None of the phthalates tested were found to induce histamine release per se. However, when crossbinding the high affinity IgE receptor (FcεRI) by stimulation with anti-IgE antibody an increased histamine release was obtained. In table 1 the results from a screening of the five mono- and five diphthalates are listed. Phthalates and monophthalates with an 8 carbon atoms alkyl side chain length turned out to be the strongest histamine release potentiators, whereas those with a four, nine or ten carbon atoms side chain length have none or low inducing effect on histamine release. These results indicate that the potentiating effect from phthalates with an 8 carbon atoms alkyl side chain length is due to a biological effect rather than a more general physical/chemical effect. At the anti-IgE concentration (1:400) producing the optimal response no differences in potentiation was observed when comparing the different phthalate preincubation periods (15, 30 or 60 min.) (fig. 1). Basophils from freshly isolated PBMCs from a non-atopic donor were preincubated with (solid symbols) or without (open symbols) MEHP (mono-(2-ethylhexyl) phthalate) (500 μM) for 15 min. (circle), 30 min. (square) or 60 min. (triangle) followed by stimulation with a dilution series of anti-IgE (1 hr) (insert panels). Data in the main figure are presented as differences in histamine release from pthalate treated and non-phthalate treated cells (HRanti-IgE,MEHP÷HRanti-IgE,buffer) (gray symbols). Histamine release in non-phthalate treated cells were 4.4–30.2% of total histamine content. The bacterial derived peptide fMLP can be used to differentiate whether the effect on histamine release is limited to the IgE-mediated signalling pathway or whether a more general mechanism is involved. Calcium mobilization is necessary for both FcεRI-dependent and -independent exocytosis of granule-stored mediators. Accordingly, PBMCs from atopic and non-atopic donors were pretreated with the two most efficacious potentiators; mono- and di-2-ethylhexyl phthalate (MEHP and DEHP) followed by washing. Cells were then stimulated with fMLP, calcium ionophore or cat hair extract, all giving rise to an average of a 88% increase in histamine release from basophils compared to controls. Stimulation with anti-IgE in particular more than tripled histamine release (fig. 2). Whereas these findings suggest that this potentiation is not restricted to the IgE-mediated pathway, the detailed mechanism is still to be outlined. Basophils from freshly isolated PBMCs pretreated with MEHP or DEHP (mono- or di-(2-ethylhexyl) phthalate) (500 μM, 15 min.) followed by stimulation with anti-IgE, fMLP (0.063 μM–4 μM), A23187 (0.063 μM–4 μM) or Cat (cat hair extract) (1 hr). The upper panel indicates the histamine release from pairs treated without phthalate (left) and with phthalate (right) and the lower panel gives the relative potentiation compared to non-phthalate treated control. Calculation of potentiation is based on the stimulus concentration producing the optimal response in the buffer experiment (e.g. %. Histamine release in non-phthalate treated cells were 10.1–37.3% of total histamine content. Cat allergic donors (solid symbols, n=3) and non-atopics donors (open symbols, n=2). PBMCs from non-atopics were treated with anti-IgE or fMLP only. In our experiments histamine release was measured as an exclusive marker of basophil granulocytes. One could suggest that other cells in the mixed leukocyte population used in this assay could be stimulated by the phthalates and subsequently affect the basophils. However, since the potentiation of histamine release from the basophils is observed within 15 min. there is a good indication that it is a fast mechanism without any influence from other leukocytes in the mixed population of cells used in this assay. An epidemiological study has shown that development of bronchial obstruction in children was associated with the presence of polyvinyl chloride (PVC) in their homes (Jaakkaola et al. 1999). Together with the findings by Clausen et al. (2003) that floor dust collected from Danish schools may contain up to 1% (w/w) DEHP, there is good evidence that the general population is exposed to phthalates by the inhalation route. Several efforts have been made to estimate the exposure and intake of phthalates in the general population by measuring both the primary metabolites in urine or the levels of non-degraded phthalates in blood samples (Faouzi et al. 1999; Koch et al. 2003a & b). When comparing the phthalate levels in blood from haemodialysis patients (Faouzi et al. 1999) with our in vitro experiments, the blood levels correspond to the lowest concentrations in the experimental cell cultures. However, since phthalates are highly insoluble in aqueous solutions one might speculate that the phthalates would be concentrated in the membrane lipid bi-layers of the cells leading to an increased local concentration in vivo. In conclusion, exposure to phthalates may affect the allergic effector cells leading to increased mediator release in susceptible individuals. This augmentation of the inflammatory response by phthalates may have impact on the risk evaluation of this widespread group of industrial chemicals. This work was sponsored by the “Foundation for Allergy Research”, Copenhagen, Denmark.