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Toxicology Letters 177 (2008) 31–37
TCDD exposure exacerbates atopic dermatitis-related inflammation in NC/Nga mice Tomohiro Ito a,b , Kaoru Inouye a,c , Keiko Nohara a,b , Chiharu Tohyama a,b , Hidekazu Fujimaki a,b,∗ a
Environmental Health Sciences Division, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan b CREST, JST (Japan Science and Technology), Kawaguchi 332-0012, Japan c Japan Society for the Promotion of Science (JSPS), Tokyo 102-8471, Japan
Received 3 September 2007; received in revised form 13 December 2007; accepted 13 December 2007 Available online 4 January 2008
Abstract Our previous study showed that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure of NC/Nga mice, a mouse model of atopic dermatitis, induces no dermal changes. In the present study, to investigate whether TCDD exacerbates atopic dermatitis-like skin lesions elicited in NC/Nga mice, NC/Nga mice were applied with picryl chloride (PC), and then were exposed to a single oral dose of 0 (control), 5, and 20 g TCDD/kg. Two weeks later, spleens, blood, and skin specimens were collected. TCDD exposure increased the production of Th1-type cytokine IFN-␥, but not Th2-type cytokine IL-4, from spleen cells stimulated with a mitogen. The plasma total IgE antibody levels of the TCDD-exposed mice remained at control levels. On the other hand, TCDD exposure markedly increased the mast cell infiltration and degranulation in PC-sensitized NC/Nga mice histologically, as compared with control mice. These results suggest that TCDD exposure exacerbates atopic dermatitis-related inflammation with no increase of IgE antibody production and that TCDD may be one of the environmental pollutants that induce exacerbations of atopic diseases. © 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: 2,3,7,8-Tetrachlorodibenzo-p-dioxin; NC/Nga mice; Atopic dermatitis; Picryl chloride; IgE; Mast cell
1. Introduction Epidemiological studies have shown an increase in the incidence of atopic dermatitis worldwide, especially in developed countries (Bj¨orkst´en, 1999; Burr et al., 1989; Grize et al., 2006; Von Mitius, 2000). The preferential deviation toward immune responses by type 2 helper T (Th2) cells is important to the pathogenesis of acute phase of atopic diseases. This deviation leads to elevation of serum IgE levels and infiltration by inflammatory cells through aberrant production of Th2-type cytokines and chemokines (Ong and Leung, 2006). On the other hand, Th1-type immune reaction becomes predominant in chronic skin lesions (Leung et al., 2004). The lesional skin of atopic dermatitis patients is histologically characterized by hypertrophy of the
∗ Corresponding author at: Environmental Health Sciences Division, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan. Fax: +81 29 850 2518. E-mail address: [email protected] (H. Fujimaki).
0378-4274/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2007.12.011
epidermis and localization of inflammatory cells in the epidermis (Horan et al., 1992; Leung, 1992). The atopic diseases are thought to be heritable (Dold et al., 1992; Schultz Larsen, 1993), but many animal studies have shown that exposure to various environmental pollutants exacerbates atopic status (Iwamura et al., 2006; Riedl and DiazSanchez, 2005; Takano et al., 2006; Walczak-Drzewiecka et al., 2003). Those studies have indicated that many chemical compounds produced by industrial development may contribute to the recent increase in incidence of atopic diseases. Therefore, searching for environmental pollutants that exacerbate atopic diseases and elucidation of the mechanism are important and urgent tasks for the health of human and wildlife. Dioxins, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are well-known environmental pollutants, and exert a number of toxicities through the arylhydrocarbon receptor (AhR) (Fernandez-Salguero et al., 1996; Vorderstrasse et al., 2001). Others and we have reported that TCDD suppresses the Th2-type immune reaction in immunized mice through the impairment of T cell functions (Ito et al., 2002; Kerkvliet,
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2002). On the other hand, TCDD has been reported to increase IgE production by B cells from atopic patients (Kimata, 2003), and to induce histamine-releasing factor production (Oikawa et al., 2002), which causes itching through the release of histamine (MacDonald et al., 1987). In addition, TCDD has been reported to affect the differentiation, proliferation, and immortalization of keratinocytes in the epidermis (Hebert et al., 1990; Loertscher et al., 2001, 2002; Ray and Swanson, 2004). Furthermore, Tauchi et al. (2005) generated transgenic mice in which a constitutive active form of AhR is specifically expressed in keratinocytes, and these transgenic mice develop atopic dermatitis-like lesions. Therefore, although the effects of dioxins vary among target cells, these reports suggest that dioxins may be included in the environmental pollutants that exacerbate the atopic skin diseases. We previously studied the effects of TCDD on immune functions of NC/Nga mice (Fujimaki et al., 2002), the most widely used animal model of atopic dermatitis (Matsuda et al., 1997). Our previous study showed that TCDD suppressed Th2type immune reactions, such as IL-4 cytokine production and antigen-specific IgE production, in NC/Nga mice immunized with ovalbumin and alum, with no obvious induction of skin lesions in the pinnae and dorsal skin. Although these results indicate that TCDD exposure alone does not provoke pathologic skin changes in NC/Nga mice, it remained unclear whether TCDD exacerbates atopic lesions of NC/Nga mice that had been sensitized and challenged with allergen, and considerable interest has centered on the timing of TCDD exposure and allergen injection. In the present study we investigated whether TCDD exposure exacerbates the skin lesions of NC/Nga mice presensitized and challenged with a hapten, picryl chloride (PC). Repeated application of PC in the skin of NC/Nga mice easily develops atopic dermatitis-like skin lesions, and this model is valuable for studying pathogenesis of atopic dermatitis (Kato et al., 2005; Matsukura et al., 2005; Taniguchi et al., 2003).
were challenged by application of 50 and 10 l of 0.8% PC in olive oil/acetone to the dorsal skin and right ear, respectively. On day 21, the mice were exposed to a single dose of TCDD (5 g/kg or 20 g/kg) by gavage before application of PC, while control mice received vehicle alone (corn oil containing 4% nonane). On day 35, the mice were euthanized by ether, and their blood, spleen, pinnae, and dorsal skin were collected. After the blood was centrifuged, the plasma was collected for measurement of total IgE by ELISA.
2.4. Flow cytometric analysis Cell suspensions were prepared from the spleen by passage through a stainless mesh as described previously (Nohara et al., 2002). B cells, CD3+ T cells, CD4+ T cells, and CD8+ T cells in the cell suspension were stained with fluorescein isothiocyanate (FITC)-labeled anti-CD45R/B220 (RA3-6B2), phycoerythrin (PE)-labeled anti-CD3 (17A2), PE-labeled anti-CD4 (GK1.5), and FITC-labeled anti-CD8 (53-6.7), respectively, and corresponding isotypematched antibodies were used as control. The cells were incubated with each antibody for 30 min on ice, and then treated with 7-aminoactinomycin D to label dead cells. Each cell population was determined with a FACSCalibur flow cytometer (BD, Mountain View, CA). FITC and PE emissions from the 488 nm excitation were collected through FL-1 (530/30 nm) and FL-2 (585/42 nm) BP filters, respectively. All antibodies were purchased from BD PharMingen (San Diego, CA).
2.5. Measurement of cytokine levels released by mitogen-stimulated spleen cells Each cell suspension (2.5 × 105 cells per well) was cultured in 200 l of RPMI-1640 medium containing 10% fetal calf serum (Invitrogen, Carlsbad, CA) with or without mitogen (25 g/ml PHA). After 48 h, the concentration of IL-4 and IFN-␥ in the supernatant was measured with commercially available mouse cytokine ELISA kits (Endogen, Woburn, MA and Amersham International, Little Chalfont, Buckinghamshire, England).
2.6. Measurement of total IgE titers in plasma Plasma total IgE titers were measured by ELISA as described previously (Fujimaki et al., 2002).
2.7. Histological analysis 2. Materials and methods 2.1. Animals Male NC/Nga mice (6 weeks old) were purchased from Charles River Laboratories Japan Inc. (Tokyo, Japan) and acclimatized to their housing environment for 1 week prior to use. They were given free access to food and water, and their room was maintained under controlled conditions at a temperature of 24 ± 1 ◦ C, a humidity of 50 ± 10%, and under a 12/12 h light/dark cycle. The mice were handled in a humane manner according to NIES guidelines
2.2. Reagents TCDD was purchased from Cambridge Isotope Laboratories (Andover, MA) and various concentrations of TCDD were prepared by diluting it with corn oil containing 4% nonane. PC was diluted with an ethanol/acetone solution (4:1 v/v) for sensitization, and with olive oil/acetone (4:1 v/v) for challenge. Phytohemagglutinin (PHA) was purchased from Sigma (St. Louis, MO).
2.3. Animal treatment On day 0, under ether anesthesia, the back of each mouse was shaved with electric clippers. The mice were sensitized by application of 150 l doses of 5% PC in ethanol/acetone to the dorsal skin. On days 7, 14, 21, and 28, the mice
The pinnae and dorsal skin of the mice were fixed with 10% buffered formalin and embedded in paraffin. Each deparaffinized section was stained with hematoxylin and eosin (HE) for histological analysis or with toluidine blue (1% aqueous solution) to stain mast cells. The mast cells were identified by their morphological features and metachromasia by toluidine blue staining, and classified as extensively degranulated (>50% of the cytoplasmic granules exhibiting fusion or discharge, and extrusion from the cell), moderately degranulated (10–50%), or normal (at 400×) (Wershil et al., 1988). The mast cells per a field were observed under a light microscope. And then, cells expressing degranulation were counted. To avoid possible bias, the cells were counted by the person who did not know the source of the material. Histological analysis of dorsal skin and pinnae of infiltrated mast cells was determined by examining at least three fields from each mouse and the total number of degranulated mast cells in the skins from six mice was averaged in each group.
2.8. Statistical analysis All data are expressed as mean ± S.E. Statistical analysis was performed by one-way ANOVA followed by post hoc analysis using Dunnett’s multiple comparison test. Significant difference of IFN-␥ between PHA- and non-stimulated groups was also evaluated by Student’s t-test. p < 0.05 was considered to be statistically significant.
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Table 1 Effects of TCDD exposure on the cellularity of spleen in PC-treated NC/Nga mice TCDD (g/kg)
B cells
CD3 T cells
CD4 T cells
CD8 T cells
0 5 20
63.6 ± 1.0 63.8 ± 2.4 69.6 ± 0.7*
25.8 ± 0.4 25.0 ± 1.3 21.2 ± 0.5*
18.8 ± 0.5 18.0 ± 0.5 16.3 ± 0.4*
7.1 ± 0.1 6.7 ± 0.3 5.9 ± 0.2*
Values are mean ± S.E. (n = 6). * p < 0.05 vs. 0 (vehicle control).
3. Results 3.1. Effect of TCDD on the weight and cellularity of immune tissue in PC-sensitized NC/Nga mice TCDD exposure at 5 and 20 g/kg had no effect on the body weight of the PC-sensitized NC/Nga mice (data not shown). Spleen weight and thymus weight did not differ significantly between the TCDD-exposed and control mice (data not shown). The percentages of CD45RB/B220+ B cells in the spleen were increased in the 20 g/kg TCDD group, whereas the percentage of CD3+ T cells was decreased at higher dose of TCDD (Table 1). Similarly, the percentages of CD4+ and CD8+ T cells were also decreased in the 20 g/kg TCDD group. 3.2. Effect of TCDD exposure on cytokine production by mitogen-stimulated spleen cells from PC-sensitized NC/Nga mice To investigate the effect of TCDD exposure on Th1/Th2 immune balance in NC/Nga mice, we examined the production of IL-4 (Th2-type cytokine) and IFN-␥ (Th1-type cytokine) by spleen cells stimulated with a T cell mitogen, PHA. TCDD exposure significantly increased PHA-induced IFN-␥ production only at the 5 g/kg dose of TCDD (Fig. 1b), while it did not induce any significant changes in IL-4 production by PHA-stimulated cells (Fig. 1a). PHA stimulation also significantly increased IFN-␥ production by spleen cells in NC/Nga mice exposed only at 5 g/kg dose of TCDD as compared with non-stimulation (Fig. 1b). On the other hand, we found that TCDD exposure induces no significant changes in IFN-␥
Fig. 1. Effect of TCDD exposure on Th1/Th2 cytokine production by mitogenstimulated spleen cells in PC-sensitized NC/Nga mice. Spleen cells from each mouse were stimulated with 25 g/ml PHA (closed column) or non-stimulated (open column). After 48 h, the concentrations of IL-4 (a) and IFN-␥ (b) in the supernatants were measured with each ELISA kit. Values are mean ± S.E. (n = 6). *p < 0.05 vs. 0 (vehicle control); † p < 0.05 vs. non-stimulated cells.
Fig. 2. Effect of TCDD exposure on plasma total IgE levels of PC-sensitized NC/Nga mice. The plasma total IgE titer of each mouse was measured with ELISA. Values are mean ± S.E. (n = 6). *p < 0.05 vs. 0 (non-treated).
production by non-stimulated cells (Fig. 1b). The amounts of IL-4 in the supernatants of non-stimulated cells from TCDDexposed and control NC/Nga mice were below the detection limit. 3.3. Effect of TCDD exposure on the plasma total IgE levels of PC-sensitized NC/Nga mice Atopic dermatitis-like skin lesions develop with IgE hyperproduction in NC/Nga mice. To investigate whether TCDD exposure affects plasma IgE levels, we measured the plasma total IgE concentration of PC-sensitized NC/Nga mice. Sensitization with PC increased the total IgE levels in the plasma of the NC/Nga mice approximately 2.5-fold compared with non-treated NC/Nga mice (Fig. 2). Exposure to TCDD at the 5 g/kg or 20 g/kg doses did not induce any significant changes in the plasma total IgE levels of PC-sensitized NC/Nga mice (Fig. 2). 3.4. TCDD exposure exacerbates PC-induced histological changes in the pinnae and dorsal skin of NC/Nga mice To explore the effect of TCDD exposure on the skin lesions of PC-sensitized NC/Nga mice, a histological analysis by HE staining was conducted. As shown in Figs. 3 and 4, PC sensitization induced mild thickening of the dermal and epidermal layers of the skin. TCDD exposure thickened them more in the back skin (Fig. 3) and pinnae (Fig. 4), and the thickening was accompanied by eosinophil infiltration and decrease of the fat tissue layer. Next, we examined the effect of TCDD exposure on mast cell infiltration and degranulation of the skin lesions by staining with toluidine blue. PC sensitization induced infiltration of the skin of the NC/Nga mice by toluidine blue-positive cells, as indicated by the arrows (mast cells) (Figs. 3e and f, 4e and f and 5a and b). TCDD exposure increased the numbers of mast cells infiltrating the pinnae and dorsal skin of PC-sensitized NC/Nga mice (Fig. 5a and b), and significantly increased the numbers of extensively degranulated mast cells in the pinnae, but not the back, of the PC-sensitized NC/Nga mice (Fig. 6a and b). These findings suggest that TCDD exposure exacerbates atopic dermatitis-like
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Fig. 3. Histological changes in the back of TCDD-exposed NC/Nga mice. The tissue section from the back was stained with HE (a–d) or 1% toluidine blue (e–h) to stain mast cells (arrow). (a and e) Non-treated mice; (b and f) PC-sensitized mice; (c and g) PC-sensitized and TCDD-exposed (5 g/kg) mice; (d and h) PC-sensitized and TCDD-exposed (20 g/kg) mice.
lesions in NC/Nga mice through mast cell infiltration and activation in the skin. 4. Discussion In this study we investigated whether TCDD exacerbates atopic dermatitis-like skin lesions in PC-sensitized NC/Nga mice. The results suggested that TCDD exacerbates the skin lesions through the enhancement of infiltration and degranulation of mast cells induced by application of PC, and that its actions are not associated with plasma IgE levels. Our findings, therefore, show that dioxins, such as TCDD, may be one of the environmental pollutants involved in the exacerbation of atopic diseases and that this experimental system is a useful model of
evaluating the effect of environmental pollutants on atopic skin diseases. Even though it is well known that IgE plays an important role in the pathogenesis of atopic diseases, our previous and present studies showed that TCDD does not increase plasma IgE levels, regardless of the timing of TCDD exposure (Fujimaki et al., 2002). Therefore, our data suggested that TCDD affects the pathway independent of IgE signaling. In disagreement with our results, it has been reported that IgE production is increased by in vitro TCDD exposure in B cells of human atopic patients (Kimata, 2003). This discrepancy suggests that the effects of TCDD on IgE production are varied by the differences of experimental conditions (in vivo vs. in vitro) and species (mouse vs. human).
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Fig. 4. Histological changes in the pinnae of TCDD-exposed NC/Nga mice. The section of pinnae was stained with HE (a–d) or 1% toluidine blue (e–h) to stain mast cells (arrow). (a and e) Non-treated mice; (b and f) PC-sensitized mice; (c and g) PC-sensitized and TCDD-exposed (5 g/kg) mice; (d and h) PC-sensitized and TCDD-exposed (20 g/kg) mice.
Our data on cytokine production clearly showed that TCDD increases IFN-␥ production by PHA-stimulated spleen cells (Fig. 1b), in accordance with our previous studies using ovalbumin-immunized mice (Fujimaki et al., 2002; Nohara et al., 2002). Our present data also showed that the higher dose of TCDD has relatively weak effect on cytokine production by stimulated splenic T cells. The production of IFN-␥ was significantly increased by TCDD exposure at the 5 g/kg dose, but not 20 g/kg dose (Fig. 1b). These could be at least partially explained by the decrease of percentage of T cell populations (CD3+ , CD4+ , and CD8+ ) in spleen cells at the 20 g/kg dose of TCDD (Table 1). On the other hand, we did not find any evidence for weaker effects of TCDD at the high dose in the results
of plasma IgE levels and histological analyses, suggesting that this weak effect at the higher dose is a characteristic feature of cytokine production by mitogen-stimulated T cells. IFN-␥ has been reported to play an important role of development of atopic dermatitis through thickening of the dermal layer (Spergel et al., 1999) and induction of neurotrophic factors (Grewe et al., 2000). Therefore, our findings suggest that TCDD may exacerbate the skin lesions elicited by allergen-challenges through the enhancement of IFN-␥ production. Since Th1-type immune reaction becomes predominant over Th2-type immune reaction in chronic skin lesions (Leung et al., 2004), the timing of TCDD exposure may be important for exacerbation of atopic dermatitis-like skin lesions.
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Our data show that TCDD increases the numbers of degranulated mast cells in the pinnae, but not the dorsal skin, of PC-sensitized NC/Nga mice (Fig. 6). The mechanism by which the effect of TCDD on the inflammatory symptoms differs between the pinnae and dorsal skin has been unknown, but this tissue specificity of TCDD’s effect may be due to the scratching behavior of pinnae. It is easy to scratch the pinnae rather than in the dorsal skin, and this hypothesis is supported by previous reports showing that scratching behavior contributes to the development of dermatitis (Mihara et al., 2004; Tanaka and Matsuda, 2005). In conclusion, we found that TCDD exposure exacerbates atopic status elicited by PC sensitization of the skin in NC/Nga mice without the enhancement of IgE production. Our data suggest that dioxins may be the cause of the increase in atopic diseases during the last decades. Other environmental pollutants, such as diesel exhaust particles (Ito et al., 2006) and cigarette smoke (Kasai et al., 2006), have a function as AhR ligands, and exposure to combinations of these pollutants may also be a risk factor for atopic diseases. Acknowledgements We thank Mrs. K. Nakazawa and K. Ohnishi for their secretarial assistance. Fig. 5. Mast cell infiltration of the back and pinnae of TCDD-exposed NC/Nga mice. The sections of the back (a) and pinnae (b) were stained with 1% toluidine blue, and the numbers of mast cells were counted. Values are mean ± S.E. (n = 6). **p < 0.01 vs. non-treated (PC−, TCDD−), † p < 0.05 vs. PC-sensitized mice (PC+, 0 g/kg), †† p < 0.01 vs. PC-sensitized mice (PC+, 0 g/kg).
Fig. 6. TCDD exposure increases degranulation of mast cells infiltrating the tissue of NC/Nga mice. The mast cell degranulation in the back (a) and pinnae (b) was evaluated as normal, moderately degranulated, and extensively degranulated. Values are mean ± S.E. (n = 6). *p < 0.05 vs. non-treated (PC−, TCDD−), **p < 0.01 vs. non-treated (PC−, TCDD−), †† p < 0.01 vs. PC-sensitized mice (PC+, 0 g/kg).
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derived cytokine production in ovalbumin (OVA)-immunized C57Bl/6 mice. Toxicology 172, 49–58. Oikawa, K., Ohbayashi, T., Mimura, J., Fujii-Kuriyama, Y., Teshima, S., Rokutan, K., Mukai, K., Kuroda, M., 2002. Dioxin stimulates synthesis and secretion of IgE-dependent histamine-releasing factor. Biochem. Biophys. Res. Commun. 290, 984–987. Ong, P.Y., Leung, D.Y., 2006. Immune dysregulation in atopic dermatitis. Curr. Allergy Asthma Rep. 6, 384–389. Ray, S.S., Swanson, H.I., 2004. Dioxin-induced immortalization of normal human keratinocytes and silencing of p53 and p16INK4a . J. Biol. Chem. 279, 27187–27193. Riedl, M., Diaz-Sanchez, D., 2005. Biology of diesel exhaust effects on respiratory function. J. Allergy Clin. Immunol. 115, 221–228. Schultz Larsen, F., 1993. Atopic dermatitis: a genetic–epidemiologic study in a population-based twin sample. J. Am. Acad. Dermatol. 28, 719–723. Spergel, J.M., Mizoguchi, E., Oettgen, H., Bhan, A.K., Geha, R.S., 1999. Roles of TH 1 and TH 2 cytokines in a murine model of allergic dermatitis. J. Clin. Invest. 103, 1103–1111. Takano, H., Yanagisawa, R., Inoue, K., Ichinose, T., Sadakane, K., Yoshikawa, T., 2006. Di-(2-ethylhexyl) phthalate enhances atopic dermatitis-like skin lesions in mice. Environ. Health Perspect. 114, 1266–1269. Tanaka, A., Matsuda, H., 2005. Expression of nerve growth factor in itchy skins of atopic NC/Nga mice. J. Vet. Med. Sci. 67, 915–919. Taniguchi, Y., Kohno, K., Inoue, S., Koya-Miyata, S., Okamoto, I., Arai, N., Iwaki, K., Ikeda, M., Kurimoto, M., 2003. Oral administration of royal jelly inhibits the development of atopic dermatitis-like skin lesions in NC/Nga mice. Int. Immunopharmacol. 3, 1313–1324. Tauchi, M., Hida, A., Negishi, T., Katsuoka, F., Noda, S., Mimura, J., Hosoya, T., Yanaka, A., Aburatani, H., Fujii-Kuriyama, Y., Motohashi, H., Yamamoto, M., 2005. Constitutive expression of aryl hydrocarbon receptor in keratinocytes causes inflammatory skin lesions. Mol. Cell. Biol. 25, 9360–9368. Von Mitius, E., 2000. The environmental predictors of allergic disease. J. Allergy Clin. Immunol. 105, 9–19. Vorderstrasse, B.A., Steppan, L.B., Silverstone, A.E., Kerkvliet, N.I., 2001. Aryl hydrocarbon receptor-deficient mice generate normal immune responses to model antigens and are resistant to TCDD-induced immune suppression. Toxicol. Appl. Pharmacol. 171, 157–164. Walczak-Drzewiecka, A., Wycz´ołkowska, J., Dastych, J., 2003. Environmental Relevant metal and transition metal enhance FcRI-mediated mast cell activation. Environ. Health Perspect. 111, 708–713. Wershil, B.K., Murakami, T., Galli, S.J., 1988. Mast cell-dependent amplification of an immunologically nonspecific inflammatory response. Mast cells are required for the full expression of cutaneous acute inflammation induced by phorbol 12-myristate 13-acetate. J. Immunol. 140, 2356–2360.
Toxicology Letters 177 (2008) 31–37
TCDD exposure exacerbates atopic dermatitis-related inflammation in NC/Nga mice Tomohiro Ito a,b , Kaoru Inouye a,c , Keiko Nohara a,b , Chiharu Tohyama a,b , Hidekazu Fujimaki a,b,∗ a
Environmental Health Sciences Division, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan b CREST, JST (Japan Science and Technology), Kawaguchi 332-0012, Japan c Japan Society for the Promotion of Science (JSPS), Tokyo 102-8471, Japan
Received 3 September 2007; received in revised form 13 December 2007; accepted 13 December 2007 Available online 4 January 2008
Abstract Our previous study showed that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure of NC/Nga mice, a mouse model of atopic dermatitis, induces no dermal changes. In the present study, to investigate whether TCDD exacerbates atopic dermatitis-like skin lesions elicited in NC/Nga mice, NC/Nga mice were applied with picryl chloride (PC), and then were exposed to a single oral dose of 0 (control), 5, and 20 g TCDD/kg. Two weeks later, spleens, blood, and skin specimens were collected. TCDD exposure increased the production of Th1-type cytokine IFN-␥, but not Th2-type cytokine IL-4, from spleen cells stimulated with a mitogen. The plasma total IgE antibody levels of the TCDD-exposed mice remained at control levels. On the other hand, TCDD exposure markedly increased the mast cell infiltration and degranulation in PC-sensitized NC/Nga mice histologically, as compared with control mice. These results suggest that TCDD exposure exacerbates atopic dermatitis-related inflammation with no increase of IgE antibody production and that TCDD may be one of the environmental pollutants that induce exacerbations of atopic diseases. © 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: 2,3,7,8-Tetrachlorodibenzo-p-dioxin; NC/Nga mice; Atopic dermatitis; Picryl chloride; IgE; Mast cell
1. Introduction Epidemiological studies have shown an increase in the incidence of atopic dermatitis worldwide, especially in developed countries (Bj¨orkst´en, 1999; Burr et al., 1989; Grize et al., 2006; Von Mitius, 2000). The preferential deviation toward immune responses by type 2 helper T (Th2) cells is important to the pathogenesis of acute phase of atopic diseases. This deviation leads to elevation of serum IgE levels and infiltration by inflammatory cells through aberrant production of Th2-type cytokines and chemokines (Ong and Leung, 2006). On the other hand, Th1-type immune reaction becomes predominant in chronic skin lesions (Leung et al., 2004). The lesional skin of atopic dermatitis patients is histologically characterized by hypertrophy of the
∗ Corresponding author at: Environmental Health Sciences Division, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan. Fax: +81 29 850 2518. E-mail address: [email protected] (H. Fujimaki).
0378-4274/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2007.12.011
epidermis and localization of inflammatory cells in the epidermis (Horan et al., 1992; Leung, 1992). The atopic diseases are thought to be heritable (Dold et al., 1992; Schultz Larsen, 1993), but many animal studies have shown that exposure to various environmental pollutants exacerbates atopic status (Iwamura et al., 2006; Riedl and DiazSanchez, 2005; Takano et al., 2006; Walczak-Drzewiecka et al., 2003). Those studies have indicated that many chemical compounds produced by industrial development may contribute to the recent increase in incidence of atopic diseases. Therefore, searching for environmental pollutants that exacerbate atopic diseases and elucidation of the mechanism are important and urgent tasks for the health of human and wildlife. Dioxins, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are well-known environmental pollutants, and exert a number of toxicities through the arylhydrocarbon receptor (AhR) (Fernandez-Salguero et al., 1996; Vorderstrasse et al., 2001). Others and we have reported that TCDD suppresses the Th2-type immune reaction in immunized mice through the impairment of T cell functions (Ito et al., 2002; Kerkvliet,
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2002). On the other hand, TCDD has been reported to increase IgE production by B cells from atopic patients (Kimata, 2003), and to induce histamine-releasing factor production (Oikawa et al., 2002), which causes itching through the release of histamine (MacDonald et al., 1987). In addition, TCDD has been reported to affect the differentiation, proliferation, and immortalization of keratinocytes in the epidermis (Hebert et al., 1990; Loertscher et al., 2001, 2002; Ray and Swanson, 2004). Furthermore, Tauchi et al. (2005) generated transgenic mice in which a constitutive active form of AhR is specifically expressed in keratinocytes, and these transgenic mice develop atopic dermatitis-like lesions. Therefore, although the effects of dioxins vary among target cells, these reports suggest that dioxins may be included in the environmental pollutants that exacerbate the atopic skin diseases. We previously studied the effects of TCDD on immune functions of NC/Nga mice (Fujimaki et al., 2002), the most widely used animal model of atopic dermatitis (Matsuda et al., 1997). Our previous study showed that TCDD suppressed Th2type immune reactions, such as IL-4 cytokine production and antigen-specific IgE production, in NC/Nga mice immunized with ovalbumin and alum, with no obvious induction of skin lesions in the pinnae and dorsal skin. Although these results indicate that TCDD exposure alone does not provoke pathologic skin changes in NC/Nga mice, it remained unclear whether TCDD exacerbates atopic lesions of NC/Nga mice that had been sensitized and challenged with allergen, and considerable interest has centered on the timing of TCDD exposure and allergen injection. In the present study we investigated whether TCDD exposure exacerbates the skin lesions of NC/Nga mice presensitized and challenged with a hapten, picryl chloride (PC). Repeated application of PC in the skin of NC/Nga mice easily develops atopic dermatitis-like skin lesions, and this model is valuable for studying pathogenesis of atopic dermatitis (Kato et al., 2005; Matsukura et al., 2005; Taniguchi et al., 2003).
were challenged by application of 50 and 10 l of 0.8% PC in olive oil/acetone to the dorsal skin and right ear, respectively. On day 21, the mice were exposed to a single dose of TCDD (5 g/kg or 20 g/kg) by gavage before application of PC, while control mice received vehicle alone (corn oil containing 4% nonane). On day 35, the mice were euthanized by ether, and their blood, spleen, pinnae, and dorsal skin were collected. After the blood was centrifuged, the plasma was collected for measurement of total IgE by ELISA.
2.4. Flow cytometric analysis Cell suspensions were prepared from the spleen by passage through a stainless mesh as described previously (Nohara et al., 2002). B cells, CD3+ T cells, CD4+ T cells, and CD8+ T cells in the cell suspension were stained with fluorescein isothiocyanate (FITC)-labeled anti-CD45R/B220 (RA3-6B2), phycoerythrin (PE)-labeled anti-CD3 (17A2), PE-labeled anti-CD4 (GK1.5), and FITC-labeled anti-CD8 (53-6.7), respectively, and corresponding isotypematched antibodies were used as control. The cells were incubated with each antibody for 30 min on ice, and then treated with 7-aminoactinomycin D to label dead cells. Each cell population was determined with a FACSCalibur flow cytometer (BD, Mountain View, CA). FITC and PE emissions from the 488 nm excitation were collected through FL-1 (530/30 nm) and FL-2 (585/42 nm) BP filters, respectively. All antibodies were purchased from BD PharMingen (San Diego, CA).
2.5. Measurement of cytokine levels released by mitogen-stimulated spleen cells Each cell suspension (2.5 × 105 cells per well) was cultured in 200 l of RPMI-1640 medium containing 10% fetal calf serum (Invitrogen, Carlsbad, CA) with or without mitogen (25 g/ml PHA). After 48 h, the concentration of IL-4 and IFN-␥ in the supernatant was measured with commercially available mouse cytokine ELISA kits (Endogen, Woburn, MA and Amersham International, Little Chalfont, Buckinghamshire, England).
2.6. Measurement of total IgE titers in plasma Plasma total IgE titers were measured by ELISA as described previously (Fujimaki et al., 2002).
2.7. Histological analysis 2. Materials and methods 2.1. Animals Male NC/Nga mice (6 weeks old) were purchased from Charles River Laboratories Japan Inc. (Tokyo, Japan) and acclimatized to their housing environment for 1 week prior to use. They were given free access to food and water, and their room was maintained under controlled conditions at a temperature of 24 ± 1 ◦ C, a humidity of 50 ± 10%, and under a 12/12 h light/dark cycle. The mice were handled in a humane manner according to NIES guidelines
2.2. Reagents TCDD was purchased from Cambridge Isotope Laboratories (Andover, MA) and various concentrations of TCDD were prepared by diluting it with corn oil containing 4% nonane. PC was diluted with an ethanol/acetone solution (4:1 v/v) for sensitization, and with olive oil/acetone (4:1 v/v) for challenge. Phytohemagglutinin (PHA) was purchased from Sigma (St. Louis, MO).
2.3. Animal treatment On day 0, under ether anesthesia, the back of each mouse was shaved with electric clippers. The mice were sensitized by application of 150 l doses of 5% PC in ethanol/acetone to the dorsal skin. On days 7, 14, 21, and 28, the mice
The pinnae and dorsal skin of the mice were fixed with 10% buffered formalin and embedded in paraffin. Each deparaffinized section was stained with hematoxylin and eosin (HE) for histological analysis or with toluidine blue (1% aqueous solution) to stain mast cells. The mast cells were identified by their morphological features and metachromasia by toluidine blue staining, and classified as extensively degranulated (>50% of the cytoplasmic granules exhibiting fusion or discharge, and extrusion from the cell), moderately degranulated (10–50%), or normal (at 400×) (Wershil et al., 1988). The mast cells per a field were observed under a light microscope. And then, cells expressing degranulation were counted. To avoid possible bias, the cells were counted by the person who did not know the source of the material. Histological analysis of dorsal skin and pinnae of infiltrated mast cells was determined by examining at least three fields from each mouse and the total number of degranulated mast cells in the skins from six mice was averaged in each group.
2.8. Statistical analysis All data are expressed as mean ± S.E. Statistical analysis was performed by one-way ANOVA followed by post hoc analysis using Dunnett’s multiple comparison test. Significant difference of IFN-␥ between PHA- and non-stimulated groups was also evaluated by Student’s t-test. p < 0.05 was considered to be statistically significant.
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Table 1 Effects of TCDD exposure on the cellularity of spleen in PC-treated NC/Nga mice TCDD (g/kg)
B cells
CD3 T cells
CD4 T cells
CD8 T cells
0 5 20
63.6 ± 1.0 63.8 ± 2.4 69.6 ± 0.7*
25.8 ± 0.4 25.0 ± 1.3 21.2 ± 0.5*
18.8 ± 0.5 18.0 ± 0.5 16.3 ± 0.4*
7.1 ± 0.1 6.7 ± 0.3 5.9 ± 0.2*
Values are mean ± S.E. (n = 6). * p < 0.05 vs. 0 (vehicle control).
3. Results 3.1. Effect of TCDD on the weight and cellularity of immune tissue in PC-sensitized NC/Nga mice TCDD exposure at 5 and 20 g/kg had no effect on the body weight of the PC-sensitized NC/Nga mice (data not shown). Spleen weight and thymus weight did not differ significantly between the TCDD-exposed and control mice (data not shown). The percentages of CD45RB/B220+ B cells in the spleen were increased in the 20 g/kg TCDD group, whereas the percentage of CD3+ T cells was decreased at higher dose of TCDD (Table 1). Similarly, the percentages of CD4+ and CD8+ T cells were also decreased in the 20 g/kg TCDD group. 3.2. Effect of TCDD exposure on cytokine production by mitogen-stimulated spleen cells from PC-sensitized NC/Nga mice To investigate the effect of TCDD exposure on Th1/Th2 immune balance in NC/Nga mice, we examined the production of IL-4 (Th2-type cytokine) and IFN-␥ (Th1-type cytokine) by spleen cells stimulated with a T cell mitogen, PHA. TCDD exposure significantly increased PHA-induced IFN-␥ production only at the 5 g/kg dose of TCDD (Fig. 1b), while it did not induce any significant changes in IL-4 production by PHA-stimulated cells (Fig. 1a). PHA stimulation also significantly increased IFN-␥ production by spleen cells in NC/Nga mice exposed only at 5 g/kg dose of TCDD as compared with non-stimulation (Fig. 1b). On the other hand, we found that TCDD exposure induces no significant changes in IFN-␥
Fig. 1. Effect of TCDD exposure on Th1/Th2 cytokine production by mitogenstimulated spleen cells in PC-sensitized NC/Nga mice. Spleen cells from each mouse were stimulated with 25 g/ml PHA (closed column) or non-stimulated (open column). After 48 h, the concentrations of IL-4 (a) and IFN-␥ (b) in the supernatants were measured with each ELISA kit. Values are mean ± S.E. (n = 6). *p < 0.05 vs. 0 (vehicle control); † p < 0.05 vs. non-stimulated cells.
Fig. 2. Effect of TCDD exposure on plasma total IgE levels of PC-sensitized NC/Nga mice. The plasma total IgE titer of each mouse was measured with ELISA. Values are mean ± S.E. (n = 6). *p < 0.05 vs. 0 (non-treated).
production by non-stimulated cells (Fig. 1b). The amounts of IL-4 in the supernatants of non-stimulated cells from TCDDexposed and control NC/Nga mice were below the detection limit. 3.3. Effect of TCDD exposure on the plasma total IgE levels of PC-sensitized NC/Nga mice Atopic dermatitis-like skin lesions develop with IgE hyperproduction in NC/Nga mice. To investigate whether TCDD exposure affects plasma IgE levels, we measured the plasma total IgE concentration of PC-sensitized NC/Nga mice. Sensitization with PC increased the total IgE levels in the plasma of the NC/Nga mice approximately 2.5-fold compared with non-treated NC/Nga mice (Fig. 2). Exposure to TCDD at the 5 g/kg or 20 g/kg doses did not induce any significant changes in the plasma total IgE levels of PC-sensitized NC/Nga mice (Fig. 2). 3.4. TCDD exposure exacerbates PC-induced histological changes in the pinnae and dorsal skin of NC/Nga mice To explore the effect of TCDD exposure on the skin lesions of PC-sensitized NC/Nga mice, a histological analysis by HE staining was conducted. As shown in Figs. 3 and 4, PC sensitization induced mild thickening of the dermal and epidermal layers of the skin. TCDD exposure thickened them more in the back skin (Fig. 3) and pinnae (Fig. 4), and the thickening was accompanied by eosinophil infiltration and decrease of the fat tissue layer. Next, we examined the effect of TCDD exposure on mast cell infiltration and degranulation of the skin lesions by staining with toluidine blue. PC sensitization induced infiltration of the skin of the NC/Nga mice by toluidine blue-positive cells, as indicated by the arrows (mast cells) (Figs. 3e and f, 4e and f and 5a and b). TCDD exposure increased the numbers of mast cells infiltrating the pinnae and dorsal skin of PC-sensitized NC/Nga mice (Fig. 5a and b), and significantly increased the numbers of extensively degranulated mast cells in the pinnae, but not the back, of the PC-sensitized NC/Nga mice (Fig. 6a and b). These findings suggest that TCDD exposure exacerbates atopic dermatitis-like
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Fig. 3. Histological changes in the back of TCDD-exposed NC/Nga mice. The tissue section from the back was stained with HE (a–d) or 1% toluidine blue (e–h) to stain mast cells (arrow). (a and e) Non-treated mice; (b and f) PC-sensitized mice; (c and g) PC-sensitized and TCDD-exposed (5 g/kg) mice; (d and h) PC-sensitized and TCDD-exposed (20 g/kg) mice.
lesions in NC/Nga mice through mast cell infiltration and activation in the skin. 4. Discussion In this study we investigated whether TCDD exacerbates atopic dermatitis-like skin lesions in PC-sensitized NC/Nga mice. The results suggested that TCDD exacerbates the skin lesions through the enhancement of infiltration and degranulation of mast cells induced by application of PC, and that its actions are not associated with plasma IgE levels. Our findings, therefore, show that dioxins, such as TCDD, may be one of the environmental pollutants involved in the exacerbation of atopic diseases and that this experimental system is a useful model of
evaluating the effect of environmental pollutants on atopic skin diseases. Even though it is well known that IgE plays an important role in the pathogenesis of atopic diseases, our previous and present studies showed that TCDD does not increase plasma IgE levels, regardless of the timing of TCDD exposure (Fujimaki et al., 2002). Therefore, our data suggested that TCDD affects the pathway independent of IgE signaling. In disagreement with our results, it has been reported that IgE production is increased by in vitro TCDD exposure in B cells of human atopic patients (Kimata, 2003). This discrepancy suggests that the effects of TCDD on IgE production are varied by the differences of experimental conditions (in vivo vs. in vitro) and species (mouse vs. human).
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Fig. 4. Histological changes in the pinnae of TCDD-exposed NC/Nga mice. The section of pinnae was stained with HE (a–d) or 1% toluidine blue (e–h) to stain mast cells (arrow). (a and e) Non-treated mice; (b and f) PC-sensitized mice; (c and g) PC-sensitized and TCDD-exposed (5 g/kg) mice; (d and h) PC-sensitized and TCDD-exposed (20 g/kg) mice.
Our data on cytokine production clearly showed that TCDD increases IFN-␥ production by PHA-stimulated spleen cells (Fig. 1b), in accordance with our previous studies using ovalbumin-immunized mice (Fujimaki et al., 2002; Nohara et al., 2002). Our present data also showed that the higher dose of TCDD has relatively weak effect on cytokine production by stimulated splenic T cells. The production of IFN-␥ was significantly increased by TCDD exposure at the 5 g/kg dose, but not 20 g/kg dose (Fig. 1b). These could be at least partially explained by the decrease of percentage of T cell populations (CD3+ , CD4+ , and CD8+ ) in spleen cells at the 20 g/kg dose of TCDD (Table 1). On the other hand, we did not find any evidence for weaker effects of TCDD at the high dose in the results
of plasma IgE levels and histological analyses, suggesting that this weak effect at the higher dose is a characteristic feature of cytokine production by mitogen-stimulated T cells. IFN-␥ has been reported to play an important role of development of atopic dermatitis through thickening of the dermal layer (Spergel et al., 1999) and induction of neurotrophic factors (Grewe et al., 2000). Therefore, our findings suggest that TCDD may exacerbate the skin lesions elicited by allergen-challenges through the enhancement of IFN-␥ production. Since Th1-type immune reaction becomes predominant over Th2-type immune reaction in chronic skin lesions (Leung et al., 2004), the timing of TCDD exposure may be important for exacerbation of atopic dermatitis-like skin lesions.
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Our data show that TCDD increases the numbers of degranulated mast cells in the pinnae, but not the dorsal skin, of PC-sensitized NC/Nga mice (Fig. 6). The mechanism by which the effect of TCDD on the inflammatory symptoms differs between the pinnae and dorsal skin has been unknown, but this tissue specificity of TCDD’s effect may be due to the scratching behavior of pinnae. It is easy to scratch the pinnae rather than in the dorsal skin, and this hypothesis is supported by previous reports showing that scratching behavior contributes to the development of dermatitis (Mihara et al., 2004; Tanaka and Matsuda, 2005). In conclusion, we found that TCDD exposure exacerbates atopic status elicited by PC sensitization of the skin in NC/Nga mice without the enhancement of IgE production. Our data suggest that dioxins may be the cause of the increase in atopic diseases during the last decades. Other environmental pollutants, such as diesel exhaust particles (Ito et al., 2006) and cigarette smoke (Kasai et al., 2006), have a function as AhR ligands, and exposure to combinations of these pollutants may also be a risk factor for atopic diseases. Acknowledgements We thank Mrs. K. Nakazawa and K. Ohnishi for their secretarial assistance. Fig. 5. Mast cell infiltration of the back and pinnae of TCDD-exposed NC/Nga mice. The sections of the back (a) and pinnae (b) were stained with 1% toluidine blue, and the numbers of mast cells were counted. Values are mean ± S.E. (n = 6). **p < 0.01 vs. non-treated (PC−, TCDD−), † p < 0.05 vs. PC-sensitized mice (PC+, 0 g/kg), †† p < 0.01 vs. PC-sensitized mice (PC+, 0 g/kg).
Fig. 6. TCDD exposure increases degranulation of mast cells infiltrating the tissue of NC/Nga mice. The mast cell degranulation in the back (a) and pinnae (b) was evaluated as normal, moderately degranulated, and extensively degranulated. Values are mean ± S.E. (n = 6). *p < 0.05 vs. non-treated (PC−, TCDD−), **p < 0.01 vs. non-treated (PC−, TCDD−), †† p < 0.01 vs. PC-sensitized mice (PC+, 0 g/kg).
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