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Low-dose ethanol aggravates allergic dermatitis in mice
Accepted Manuscript Low-dose ethanol aggravates allergic dermatitis in mice Fumitoshi Sakazaki , Hirofumi Ogino , Tomohiro Arakawa , Tomofumi Okuno , Hitoshi Ueno PII:
S0741-8329(14)00100-1
DOI:
10.1016/j.alcohol.2014.05.001
Reference:
ALC 6406
To appear in:
Alcohol
Please cite this article as: SakazakiF., OginoH., ArakawaT., OkunoT. & UenoH., Low-dose ethanol aggravates allergic dermatitis in mice, Alcohol (2014), doi: 10.1016/j.alcohol.2014.05.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Low-dose ethanol aggravates allergic dermatitis in mice Fumitoshi Sakazaki1, Hirofumi Ogino2, Tomohiro Arakawa2, Tomofumi Okuno2, and Hitoshi Ueno2 1
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Laboratory of Toxicology, Faculty of Pharmacy, Osaka Ohtani University, 3-11-1 Nishikiori-kita, Tondabayashi, Osaka 584-8540, Japan
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Address correspondence to: Fumitoshi Sakazaki, Ph.D. Laboratory of Toxicology Faculty of Pharmacy Osaka Ohtani University 3-11-1 Nishikiori-kita Tondabayashi, Osaka 584-8540, Japan Telephone: +81 721 24 9986 Fax: +81 721 24 9986 Email: [email protected]
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Department of Public Health & Preventive Pharmacology, Faculty of Pharmaceutical Sciences, Setsunan University, 45-1 Nagaotoge-cho, Hirakata Osaka 573-0101, Japan
ACCEPTED MANUSCRIPT Abstract Alcohol injures dendritic cells and suppresses cellular immunity, while some evidence indicates that drinking alcohol aggravates allergic asthma. This study investigated the effect of low doses of ethanol in enhancing allergic reactions in the
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skin of mice. Liquid food containing alcohol was administered to conventional NC/Nga mice to induce alcoholic hepatic steatosis, and spontaneous dermatitis was evaluated. BALB/c mice were administered approximately 1 g/kg body weight of
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ethanol by gavage, and contact hypersensitivity (CHS) or active cutaneous anaphylaxis (ACA) was induced. Spleens were collected 24 h after the elicitation of CHS and
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mRNA expressions of interferon (IFN)-γ, interleukin (IL)-4, IL-6, IL-10, and IL-18 were measured by quantitative RT-PCR. Alcohol-containing diet exaggerated spontaneous dermatitis in conventional NC/Nga mice and contact hypersensitivity in BALB/c mice. Ethanol administered by gavage for 5 days enhanced contact
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hypersensitivity in BALB/c mice. Ethanol administration with gavage also enhanced ACA of BALB/c mice. Ethanol did not affect mRNA expression of IFN-γ and IL-4, but did enhance IL-6, IL-10, and IL-18 mRNA expression. Histological evaluation
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revealed an absence of hepatic steatosis in mice administered ethanol by gavage for 5 days. In ethanol-administered mice, inflamed areas presented as lesions or a local
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extreme accumulation of mononuclear cells in the epidermis. These findings suggest that ethanol enhances the expression of inflammatory cytokines independently from T helper (Th)1/Th2 cytokine phenotypes, causing abnormalities in the epidermis resulting in exacerbated allergic reactivity. Keywords: alcohol, ethanol, allergy, dermatitis
ACCEPTED MANUSCRIPT Introduction Previous studies reported that drinking alcohol aggravates allergic asthma; however, the mechanism has not been fully elucidated (Sisson, 2007; Waldschmidt, Cook, & Kovacs, 2008). The effects of ethanol on skin allergy have been investigated
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but are not fully understood (Lachenmeier, 2008). Alcohol dependence causes the inhibition of cell-mediated immunity (Dehne, Mendenhall, Roselle, & Grossman, 1989), and ethanol impairs the function of dendritic cells (Heinz & Waltenbaugh,
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2007) and macrophages (Pruett & Fan, 2009). However, the relationship between
immunosuppression by alcohol and aggravation of allergy by drinking alcohol has not
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been investigated. Furthermore, a dose-dependent experiment showed that low-dose ethanol enhanced cell-mediated immunity (Dehne et al., 1989). According to the T helper cell (Th)1/Th2 cytokine balance hypothesis (Akahoshi et al., 1999; Girón-González et al., 2000; Wilczyński, 2005), ethanol
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suppresses Th1 cytokines and might enhance Th2 cytokine responses that exacerbate allergic asthma. However, some reports have suggested that the Th1/Th2 balance hypothesis does not explain every immune response (Kitagaki et al., 1997), and recent
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reports have identified additional types of helper T cells, including interleukin (IL)-17producing helper T cells (Th17) and regulatory helper T cells (Treg). To investigate the
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immunotoxicity of ethanol, Th1, Th2, Th17, and Treg should be taken into consideration.
The NC/Nga mouse model develops dermatitis under conventional conditions,
and the symptoms resemble atopic dermatitis in humans. Thus, the NC/Nga mouse is useful for the investigation of allergic dermatitis (Jin, He, Oyoshi, & Geha, 2009). For type I allergy models, passive cutaneous anaphylaxis (PCA) and active cutaneous anaphylaxis (ACA) are used (Inagaki, Miura, Nagai, & Koda, 1992; McCamish & Benedict, 1963). In PCA experiments, immunoglobulin E is artificially administered
ACCEPTED MANUSCRIPT and the permeability of blood vessels is measured. PCA focuses on histamine release from mast cells. In ACA experiments, antigen is administered and the permeability of blood vessels is measured. ACA focuses on immunoglobulin E secretion from B lymphocytes, histamine release from mast cells, and T cell activation that induces
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B cell functions. For type IV allergy models, contact hypersensitivity (CHS) is used (Inagaki & Nagai, 2009). BALB/c or C57BL/6 mice are widely used for ACA and CHS experiments.
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To elucidate the effects of a low dose of ethanol on skin allergies, the present study determined whether a small amount of ethanol enhanced CHS in mice and
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investigated the effects of ethanol on ACA. Materials and Methods Animal treatment
All experimental protocols used in this study were in accordance with the
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animal experiment guidelines of Setsunan University, Japan. These guidelines were adapted from the guidelines of the Japanese Society for Pharmacology. The Committee for the Ethical Use of Experimental Animals of Setsunan University
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approved the study. Efforts were made to minimize animal suffering as much as possible by limiting the number of animals used in experimentation and using
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alternatives for in vivo techniques. A total of 20 to 30 mice of each strain (NC/Nga and BALB/c mice; Japan SLC,
Inc., Hamamatsu, Japan) were housed 5 per cage and allowed 1 week for acclimation while fed a normal diet (MF Certified Diet, Oriental Yeast Co., Ltd.) ad libitum with full access to water. After acclimation, mice were randomly assigned to one of six treatment groups (described below). The mice were kept in a pathogen-free room maintained on a 12-h light-dark cycle (lights on at 7:00 AM), at an ambient
ACCEPTED MANUSCRIPT temperature of 23 ± 1 °C and relative humidity level of 47–67%. Ethanol administration Ethanol (99.5%, Extra Pure Reagent grade, Nacalai Tesque Inc., Kyoto, Japan) was administered in three different ways. For subcutaneous administration, 10 µL of
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ethanol was added to 1 mL of olive oil, vortexed, and sterilized by filtration. An
aliquot of 0.1 mL was injected into mouse dorsal skin. A model diet for alcoholic
hepatic steatosis and its isocaloric control diet were purchased from Oriental Yeast
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Co., Ltd. The ethanol diet and the control diet were mixed to prepare 0, 12.5, 25, and 50 g/L ethanol diets. Each liquid diet was fed to mice ad libitum with a food-water
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bottle. For oral administration with gavage, 0.25 mL of 0, 2.5, 5, 10, 30, and 50% ethanol aqueous solutions were administered. Disulfiram (LKT Laboratories, Inc., St. Paul, MN, USA) was dissolved in olive oil (Kenei Pharmaceutical, Osaka, Japan) and administered intraperitoneally at a dose of 90 mg/kg body weight, one time prior to the
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sensitization of mice. Olive oil was administered as a control for disulfiram. Spontaneous dermatitis in NC/Nga mice
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Conventional 3-week-old female NC/Nga mice weighing 18−20 g (Japan SLC Co., Shizuoka, Japan) received one of four different concentrations of ethanol liquid
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diets (described above) for a period of 8 weeks. The degree of dermatitis observed in mice was scored depending on severity according to a previous report (Sakazaki et al., 2013) with some modifications as follows: 1) presence of scratching behavior, 2) presence of blushing, 3) presence of hair loss, and 4) presence of bleeding and crust. After a 5-week intervention period, we anesthetized mice by intraperitoneal administration of 0.1 mL physiological saline with 10 mg/mL sodium pentobarbital (approximately 50 mg/kg body weight, 1 mg/mouse), and inflamed skin was collected for histological evaluation.
ACCEPTED MANUSCRIPT The mice usually slept and did not scratch their bodies. After cage exchange they started to wander around and to scratch their bodies, and they gradually stopped wandering and scratching and started sleeping again. Frequency of scratching over a few seconds was counted over 1 h after cage exchange by visual observation. Although
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Mihara et al. took video movies of mice from above with a tripod (Mihara, Kuratani, Matsui, Nakamura, & Yokota, 2004), we did not take videos of mice because mice prefer to stay in the shadows rather than being exposed to light. The cages of mice
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were set in a rack and we observed the behavior of mice from the side. CHS
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After administration of ethanol, mice were sensitized by the topical application of 50 µL of 3% 4-ethoxymethylene-2-phenyl-2-oxazoline-5-one (OXA) (SigmaAldrich, Inc., St. Louis, MO, USA) in acetone or a 3:1 (v/v) mixture of ethanol and acetone on the dorsal skin. After 7 days, the mice were challenged for the elicitation of
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allergy by applying 7.5 µL of 0.1% OXA in acetone or ethanol to both sides of the right auricle. The auricle thickness was measured under ether anesthesia using a digital thickness gauge (Ozaki MFG Co. Ltd., Tokyo, Japan) while taking care to avoid
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denting the edematous skin. The degree of ear swelling was calculated by subtracting
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the thickness of the left ear from that of the right ear.
Seven-week-old female BALB/c mice weighing 18–20 g (Japan SLC Co.)
were fed one of the four alcohol liquid diets for the indicated period before we induced ACA responses with subcutaneous administration of 1 µg ovalbumin (OVA) (SigmaAldrich) and 1 mg alum (Sigma-Aldrich). Two weeks after the initial administration of OVA and alum, the mice were anesthetized by intraperitoneal administration of 0.1 mL of physiological saline with 10 mg/mL sodium pentobarbital (approximately
ACCEPTED MANUSCRIPT 50 mg/kg body weight, 1 mg/mouse). Then, 250 µL of 0.5% Evan’s blue solution was injected into the tail vein, and 10 µL of 0.1 mg/mL OVA was injected subcutaneously into the right ear lobe. As a control, physiological saline was injected into the left ear lobe. To measure the exudate of Evan’s blue, the ear lobes of mice were excised
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30 min post injection, incubated overnight in 0.3 mL KOH solution at 37 °C,
neutralized with 10 mol/L phosphoric acid-acetone (3:67) solution (0.7 mL), and centrifuged at 15,000 × g for 10 min at room temperature. The absorbance of the
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supernatant was measured at 620 nm. Histological evaluation
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The skin, including areas of inflammation, was removed surgically and fixed in 4% formalin, containing 0.1 mol/L phosphate buffer, overnight. The skin was then embedded in paraffin and sectioned. Sections on glass slides were dried at 37 °C overnight and stained with hematoxylin and eosin to evaluate the general structure, and
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slides were examined by microscopy.
Reverse transcription-polymerase chain reaction (RT-PCR) of cytokine mRNAs
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The right auricles and spleens were excised and immersed in TRIzol reagent (Gibco BRL Invitrogen Co., Carlsbad, CA, USA). The samples were stored at −80 °C
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until RNA extraction using the guanidinium isothiocyanate method. An aliquot (1 µg) of each total RNA sample was reverse-transcribed using the high-capacity reverse transcription kit (Applied Biosystems, Inc., Foster City, CA, USA), and 2 µL of the obtained cDNA solution was analyzed by real-time PCR with SYBR Green I (Roche Diagnostics GmbH, Mannheim, Germany) and primer sets obtained from Takara Bio Inc., (Otsu, Japan). Primer sequences are: Mouse IL-4 primers, 5’TCTCGAATGTACCAGGAGCCATATC-3’, 5’AGCACCTTGGAAGCCCTACAGA-3’; Mouse IL-6 primers, 5’-
ACCEPTED MANUSCRIPT CCACTTCACAAGTCGGAGGCTTA-3’, 5’GCAAGTGCATCATCGTTGTTCATAC-3’; Mouse IL-10 primers, 5’GACCAGCTGGACAACATACTGCTAA-3’, 5’GATAAGGCTTGGCAACCCAAGTAA-3’; Mouse IL-18 primers, 5’-
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AAGACTCTTGCGTCAACTTCAAGGA-3’, 5’-
AGTCGGCCAAAGTTGTCTGATTC-3’, Mouse INF-γ primers, 5’-
CGGCACAGTCATTGAAAGCCTA-3’, 5’-GTTGCTGATGGCCTGATTGTC-3’;
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Mouse ribosomal protein S18 (Rps18) primers, 5’-
TTCTGGCCAACGGTCTAGACAAC-3’, 5’-CCAGTGGTCTTGGTGTGCTGA-3’.
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By PCR, the samples were pre-denatured at 95 °C for 5 min, denatured at 95 °C for 10 sec, annealed at 60 °C for 10 sec, and elongated at 72 °C for 10 sec for 55 cycles, followed by melting curve analysis between 65 and 95 °C to confirm the absence of nonspecific amplification. The mRNA levels were calculated as ratios relative to the
Statistical analysis
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corresponding ribosome protein S18 (RPS18) mRNA levels.
We calculated the means ± standard deviation (SD) of values for 5 mice in each
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group, and compared differences between the means using 1-way analysis of variance
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(ANOVA) and t tests with Bonferroni correction. We calculated the mean ± standard error (SE) of values for 4 or 5 mice in each group for the inflammation score. Differences in scratching behavior or inflammation scores were analyzed using Mann-Whitney U tests. Differences in dye exudation of ACA were analyzed by Student’s t tests. p values < 0.05 were considered statistically significant. Results Subcutaneous and topical administration of ethanol exacerbates CHS We previously investigated the effects of environmental pollutants on mouse
ACCEPTED MANUSCRIPT allergy models, and compared solvents to consider the possibility of contamination. A subcutaneous injection of olive oil containing ethanol significantly increased ear thickness in mice with CHS compared with olive oil alone (p < 0.05, Bonferroni’s t test) (Fig. 1A). The solvents for topical application of the hapten were also compared.
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Acetone with ethanol enhanced thickening of the ear when compared with acetone
alone (p < 0.05, Bonferroni’s t test) (Fig. 1B). To focus on ethanol as a beverage, the
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oral administration of ethanol was investigated.
Ethanol-containing diets enhance spontaneous dermatitis of NC/Nga mice An ethanol-containing diet induced alcoholic hepatic steatosis in NC/Nga mice
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(data not shown). The inflammation score and enhanced scratching behavior of the 50 g/L ethanol-containing diet group were significantly greater than that of the control group (p < 0.05, Mann-Whitney test) (Fig. 2A & B). The inflammation score but not scratching behavior of the 25 g/L ethanol diet group was greater than that of the
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control group (p < 0.05, Mann-Whitney test). Histology demonstrated that ethanol caused lesions of the epidermis (Fig. 2D, E, & H), with cell infiltration in hair follicles,
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and sometimes fasces (Fig. 2F, I, & J).
Oral administration of ethanol aggravates experimental allergy models
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CHS in mice fed ethanol-containing diets for one week or two weeks was significantly greater than that of control mice (p < 0.05, Bonferroni’s t test) (Fig. 3A). For the accurate administration of ethanol, an aqueous ethanol solution was administered by daily gavage for 5 days after sensitization and before the elicitation of CHS inflammation. Earlobe thickness of mice administered 5 and 10% alcohol solution was significantly increased compared with control mice, while CHS from mice administered > 30% ethanol was not (Fig. 3B & C). A single administration of ethanol before the elicitation of allergy did not exacerbate inflammation compared
ACCEPTED MANUSCRIPT with no administration, while dosing for 5 days to 3 weeks aggravated allergy (Fig. 3D & E). ACA, a type I allergic response, was also enhanced by daily administration of 10% ethanol for 5 days before the elicitation of inflammation (p < 0.05, Student’s
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t test) (Fig. 3F). Histological evaluation of mice administered ethanol with gavage
The livers of NC/Nga mice fed with an ethanol-containing diet developed
many small void areas, probably due to lipid droplets (data not shown). The livers of
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BALB/c mice administered ethanol by gavage for 5 days did not present such void
areas (data not shown). The auricles from mice administered ethanol that developed
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CHS presented with hyperplasia of the epidermis and cell accumulation in limited areas of the epidermis (Fig. 4B, C, & D). Effects of acetaldehyde on CHS
Disulfiram was administered intraperitoneally before the administration of
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ethanol to suppress ethanol metabolism and to increase acetaldehyde. The ear thickness of mice that received a co-administration of disulfiram and ethanol was not
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greater than that of vehicle-control mice (Fig. 5A). Histological evaluation showed disulfiram caused extreme cell infiltration to the dermis but not the epidermis
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(Fig. 5B).
Cytokine expression in mice with CHS The mRNA expressions of cytokines in the spleens were determined by
RT-PCR. The mRNA expression of IFN-γ and IL-4 from ethanol-administered mice tended to be larger than that of water-administered mice; however, the differences were not statistically significant. The expression of IL-6, IL-10, and IL-18 of ethanoladministered mice were significantly larger than those of water-administered mice. Ethanol increased the expression of IL-6, IL-10, and IL-18 mRNA (Fig. 6).
ACCEPTED MANUSCRIPT Discussion Ethanol can provoke contact hypersensitive reactions in humans (Lachenmeier, 2008; Stotts & Ely, 1977), and some ethanol compounds cause irritant contact dermatitis (Berlin, Johanson, & Lindberg, 1995; Jensen, 1981); however, ethanol
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rarely causes allergic dermatitis. Our aim was to investigate whether ethanol enhanced allergies against other allergens or haptens. Previously we found that the subcutaneous administration of ethanol enhanced CHS when investigating the effects of solvents on
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allergy (Fig. 1). When ethanol was used as a solvent for the hapten, it also enhanced
CHS. Alcohol is imbibed in daily life; therefore, it is important to determine whether
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ethanol exaggerates allergic reactions. This prompted us to investigate the effects of ethanol on skin hypersensitivity in mouse models of allergy.
Dehne et al. reported that oral administration of ethanol suppressed inflammation induced by intracutaneous injection of phytohemagglutinin, and
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represented a downward-sloping dose-response curve (Dehne et al., 1989). In that article, the lower-dose end of the curve is located above the control. They showed that low doses of ethanol enhanced inflammation compared with the control but did not
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discuss this point further. We observed a similar effect, as ethanol enhanced CHS reactions against OXA, thus confirming that the amount of ethanol imbibed can
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exacerbate skin allergy (Fig. 3C). Moreover, ethanol enhanced contact allergy in a dose-dependent manner when doses lower than 1 mg/kg body weight were used (Fig. 3B). Ethanol concentrations of 30% and 50% did not enhance inflammation (Fig. 3C). These findings are in accord with the downward-sloping dose-response curve of Dehne et al. Our results confirmed that low-dose ethanol enhanced allergy and excessive doses of ethanol reduced cell-mediated allergies of skin. The Th1/Th2 balance hypothesis states allergies result from an imbalance of Th1 and Th2 activity. CHS is classified as a Th1-type response and ACA is classified
ACCEPTED MANUSCRIPT as a Th2-type response. In our study, the same dose of ethanol enhanced both CHS and ACA (Fig. 3). IFN-γ, a typical Th1 cytokine, and IL-4, a typical Th2 cytokine, tended to be induced by ethanol, but this effect was not statistically significant compared with controls (Fig. 6A & B). This might be because of the inverse effects of the two
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cytokines, IL-18 and IL-10. The increase of IL-10, a regulatory cytokine, seems to
oppose the exaggeration of allergy observed; however, IL-10 might be increased to
compensate for the enhanced inflammation. Ethanol enhanced the mRNA expression
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of IL-6, a cytokine that induces Th17 cells, from the spleen (Fig. 6C). Recently, much evidence that Th17 in involved in inflammation including CHS has been reported
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(Iwakura, Nakae, Saijo, & Ishigame, 2008). Thus, ethanol might mediate its effect independently from Th1 and Th2. Our RT-PCR experiment indicated that ethanol enhanced the expression of IL-18 mRNA (Fig. 6E). IL-18, one of the proinflammatory cytokines that includes IL-1β and TNF-α, is produced by antigen-
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presenting cells such as macrophages and dendritic cells, to induce IFN-γ and IL-4 production from Th1 and Th2 cells. Ethanol impairs dendritic cell functions (Heinz & Waltenbaugh, 2007) and inhibits (Pruett & Fan, 2009) or modifies (Pruett & Fan,
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2009; Yeligar, Harris, Hart, & Brown, 2012) the function of macrophages. These
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observations suggest ethanol affects antigen-presenting cells independently from either Th1 or Th2 cells.
High-dose ethanol injures dendritic cells and restricts immunity (Dehne et al.,
1989; Heinz & Waltenbaugh, 2007). In our study, administration of 30% or 50% ethanol solution did not enhance CHS. The observation that low-dose ethanol enhanced allergy in mice suggests that a detailed examination might reveal that low concentrations of ethanol enhanced dendritic cell functions. We previously reported that a small dose of selenomethionine (approximately 0.13 mg selenomethionine/kg
ACCEPTED MANUSCRIPT body weight/day) activated inflammation and a supplemental dose of selenomethionine (approximately 0.37 mg selenomethionine/kg body weight/day) suppressed allergy (Sakazaki et al., 2013). A reverse U-shape dose-response curve should also be considered in investigating the toxic effect of ethanol.
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Histological evaluation revealed an accumulation of cells in a limited area of
the epidermis (Fig. 4). This local accumulation might result from an abnormal action of dendritic cells distributed in the epidermis. Administration of disulfiram, which
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suppresses ethanol metabolism and induces acetaldehyde accumulation, inhibited the exacerbation of allergy by ethanol and enhanced the infiltration of cells into the dermis
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(Fig. 5). The different pattern of cell infiltration observed between ethanol and disulfiram treatment suggests that allergy exacerbation by ethanol is dissimilar to that of acetaldehyde. Novitskiy showed that 5−75 µM of acetaldehyde induced oxidative stress in cultured hepatic stellate cells, but up to 20 mM of ethanol did not (Novitskiy,
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Traore, Wang, Trush, & Mezey, 2006). Yeligar reported that 0.08% ethanol induced oxidative stress in cultured alveolus macrophages (Yeligar, Harris, Hart, & Brown, 2012). Those reports indicate a difference of action between ethanol and acetaldehyde,
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and a different sensitivity among cells to these stimuli. Dendritic cells are present in
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the epidermis of hair follicles (Yoneda et al., 2003). Thus, ethanol can affect certain cell types including dendritic cells, in hair follicle epidermis, resulting in cell accumulation in a limited area of the epidermis that then exacerbates allergy. Further investigations of dendritic cells with attention to dose-response relationships with lower concentrations are required. In NC/Nga mice fed conventional food, the onset of spontaneous lesions occurred at 8 weeks of age as previously described (Jin et al., 2009; Yamaguchi et al., 2001) (data not shown). The onset of spontaneous dermatitis was hastened in NC/Nga
ACCEPTED MANUSCRIPT mice given an ethanol diet (Fig. 2). Itch and scratching behavior are involved in the development of dermatitis in NC/Nga mice (Inagaki et al., 2006; Yamaguchi et al., 2001) and ethanol enhances itch (Fujii et al., 2009). However, in the current study, the increases in inflammation scores preceded the increases in scratching behavior
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(Fig. 2A & B). Moreover, we did not observe scratching behavior after the elicitation of inflammation by CHS or in mice anesthetized between 30 min of allergen challenge and allergy evaluation in the ACA experiment (data not shown). Thus, ethanol-
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exacerbated inflammation was not mediated by enhancing itch or scratching, suggesting ethanol enhanced a mechanism related to the onset of allergy.
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A single drink of alcohol aggravated human allergic asthma (Sisson, 2007; Waldschmidt et al., 2008). One subcutaneous or one topical administration of ethanol enhanced CHS (Fig. 1). However, one oral administration of ethanol did not aggravate allergy (Fig. 3D). This difference might be due to the pharmacokinetic properties of
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the administration methods. For example, a dose of 0.2 mL of 10% ethanol for a mouse with a body weight of 20 g corresponds to one liter of beer for a human with a body weight of 50 kg. The intake amount of ethanol-containing liquid diet by a mouse
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(about 100 mL per day) is extreme for a human. We previously presented an example
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of the dose differences between administration with gavage and ad libitum intake by diet in experiments of allergy suppression by selenomethionine (Arakawa et al., 2013; Sakazaki et al., 2013). Moreover, the difference of alcohol metabolism between mouse and human influences the different kinetics observed. Thus, the pharmacokinetic character of ethanol is complicated. Furthermore, human allergic asthma and dermatitis are usually chronic whereas the mouse models are acute. Therefore, a chronic allergic mouse model is required to investigate the effects of alcohol on human chronic allergy, and a clinical trial would be necessary to extrapolate the current results
ACCEPTED MANUSCRIPT to humans. In conclusion, our results suggest that the habit of drinking alcohol might exaggerate skin allergies. Further investigations including mechanistic studies with
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dendritic cells, cytokine knockout mice, and human clinical studies are needed. Acknowledgments We wish to thank Takashi Iguchi and Masatoshi Sumitani for their technical assistance. We appreciate Dr. J. Ludovic Croxford for helpful reviewing and useful
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advice on editing this article.
ACCEPTED MANUSCRIPT References Akahoshi, M., Nakashima, H., Tanaka, Y., Kohsaka, T., Nagano, S., Ohgami, E., et al. (1999). Th1/Th2 balance of peripheral T helper cells in systemic lupus erythematosus. Arthritis and Rheumatism, 42, 1644–1648. Arakawa, T., Deguchi, T., Sakazaki, F., Ogino, H., Okuno, T., & Ueno, H. (2013). Supplementary seleno-L-methionine suppresses active cutaneous anaphylaxis reaction. Biological & Pharmaceutical Bulletin, 36, 1969–1974.
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Berlin, K., Johanson, G., & Lindberg, M. (1995). Hypersensitivity to 2-(2butoxyethoxy)ethanol. Contact Dermatitis, 32, 54.
Dehne, N. E., Mendenhall, C. L., Roselle, G. A., & Grossman, C. J. (1989). Cellmediated immune responses associated with short term alcohol intake: time course and dose dependency. Alcoholism: Clinical and Experimental Research, 13, 201–205.
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Fujii, M., Nakamura, T., Fukuno, S., Mizutani, N., Nabe, T., & Kohno, S. (2009). Ethanol aggravates itch-related scratching in hairless mice developing atopic dermatitis. European Journal of Pharmacology, 611, 92–99.
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Girón-González, J. A., Moral, F. J., Elvira, J., García-Gil, D., Guerrero, F., Gavilán, I., et al. (2000). Consistent production of a higher TH1:TH2 cytokine ratio by stimulated T cells in men compared with women. European Journal of Endocrinology, 143, 31–36. Heinz, R., & Waltenbaugh, C. (2007). Ethanol consumption modifies dendritic cell antigen presentation in mice. Alcoholism: Clinical and Experimental Research, 31, 1759–1771.
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Inagaki, N., Miura, T., Nagai, H., & Koda, A. (1992). Active cutaneous anaphylaxis (ACA) in the mouse ear. Japanese Journal of Pharmacology, 59, 201–208.
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Inagaki, N., & Nagai, H. (2009). Analysis of the mechanism for the development of allergic skin inflammation and the application for its treatment: mouse models for the development of remedies for human allergic dermatitis. Journal of Pharmacological Sciences, 110, 251–259.
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Inagaki, N., Shiraishi, N., Igeta, K., Itoh, T., Chikumoto, T., Nagao, M., et al. (2006). Inhibition of scratching behavior associated with allergic dermatitis in mice by tacrolimus, but not by dexamethasone. European Journal of Pharmacology, 546, 189–196. Iwakura, Y., Nakae, S., Saijo, S., & Ishigame, H. (2008). The roles of IL-17A in inflammatory immune responses and host defense against pathogens. Immunological Reviews, 226, 57–79. Jensen, O. (1981). Contact allergy to propylene oxide and isopropyl alcohol in a skin disinfectant swab. Contact Dermatitis, 7, 148–150. Jin, H., He, R., Oyoshi, M., & Geha, R. S. (2009). Animal models of atopic dermatitis. The Journal of Investigative Dermatology, 129, 31–40. Kitagaki, H., Ono, N., Hayakawa, K., Kitazawa, T., Watanabe, K., & Shiohara, T. (1997). Repeated elicitation of contact hypersensitivity induces a shift in cutaneous cytokine milieu from a T helper cell type 1 to a T helper cell type 2 profile. Journal of Immunology, 159, 2484–2491.
ACCEPTED MANUSCRIPT Lachenmeier, D. W. (2008). Safety evaluation of topical applications of ethanol on the skin and inside the oral cavity. Journal of Occupational Medicine and Toxicology, 3, 26. McCamish, J., & Benedict, A. A. (1963). Studies on immediate cutaneous hypersensitivity in mice: I. Active cutaneous hypersensitivity. Journal of Immunology, 91, 651.
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Mihara, K., Kuratani, K., Matsui, T., Nakamura, M., & Yokota, K. (2004). Vital role of the itch-scratch response in development of spontaneous dermatitis in NC⁄Nga mice. The British Journal of Dermatology, 151, 335–345. Novitskiy, G., Traore, K., Wang, L., Trush, M. A., & Mezey, E. (2006). Effects of ethanol and acetaldehyde on reactive oxygen species production in rat hepatic stellate cells. Alcohol: Clinical and Experimental Research, 30, 1429–1435.
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Pruett, S. B., & Fan, R. (2009). Ethanol inhibits LPS-induced signaling and modulates cytokine production in peritoneal macrophages in vivo in a model for binge drinking. BMC Immunology, 10, 49.
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Sakazaki, F., Arakawa, T., Shimizu, R., Ogino, H., Okuno, T., & Ueno, H. (2013). Allergies are aggravated by mild selenium deficiency and abrogated by supplementation with selenomethionine. Food and Agricultural Immunology, http://dx.doi.org/10.1080/09540105.2013.837866 (Published online: 19 Sep 2013). Sisson, J. H. (2007). Alcohol and airways function in health and disease. Alcohol, 41, 293–307.
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Stotts, J., & Ely, W. J. (1977). Induction of human skin sensitization to ethanol. The Journal of Investigative Dermatology, 69, 219–222. Waldschmidt, T. J., Cook, R. T., & Kovacs, E. J. (2008). Alcohol and inflammation and immune responses: summary of the 2006 Alcohol and Immunology Research Interest Group (AIRIG) meeting. Alcohol, 42, 137–142.
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Wilczyński, J. (2005). Th1/Th2 cytokines balance – yin and yang of reproductive immunology. European Journal of Obstetrics, Gynecology, and Reproductive Biology, 122, 136–143.
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Yamaguchi, T., Maekawa, T., Nishikawa, Y., Nojima, H., Kaneko, M., Kawakita, T., et al. (2001). Characterization of itch-associated responses of NC mice with mite-induced chronic dermatitis. Journal of Dermatological Science, 25, 20– 28. Yeligar, S. M., Harris, F. L., Hart, C. M., & Brown, L. A. (2012). Ethanol induces oxidative stress in alveolar macrophages via upregulation of NADPH oxidases. Journal of Immunology, 188, 3648–3657. Yoneda, Y., Hirota, R., Tashiro, J., Okada, M., Sakurai, K., Lee, K., et al. (2003). Cellular origin of IFN-gamma essential for hair cycle in normal skin. Journal of Interferon & Cytokine Research, 23, 299–305.
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Figure 1. Subcutaneous and topical administration of ethanol enhanced CHS. a) Subcutaneous administration of 1 µL ethanol with 100 µL olive oil. b) Topical administration of 100 µL or 15 µL ethanol as a solvent for hapten at the sensitization of mice or at the elicitation of allergy, respectively. The results are presented as mean ± SD (n = 5 per group). The mean values of the different groups were compared with the mean value for mice that were not administered ethanol. *p < 0.05; **p < 0.01.
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Figure 2. The degree of inflammation in NC/Nga mice fed diets containing various concentrations of ethanol. a) Inflammation scores. The severity of dermatitis was scored (see Methods) and the results are presented as mean ± SE (n = 5 per group). The mean values of the different groups were compared with the mean values observed in mice fed without ethanol. b) The frequency of scratching behavior. *p < 0.05 compared with the group fed a diet without ethanol. c to f) Appearance of mice. The images show the typical appearance of mice. c) A mouse fed a diet without ethanol. d) Mouse fed a diet containing 12.5 g/L ethanol. e) Mouse fed a diet containing 25 g/L ethanol. f) Mouse with abscess. g to j) Histological evaluation of inflamed areas of skin adjacent to the nose. g) Normal skin. h) Inflamed skin with lesions. i) Excess cell infiltration around a hair follicle. j) Inflamed skin with abscess. The letters E, D, and F represent the epidermis, dermis, and hair follicle, respectively. Scale bars indicate 50 µm. Representative data are shown.
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Figure 3. Ear swelling and CHS increases in mice administered ethanol orally. Mice were administered ethanol under various conditions. CHS in the ear was elicited by OXA and the ear thickness was measured 24 h or at the indicated time after the elicitation of allergy. a) CHS time-course studies of ear swelling in mice administered a 25 g/L ethanol liquid diet for 1 week. b and c) Dose-dependent study of CHS, 24 h after elicitation of allergy. Indicated concentrations of ethanol aqueous solution were administered by gavage for 5 days. d) CHS and duration of ethanol administration with ethanol aqueous solution administered by gavage. Mice were administered 10% ethanol for indicated days before the elicitation of CHS. e) ACA in mice administered ethanol. Mice were administered 10% ethanol for 5 days before the elicitation of ACA. The results are presented as mean ± SD (n = 5 per group). The mean values of the different groups were compared with the mean value for mice administered vehicle control. *p < 0.05.
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Figure 4. Histological evaluation of inflamed skin in CHS mice. Ear auricles were excised 24 h after the elicitation of inflammation. Histology from the ear of: a) a mouse administered vehicle for 5 days, b) a mouse administered 10% ethanol for 5 days, c) a mouse administered 10% ethanol for 21 days, d) a mouse administered 50% ethanol for 5 days. Letters E, D, and C represent the epidermis, dermis, and cartilage, respectively. Scale bars indicate 50 µm. Representative data are shown. Figure 5. Effects of ethanol are different from effects of acetaldehyde. Disulfiram was administered intraperitoneally before the administration of ethanol and effects on CHS was observed. a) Thickness of ear lobes 24 h after the elicitation of allergy. The results are presented as mean ± SD (n = 5 per group). The mean values of the different groups were compared with the mean value for vehicle-control mice. *p < 0.05. b) Histology from the ear of a mouse administered 10% ethanol for 5 days after administration of disulfiram. Ear auricles were excised 24 h after the elicitation of inflammation. Letters E, D, and C represent the epidermis, dermis, and cartilage, respectively. Scale bars
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Figure 6. Cytokine expressions from ethanol-administered mice. Quantification of mRNA levels in the spleen was evaluated 6 h after the elicitation of CHS using realtime RT-PCR relative to that of RPS18. a) IFN-γ levels, b) IL-4 levels, c) IL-6 levels, d) IL-10 levels, e) IL-18 levels. The results are presented as mean ± SD (n = 5 per group). The mean values of the different groups were compared with the mean value for vehicle-control mice. *p < 0.05.
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S0741-8329(14)00100-1
DOI:
10.1016/j.alcohol.2014.05.001
Reference:
ALC 6406
To appear in:
Alcohol
Please cite this article as: SakazakiF., OginoH., ArakawaT., OkunoT. & UenoH., Low-dose ethanol aggravates allergic dermatitis in mice, Alcohol (2014), doi: 10.1016/j.alcohol.2014.05.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Low-dose ethanol aggravates allergic dermatitis in mice Fumitoshi Sakazaki1, Hirofumi Ogino2, Tomohiro Arakawa2, Tomofumi Okuno2, and Hitoshi Ueno2 1
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Laboratory of Toxicology, Faculty of Pharmacy, Osaka Ohtani University, 3-11-1 Nishikiori-kita, Tondabayashi, Osaka 584-8540, Japan
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Address correspondence to: Fumitoshi Sakazaki, Ph.D. Laboratory of Toxicology Faculty of Pharmacy Osaka Ohtani University 3-11-1 Nishikiori-kita Tondabayashi, Osaka 584-8540, Japan Telephone: +81 721 24 9986 Fax: +81 721 24 9986 Email: [email protected]
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Department of Public Health & Preventive Pharmacology, Faculty of Pharmaceutical Sciences, Setsunan University, 45-1 Nagaotoge-cho, Hirakata Osaka 573-0101, Japan
ACCEPTED MANUSCRIPT Abstract Alcohol injures dendritic cells and suppresses cellular immunity, while some evidence indicates that drinking alcohol aggravates allergic asthma. This study investigated the effect of low doses of ethanol in enhancing allergic reactions in the
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skin of mice. Liquid food containing alcohol was administered to conventional NC/Nga mice to induce alcoholic hepatic steatosis, and spontaneous dermatitis was evaluated. BALB/c mice were administered approximately 1 g/kg body weight of
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ethanol by gavage, and contact hypersensitivity (CHS) or active cutaneous anaphylaxis (ACA) was induced. Spleens were collected 24 h after the elicitation of CHS and
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mRNA expressions of interferon (IFN)-γ, interleukin (IL)-4, IL-6, IL-10, and IL-18 were measured by quantitative RT-PCR. Alcohol-containing diet exaggerated spontaneous dermatitis in conventional NC/Nga mice and contact hypersensitivity in BALB/c mice. Ethanol administered by gavage for 5 days enhanced contact
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hypersensitivity in BALB/c mice. Ethanol administration with gavage also enhanced ACA of BALB/c mice. Ethanol did not affect mRNA expression of IFN-γ and IL-4, but did enhance IL-6, IL-10, and IL-18 mRNA expression. Histological evaluation
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revealed an absence of hepatic steatosis in mice administered ethanol by gavage for 5 days. In ethanol-administered mice, inflamed areas presented as lesions or a local
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extreme accumulation of mononuclear cells in the epidermis. These findings suggest that ethanol enhances the expression of inflammatory cytokines independently from T helper (Th)1/Th2 cytokine phenotypes, causing abnormalities in the epidermis resulting in exacerbated allergic reactivity. Keywords: alcohol, ethanol, allergy, dermatitis
ACCEPTED MANUSCRIPT Introduction Previous studies reported that drinking alcohol aggravates allergic asthma; however, the mechanism has not been fully elucidated (Sisson, 2007; Waldschmidt, Cook, & Kovacs, 2008). The effects of ethanol on skin allergy have been investigated
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but are not fully understood (Lachenmeier, 2008). Alcohol dependence causes the inhibition of cell-mediated immunity (Dehne, Mendenhall, Roselle, & Grossman, 1989), and ethanol impairs the function of dendritic cells (Heinz & Waltenbaugh,
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2007) and macrophages (Pruett & Fan, 2009). However, the relationship between
immunosuppression by alcohol and aggravation of allergy by drinking alcohol has not
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been investigated. Furthermore, a dose-dependent experiment showed that low-dose ethanol enhanced cell-mediated immunity (Dehne et al., 1989). According to the T helper cell (Th)1/Th2 cytokine balance hypothesis (Akahoshi et al., 1999; Girón-González et al., 2000; Wilczyński, 2005), ethanol
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suppresses Th1 cytokines and might enhance Th2 cytokine responses that exacerbate allergic asthma. However, some reports have suggested that the Th1/Th2 balance hypothesis does not explain every immune response (Kitagaki et al., 1997), and recent
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reports have identified additional types of helper T cells, including interleukin (IL)-17producing helper T cells (Th17) and regulatory helper T cells (Treg). To investigate the
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immunotoxicity of ethanol, Th1, Th2, Th17, and Treg should be taken into consideration.
The NC/Nga mouse model develops dermatitis under conventional conditions,
and the symptoms resemble atopic dermatitis in humans. Thus, the NC/Nga mouse is useful for the investigation of allergic dermatitis (Jin, He, Oyoshi, & Geha, 2009). For type I allergy models, passive cutaneous anaphylaxis (PCA) and active cutaneous anaphylaxis (ACA) are used (Inagaki, Miura, Nagai, & Koda, 1992; McCamish & Benedict, 1963). In PCA experiments, immunoglobulin E is artificially administered
ACCEPTED MANUSCRIPT and the permeability of blood vessels is measured. PCA focuses on histamine release from mast cells. In ACA experiments, antigen is administered and the permeability of blood vessels is measured. ACA focuses on immunoglobulin E secretion from B lymphocytes, histamine release from mast cells, and T cell activation that induces
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B cell functions. For type IV allergy models, contact hypersensitivity (CHS) is used (Inagaki & Nagai, 2009). BALB/c or C57BL/6 mice are widely used for ACA and CHS experiments.
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To elucidate the effects of a low dose of ethanol on skin allergies, the present study determined whether a small amount of ethanol enhanced CHS in mice and
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investigated the effects of ethanol on ACA. Materials and Methods Animal treatment
All experimental protocols used in this study were in accordance with the
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animal experiment guidelines of Setsunan University, Japan. These guidelines were adapted from the guidelines of the Japanese Society for Pharmacology. The Committee for the Ethical Use of Experimental Animals of Setsunan University
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approved the study. Efforts were made to minimize animal suffering as much as possible by limiting the number of animals used in experimentation and using
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alternatives for in vivo techniques. A total of 20 to 30 mice of each strain (NC/Nga and BALB/c mice; Japan SLC,
Inc., Hamamatsu, Japan) were housed 5 per cage and allowed 1 week for acclimation while fed a normal diet (MF Certified Diet, Oriental Yeast Co., Ltd.) ad libitum with full access to water. After acclimation, mice were randomly assigned to one of six treatment groups (described below). The mice were kept in a pathogen-free room maintained on a 12-h light-dark cycle (lights on at 7:00 AM), at an ambient
ACCEPTED MANUSCRIPT temperature of 23 ± 1 °C and relative humidity level of 47–67%. Ethanol administration Ethanol (99.5%, Extra Pure Reagent grade, Nacalai Tesque Inc., Kyoto, Japan) was administered in three different ways. For subcutaneous administration, 10 µL of
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ethanol was added to 1 mL of olive oil, vortexed, and sterilized by filtration. An
aliquot of 0.1 mL was injected into mouse dorsal skin. A model diet for alcoholic
hepatic steatosis and its isocaloric control diet were purchased from Oriental Yeast
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Co., Ltd. The ethanol diet and the control diet were mixed to prepare 0, 12.5, 25, and 50 g/L ethanol diets. Each liquid diet was fed to mice ad libitum with a food-water
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bottle. For oral administration with gavage, 0.25 mL of 0, 2.5, 5, 10, 30, and 50% ethanol aqueous solutions were administered. Disulfiram (LKT Laboratories, Inc., St. Paul, MN, USA) was dissolved in olive oil (Kenei Pharmaceutical, Osaka, Japan) and administered intraperitoneally at a dose of 90 mg/kg body weight, one time prior to the
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sensitization of mice. Olive oil was administered as a control for disulfiram. Spontaneous dermatitis in NC/Nga mice
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Conventional 3-week-old female NC/Nga mice weighing 18−20 g (Japan SLC Co., Shizuoka, Japan) received one of four different concentrations of ethanol liquid
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diets (described above) for a period of 8 weeks. The degree of dermatitis observed in mice was scored depending on severity according to a previous report (Sakazaki et al., 2013) with some modifications as follows: 1) presence of scratching behavior, 2) presence of blushing, 3) presence of hair loss, and 4) presence of bleeding and crust. After a 5-week intervention period, we anesthetized mice by intraperitoneal administration of 0.1 mL physiological saline with 10 mg/mL sodium pentobarbital (approximately 50 mg/kg body weight, 1 mg/mouse), and inflamed skin was collected for histological evaluation.
ACCEPTED MANUSCRIPT The mice usually slept and did not scratch their bodies. After cage exchange they started to wander around and to scratch their bodies, and they gradually stopped wandering and scratching and started sleeping again. Frequency of scratching over a few seconds was counted over 1 h after cage exchange by visual observation. Although
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Mihara et al. took video movies of mice from above with a tripod (Mihara, Kuratani, Matsui, Nakamura, & Yokota, 2004), we did not take videos of mice because mice prefer to stay in the shadows rather than being exposed to light. The cages of mice
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were set in a rack and we observed the behavior of mice from the side. CHS
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After administration of ethanol, mice were sensitized by the topical application of 50 µL of 3% 4-ethoxymethylene-2-phenyl-2-oxazoline-5-one (OXA) (SigmaAldrich, Inc., St. Louis, MO, USA) in acetone or a 3:1 (v/v) mixture of ethanol and acetone on the dorsal skin. After 7 days, the mice were challenged for the elicitation of
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allergy by applying 7.5 µL of 0.1% OXA in acetone or ethanol to both sides of the right auricle. The auricle thickness was measured under ether anesthesia using a digital thickness gauge (Ozaki MFG Co. Ltd., Tokyo, Japan) while taking care to avoid
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denting the edematous skin. The degree of ear swelling was calculated by subtracting
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the thickness of the left ear from that of the right ear.
Seven-week-old female BALB/c mice weighing 18–20 g (Japan SLC Co.)
were fed one of the four alcohol liquid diets for the indicated period before we induced ACA responses with subcutaneous administration of 1 µg ovalbumin (OVA) (SigmaAldrich) and 1 mg alum (Sigma-Aldrich). Two weeks after the initial administration of OVA and alum, the mice were anesthetized by intraperitoneal administration of 0.1 mL of physiological saline with 10 mg/mL sodium pentobarbital (approximately
ACCEPTED MANUSCRIPT 50 mg/kg body weight, 1 mg/mouse). Then, 250 µL of 0.5% Evan’s blue solution was injected into the tail vein, and 10 µL of 0.1 mg/mL OVA was injected subcutaneously into the right ear lobe. As a control, physiological saline was injected into the left ear lobe. To measure the exudate of Evan’s blue, the ear lobes of mice were excised
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30 min post injection, incubated overnight in 0.3 mL KOH solution at 37 °C,
neutralized with 10 mol/L phosphoric acid-acetone (3:67) solution (0.7 mL), and centrifuged at 15,000 × g for 10 min at room temperature. The absorbance of the
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supernatant was measured at 620 nm. Histological evaluation
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The skin, including areas of inflammation, was removed surgically and fixed in 4% formalin, containing 0.1 mol/L phosphate buffer, overnight. The skin was then embedded in paraffin and sectioned. Sections on glass slides were dried at 37 °C overnight and stained with hematoxylin and eosin to evaluate the general structure, and
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slides were examined by microscopy.
Reverse transcription-polymerase chain reaction (RT-PCR) of cytokine mRNAs
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The right auricles and spleens were excised and immersed in TRIzol reagent (Gibco BRL Invitrogen Co., Carlsbad, CA, USA). The samples were stored at −80 °C
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until RNA extraction using the guanidinium isothiocyanate method. An aliquot (1 µg) of each total RNA sample was reverse-transcribed using the high-capacity reverse transcription kit (Applied Biosystems, Inc., Foster City, CA, USA), and 2 µL of the obtained cDNA solution was analyzed by real-time PCR with SYBR Green I (Roche Diagnostics GmbH, Mannheim, Germany) and primer sets obtained from Takara Bio Inc., (Otsu, Japan). Primer sequences are: Mouse IL-4 primers, 5’TCTCGAATGTACCAGGAGCCATATC-3’, 5’AGCACCTTGGAAGCCCTACAGA-3’; Mouse IL-6 primers, 5’-
ACCEPTED MANUSCRIPT CCACTTCACAAGTCGGAGGCTTA-3’, 5’GCAAGTGCATCATCGTTGTTCATAC-3’; Mouse IL-10 primers, 5’GACCAGCTGGACAACATACTGCTAA-3’, 5’GATAAGGCTTGGCAACCCAAGTAA-3’; Mouse IL-18 primers, 5’-
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AAGACTCTTGCGTCAACTTCAAGGA-3’, 5’-
AGTCGGCCAAAGTTGTCTGATTC-3’, Mouse INF-γ primers, 5’-
CGGCACAGTCATTGAAAGCCTA-3’, 5’-GTTGCTGATGGCCTGATTGTC-3’;
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Mouse ribosomal protein S18 (Rps18) primers, 5’-
TTCTGGCCAACGGTCTAGACAAC-3’, 5’-CCAGTGGTCTTGGTGTGCTGA-3’.
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By PCR, the samples were pre-denatured at 95 °C for 5 min, denatured at 95 °C for 10 sec, annealed at 60 °C for 10 sec, and elongated at 72 °C for 10 sec for 55 cycles, followed by melting curve analysis between 65 and 95 °C to confirm the absence of nonspecific amplification. The mRNA levels were calculated as ratios relative to the
Statistical analysis
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corresponding ribosome protein S18 (RPS18) mRNA levels.
We calculated the means ± standard deviation (SD) of values for 5 mice in each
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group, and compared differences between the means using 1-way analysis of variance
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(ANOVA) and t tests with Bonferroni correction. We calculated the mean ± standard error (SE) of values for 4 or 5 mice in each group for the inflammation score. Differences in scratching behavior or inflammation scores were analyzed using Mann-Whitney U tests. Differences in dye exudation of ACA were analyzed by Student’s t tests. p values < 0.05 were considered statistically significant. Results Subcutaneous and topical administration of ethanol exacerbates CHS We previously investigated the effects of environmental pollutants on mouse
ACCEPTED MANUSCRIPT allergy models, and compared solvents to consider the possibility of contamination. A subcutaneous injection of olive oil containing ethanol significantly increased ear thickness in mice with CHS compared with olive oil alone (p < 0.05, Bonferroni’s t test) (Fig. 1A). The solvents for topical application of the hapten were also compared.
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Acetone with ethanol enhanced thickening of the ear when compared with acetone
alone (p < 0.05, Bonferroni’s t test) (Fig. 1B). To focus on ethanol as a beverage, the
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oral administration of ethanol was investigated.
Ethanol-containing diets enhance spontaneous dermatitis of NC/Nga mice An ethanol-containing diet induced alcoholic hepatic steatosis in NC/Nga mice
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(data not shown). The inflammation score and enhanced scratching behavior of the 50 g/L ethanol-containing diet group were significantly greater than that of the control group (p < 0.05, Mann-Whitney test) (Fig. 2A & B). The inflammation score but not scratching behavior of the 25 g/L ethanol diet group was greater than that of the
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control group (p < 0.05, Mann-Whitney test). Histology demonstrated that ethanol caused lesions of the epidermis (Fig. 2D, E, & H), with cell infiltration in hair follicles,
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and sometimes fasces (Fig. 2F, I, & J).
Oral administration of ethanol aggravates experimental allergy models
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CHS in mice fed ethanol-containing diets for one week or two weeks was significantly greater than that of control mice (p < 0.05, Bonferroni’s t test) (Fig. 3A). For the accurate administration of ethanol, an aqueous ethanol solution was administered by daily gavage for 5 days after sensitization and before the elicitation of CHS inflammation. Earlobe thickness of mice administered 5 and 10% alcohol solution was significantly increased compared with control mice, while CHS from mice administered > 30% ethanol was not (Fig. 3B & C). A single administration of ethanol before the elicitation of allergy did not exacerbate inflammation compared
ACCEPTED MANUSCRIPT with no administration, while dosing for 5 days to 3 weeks aggravated allergy (Fig. 3D & E). ACA, a type I allergic response, was also enhanced by daily administration of 10% ethanol for 5 days before the elicitation of inflammation (p < 0.05, Student’s
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t test) (Fig. 3F). Histological evaluation of mice administered ethanol with gavage
The livers of NC/Nga mice fed with an ethanol-containing diet developed
many small void areas, probably due to lipid droplets (data not shown). The livers of
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BALB/c mice administered ethanol by gavage for 5 days did not present such void
areas (data not shown). The auricles from mice administered ethanol that developed
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CHS presented with hyperplasia of the epidermis and cell accumulation in limited areas of the epidermis (Fig. 4B, C, & D). Effects of acetaldehyde on CHS
Disulfiram was administered intraperitoneally before the administration of
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ethanol to suppress ethanol metabolism and to increase acetaldehyde. The ear thickness of mice that received a co-administration of disulfiram and ethanol was not
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greater than that of vehicle-control mice (Fig. 5A). Histological evaluation showed disulfiram caused extreme cell infiltration to the dermis but not the epidermis
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(Fig. 5B).
Cytokine expression in mice with CHS The mRNA expressions of cytokines in the spleens were determined by
RT-PCR. The mRNA expression of IFN-γ and IL-4 from ethanol-administered mice tended to be larger than that of water-administered mice; however, the differences were not statistically significant. The expression of IL-6, IL-10, and IL-18 of ethanoladministered mice were significantly larger than those of water-administered mice. Ethanol increased the expression of IL-6, IL-10, and IL-18 mRNA (Fig. 6).
ACCEPTED MANUSCRIPT Discussion Ethanol can provoke contact hypersensitive reactions in humans (Lachenmeier, 2008; Stotts & Ely, 1977), and some ethanol compounds cause irritant contact dermatitis (Berlin, Johanson, & Lindberg, 1995; Jensen, 1981); however, ethanol
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rarely causes allergic dermatitis. Our aim was to investigate whether ethanol enhanced allergies against other allergens or haptens. Previously we found that the subcutaneous administration of ethanol enhanced CHS when investigating the effects of solvents on
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allergy (Fig. 1). When ethanol was used as a solvent for the hapten, it also enhanced
CHS. Alcohol is imbibed in daily life; therefore, it is important to determine whether
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ethanol exaggerates allergic reactions. This prompted us to investigate the effects of ethanol on skin hypersensitivity in mouse models of allergy.
Dehne et al. reported that oral administration of ethanol suppressed inflammation induced by intracutaneous injection of phytohemagglutinin, and
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represented a downward-sloping dose-response curve (Dehne et al., 1989). In that article, the lower-dose end of the curve is located above the control. They showed that low doses of ethanol enhanced inflammation compared with the control but did not
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discuss this point further. We observed a similar effect, as ethanol enhanced CHS reactions against OXA, thus confirming that the amount of ethanol imbibed can
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exacerbate skin allergy (Fig. 3C). Moreover, ethanol enhanced contact allergy in a dose-dependent manner when doses lower than 1 mg/kg body weight were used (Fig. 3B). Ethanol concentrations of 30% and 50% did not enhance inflammation (Fig. 3C). These findings are in accord with the downward-sloping dose-response curve of Dehne et al. Our results confirmed that low-dose ethanol enhanced allergy and excessive doses of ethanol reduced cell-mediated allergies of skin. The Th1/Th2 balance hypothesis states allergies result from an imbalance of Th1 and Th2 activity. CHS is classified as a Th1-type response and ACA is classified
ACCEPTED MANUSCRIPT as a Th2-type response. In our study, the same dose of ethanol enhanced both CHS and ACA (Fig. 3). IFN-γ, a typical Th1 cytokine, and IL-4, a typical Th2 cytokine, tended to be induced by ethanol, but this effect was not statistically significant compared with controls (Fig. 6A & B). This might be because of the inverse effects of the two
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cytokines, IL-18 and IL-10. The increase of IL-10, a regulatory cytokine, seems to
oppose the exaggeration of allergy observed; however, IL-10 might be increased to
compensate for the enhanced inflammation. Ethanol enhanced the mRNA expression
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of IL-6, a cytokine that induces Th17 cells, from the spleen (Fig. 6C). Recently, much evidence that Th17 in involved in inflammation including CHS has been reported
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(Iwakura, Nakae, Saijo, & Ishigame, 2008). Thus, ethanol might mediate its effect independently from Th1 and Th2. Our RT-PCR experiment indicated that ethanol enhanced the expression of IL-18 mRNA (Fig. 6E). IL-18, one of the proinflammatory cytokines that includes IL-1β and TNF-α, is produced by antigen-
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presenting cells such as macrophages and dendritic cells, to induce IFN-γ and IL-4 production from Th1 and Th2 cells. Ethanol impairs dendritic cell functions (Heinz & Waltenbaugh, 2007) and inhibits (Pruett & Fan, 2009) or modifies (Pruett & Fan,
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2009; Yeligar, Harris, Hart, & Brown, 2012) the function of macrophages. These
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observations suggest ethanol affects antigen-presenting cells independently from either Th1 or Th2 cells.
High-dose ethanol injures dendritic cells and restricts immunity (Dehne et al.,
1989; Heinz & Waltenbaugh, 2007). In our study, administration of 30% or 50% ethanol solution did not enhance CHS. The observation that low-dose ethanol enhanced allergy in mice suggests that a detailed examination might reveal that low concentrations of ethanol enhanced dendritic cell functions. We previously reported that a small dose of selenomethionine (approximately 0.13 mg selenomethionine/kg
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Histological evaluation revealed an accumulation of cells in a limited area of
the epidermis (Fig. 4). This local accumulation might result from an abnormal action of dendritic cells distributed in the epidermis. Administration of disulfiram, which
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suppresses ethanol metabolism and induces acetaldehyde accumulation, inhibited the exacerbation of allergy by ethanol and enhanced the infiltration of cells into the dermis
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(Fig. 5). The different pattern of cell infiltration observed between ethanol and disulfiram treatment suggests that allergy exacerbation by ethanol is dissimilar to that of acetaldehyde. Novitskiy showed that 5−75 µM of acetaldehyde induced oxidative stress in cultured hepatic stellate cells, but up to 20 mM of ethanol did not (Novitskiy,
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Traore, Wang, Trush, & Mezey, 2006). Yeligar reported that 0.08% ethanol induced oxidative stress in cultured alveolus macrophages (Yeligar, Harris, Hart, & Brown, 2012). Those reports indicate a difference of action between ethanol and acetaldehyde,
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and a different sensitivity among cells to these stimuli. Dendritic cells are present in
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the epidermis of hair follicles (Yoneda et al., 2003). Thus, ethanol can affect certain cell types including dendritic cells, in hair follicle epidermis, resulting in cell accumulation in a limited area of the epidermis that then exacerbates allergy. Further investigations of dendritic cells with attention to dose-response relationships with lower concentrations are required. In NC/Nga mice fed conventional food, the onset of spontaneous lesions occurred at 8 weeks of age as previously described (Jin et al., 2009; Yamaguchi et al., 2001) (data not shown). The onset of spontaneous dermatitis was hastened in NC/Nga
ACCEPTED MANUSCRIPT mice given an ethanol diet (Fig. 2). Itch and scratching behavior are involved in the development of dermatitis in NC/Nga mice (Inagaki et al., 2006; Yamaguchi et al., 2001) and ethanol enhances itch (Fujii et al., 2009). However, in the current study, the increases in inflammation scores preceded the increases in scratching behavior
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(Fig. 2A & B). Moreover, we did not observe scratching behavior after the elicitation of inflammation by CHS or in mice anesthetized between 30 min of allergen challenge and allergy evaluation in the ACA experiment (data not shown). Thus, ethanol-
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exacerbated inflammation was not mediated by enhancing itch or scratching, suggesting ethanol enhanced a mechanism related to the onset of allergy.
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A single drink of alcohol aggravated human allergic asthma (Sisson, 2007; Waldschmidt et al., 2008). One subcutaneous or one topical administration of ethanol enhanced CHS (Fig. 1). However, one oral administration of ethanol did not aggravate allergy (Fig. 3D). This difference might be due to the pharmacokinetic properties of
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the administration methods. For example, a dose of 0.2 mL of 10% ethanol for a mouse with a body weight of 20 g corresponds to one liter of beer for a human with a body weight of 50 kg. The intake amount of ethanol-containing liquid diet by a mouse
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(about 100 mL per day) is extreme for a human. We previously presented an example
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of the dose differences between administration with gavage and ad libitum intake by diet in experiments of allergy suppression by selenomethionine (Arakawa et al., 2013; Sakazaki et al., 2013). Moreover, the difference of alcohol metabolism between mouse and human influences the different kinetics observed. Thus, the pharmacokinetic character of ethanol is complicated. Furthermore, human allergic asthma and dermatitis are usually chronic whereas the mouse models are acute. Therefore, a chronic allergic mouse model is required to investigate the effects of alcohol on human chronic allergy, and a clinical trial would be necessary to extrapolate the current results
ACCEPTED MANUSCRIPT to humans. In conclusion, our results suggest that the habit of drinking alcohol might exaggerate skin allergies. Further investigations including mechanistic studies with
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dendritic cells, cytokine knockout mice, and human clinical studies are needed. Acknowledgments We wish to thank Takashi Iguchi and Masatoshi Sumitani for their technical assistance. We appreciate Dr. J. Ludovic Croxford for helpful reviewing and useful
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advice on editing this article.
ACCEPTED MANUSCRIPT References Akahoshi, M., Nakashima, H., Tanaka, Y., Kohsaka, T., Nagano, S., Ohgami, E., et al. (1999). Th1/Th2 balance of peripheral T helper cells in systemic lupus erythematosus. Arthritis and Rheumatism, 42, 1644–1648. Arakawa, T., Deguchi, T., Sakazaki, F., Ogino, H., Okuno, T., & Ueno, H. (2013). Supplementary seleno-L-methionine suppresses active cutaneous anaphylaxis reaction. Biological & Pharmaceutical Bulletin, 36, 1969–1974.
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Berlin, K., Johanson, G., & Lindberg, M. (1995). Hypersensitivity to 2-(2butoxyethoxy)ethanol. Contact Dermatitis, 32, 54.
Dehne, N. E., Mendenhall, C. L., Roselle, G. A., & Grossman, C. J. (1989). Cellmediated immune responses associated with short term alcohol intake: time course and dose dependency. Alcoholism: Clinical and Experimental Research, 13, 201–205.
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Fujii, M., Nakamura, T., Fukuno, S., Mizutani, N., Nabe, T., & Kohno, S. (2009). Ethanol aggravates itch-related scratching in hairless mice developing atopic dermatitis. European Journal of Pharmacology, 611, 92–99.
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Girón-González, J. A., Moral, F. J., Elvira, J., García-Gil, D., Guerrero, F., Gavilán, I., et al. (2000). Consistent production of a higher TH1:TH2 cytokine ratio by stimulated T cells in men compared with women. European Journal of Endocrinology, 143, 31–36. Heinz, R., & Waltenbaugh, C. (2007). Ethanol consumption modifies dendritic cell antigen presentation in mice. Alcoholism: Clinical and Experimental Research, 31, 1759–1771.
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Inagaki, N., Miura, T., Nagai, H., & Koda, A. (1992). Active cutaneous anaphylaxis (ACA) in the mouse ear. Japanese Journal of Pharmacology, 59, 201–208.
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Inagaki, N., & Nagai, H. (2009). Analysis of the mechanism for the development of allergic skin inflammation and the application for its treatment: mouse models for the development of remedies for human allergic dermatitis. Journal of Pharmacological Sciences, 110, 251–259.
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Inagaki, N., Shiraishi, N., Igeta, K., Itoh, T., Chikumoto, T., Nagao, M., et al. (2006). Inhibition of scratching behavior associated with allergic dermatitis in mice by tacrolimus, but not by dexamethasone. European Journal of Pharmacology, 546, 189–196. Iwakura, Y., Nakae, S., Saijo, S., & Ishigame, H. (2008). The roles of IL-17A in inflammatory immune responses and host defense against pathogens. Immunological Reviews, 226, 57–79. Jensen, O. (1981). Contact allergy to propylene oxide and isopropyl alcohol in a skin disinfectant swab. Contact Dermatitis, 7, 148–150. Jin, H., He, R., Oyoshi, M., & Geha, R. S. (2009). Animal models of atopic dermatitis. The Journal of Investigative Dermatology, 129, 31–40. Kitagaki, H., Ono, N., Hayakawa, K., Kitazawa, T., Watanabe, K., & Shiohara, T. (1997). Repeated elicitation of contact hypersensitivity induces a shift in cutaneous cytokine milieu from a T helper cell type 1 to a T helper cell type 2 profile. Journal of Immunology, 159, 2484–2491.
ACCEPTED MANUSCRIPT Lachenmeier, D. W. (2008). Safety evaluation of topical applications of ethanol on the skin and inside the oral cavity. Journal of Occupational Medicine and Toxicology, 3, 26. McCamish, J., & Benedict, A. A. (1963). Studies on immediate cutaneous hypersensitivity in mice: I. Active cutaneous hypersensitivity. Journal of Immunology, 91, 651.
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Mihara, K., Kuratani, K., Matsui, T., Nakamura, M., & Yokota, K. (2004). Vital role of the itch-scratch response in development of spontaneous dermatitis in NC⁄Nga mice. The British Journal of Dermatology, 151, 335–345. Novitskiy, G., Traore, K., Wang, L., Trush, M. A., & Mezey, E. (2006). Effects of ethanol and acetaldehyde on reactive oxygen species production in rat hepatic stellate cells. Alcohol: Clinical and Experimental Research, 30, 1429–1435.
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Pruett, S. B., & Fan, R. (2009). Ethanol inhibits LPS-induced signaling and modulates cytokine production in peritoneal macrophages in vivo in a model for binge drinking. BMC Immunology, 10, 49.
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Sakazaki, F., Arakawa, T., Shimizu, R., Ogino, H., Okuno, T., & Ueno, H. (2013). Allergies are aggravated by mild selenium deficiency and abrogated by supplementation with selenomethionine. Food and Agricultural Immunology, http://dx.doi.org/10.1080/09540105.2013.837866 (Published online: 19 Sep 2013). Sisson, J. H. (2007). Alcohol and airways function in health and disease. Alcohol, 41, 293–307.
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Stotts, J., & Ely, W. J. (1977). Induction of human skin sensitization to ethanol. The Journal of Investigative Dermatology, 69, 219–222. Waldschmidt, T. J., Cook, R. T., & Kovacs, E. J. (2008). Alcohol and inflammation and immune responses: summary of the 2006 Alcohol and Immunology Research Interest Group (AIRIG) meeting. Alcohol, 42, 137–142.
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Wilczyński, J. (2005). Th1/Th2 cytokines balance – yin and yang of reproductive immunology. European Journal of Obstetrics, Gynecology, and Reproductive Biology, 122, 136–143.
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Yamaguchi, T., Maekawa, T., Nishikawa, Y., Nojima, H., Kaneko, M., Kawakita, T., et al. (2001). Characterization of itch-associated responses of NC mice with mite-induced chronic dermatitis. Journal of Dermatological Science, 25, 20– 28. Yeligar, S. M., Harris, F. L., Hart, C. M., & Brown, L. A. (2012). Ethanol induces oxidative stress in alveolar macrophages via upregulation of NADPH oxidases. Journal of Immunology, 188, 3648–3657. Yoneda, Y., Hirota, R., Tashiro, J., Okada, M., Sakurai, K., Lee, K., et al. (2003). Cellular origin of IFN-gamma essential for hair cycle in normal skin. Journal of Interferon & Cytokine Research, 23, 299–305.
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Figure 1. Subcutaneous and topical administration of ethanol enhanced CHS. a) Subcutaneous administration of 1 µL ethanol with 100 µL olive oil. b) Topical administration of 100 µL or 15 µL ethanol as a solvent for hapten at the sensitization of mice or at the elicitation of allergy, respectively. The results are presented as mean ± SD (n = 5 per group). The mean values of the different groups were compared with the mean value for mice that were not administered ethanol. *p < 0.05; **p < 0.01.
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Figure 2. The degree of inflammation in NC/Nga mice fed diets containing various concentrations of ethanol. a) Inflammation scores. The severity of dermatitis was scored (see Methods) and the results are presented as mean ± SE (n = 5 per group). The mean values of the different groups were compared with the mean values observed in mice fed without ethanol. b) The frequency of scratching behavior. *p < 0.05 compared with the group fed a diet without ethanol. c to f) Appearance of mice. The images show the typical appearance of mice. c) A mouse fed a diet without ethanol. d) Mouse fed a diet containing 12.5 g/L ethanol. e) Mouse fed a diet containing 25 g/L ethanol. f) Mouse with abscess. g to j) Histological evaluation of inflamed areas of skin adjacent to the nose. g) Normal skin. h) Inflamed skin with lesions. i) Excess cell infiltration around a hair follicle. j) Inflamed skin with abscess. The letters E, D, and F represent the epidermis, dermis, and hair follicle, respectively. Scale bars indicate 50 µm. Representative data are shown.
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Figure 3. Ear swelling and CHS increases in mice administered ethanol orally. Mice were administered ethanol under various conditions. CHS in the ear was elicited by OXA and the ear thickness was measured 24 h or at the indicated time after the elicitation of allergy. a) CHS time-course studies of ear swelling in mice administered a 25 g/L ethanol liquid diet for 1 week. b and c) Dose-dependent study of CHS, 24 h after elicitation of allergy. Indicated concentrations of ethanol aqueous solution were administered by gavage for 5 days. d) CHS and duration of ethanol administration with ethanol aqueous solution administered by gavage. Mice were administered 10% ethanol for indicated days before the elicitation of CHS. e) ACA in mice administered ethanol. Mice were administered 10% ethanol for 5 days before the elicitation of ACA. The results are presented as mean ± SD (n = 5 per group). The mean values of the different groups were compared with the mean value for mice administered vehicle control. *p < 0.05.
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Figure 4. Histological evaluation of inflamed skin in CHS mice. Ear auricles were excised 24 h after the elicitation of inflammation. Histology from the ear of: a) a mouse administered vehicle for 5 days, b) a mouse administered 10% ethanol for 5 days, c) a mouse administered 10% ethanol for 21 days, d) a mouse administered 50% ethanol for 5 days. Letters E, D, and C represent the epidermis, dermis, and cartilage, respectively. Scale bars indicate 50 µm. Representative data are shown. Figure 5. Effects of ethanol are different from effects of acetaldehyde. Disulfiram was administered intraperitoneally before the administration of ethanol and effects on CHS was observed. a) Thickness of ear lobes 24 h after the elicitation of allergy. The results are presented as mean ± SD (n = 5 per group). The mean values of the different groups were compared with the mean value for vehicle-control mice. *p < 0.05. b) Histology from the ear of a mouse administered 10% ethanol for 5 days after administration of disulfiram. Ear auricles were excised 24 h after the elicitation of inflammation. Letters E, D, and C represent the epidermis, dermis, and cartilage, respectively. Scale bars
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Figure 6. Cytokine expressions from ethanol-administered mice. Quantification of mRNA levels in the spleen was evaluated 6 h after the elicitation of CHS using realtime RT-PCR relative to that of RPS18. a) IFN-γ levels, b) IL-4 levels, c) IL-6 levels, d) IL-10 levels, e) IL-18 levels. The results are presented as mean ± SD (n = 5 per group). The mean values of the different groups were compared with the mean value for vehicle-control mice. *p < 0.05.
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