The skin is an important immunologic tissue and acts as the first barrier to protect against invasive factors, including viruses, chemicals and insects. The inflammatory stimulus, which results from exposure to a variety of chemical agents, generates the irritation and itching and can lead to severe contact dermatitis. The contact dermatitis provoked by chemical agents, such as picryl chloride (PC) and 2,4-dinitrofluorobenzene (DNFB), is an inflammatory disorder generated in the local skin. In contact dermatitis, the proliferation of keratinocytes and marked infiltration of inflammatory cells, including neutrophils, monocytes and lymphocytes, to the skin are observed.1 In addition, there is some evidence to indicate that various kinds of cytokines including interleukin (IL)-4, interferon (IFN)-γ, IL-6 and IL-1 play crucial roles for the initiation and progression of contact dermatitis.2-5 Among these cytokines, the importance of IFN-γ in the elicitation and progression of contact dermatitis has been verified using IFN-γ-overexpressing mice and IFN-γ receptor-deficient mice.6, 7 Carroll et al. have reported that IFN-γ transgenic mice show an augmentation of contact hypersensitivity to topical application of DNFB, compared with wild-type mice.6 Saulnier et al. have reported that dermal infiltration of mononuclear cells and epidermal microabscesses induced by the 2,4,6-trinitrochlorobenzene (TNCB) is diminished in IFN-γ receptor-deficient mice.7 In addition to this evidence, some reports have shown that retinoids regulate transcription of cytokines such as IL-6 and IFN-γ.8, 9 Cippitelli et al. have demonstrated that retinoic acid inhibits IFN-γ gene expression through the down-modulation of activation of the IFN-γ promoter.8

Retinoid is a potent ligand for retinoic acid receptors (RAR), which belong to the steroid/thyroid receptor superfamily.10 Three RAR subtypes, including α , β and γ, have been identified.11 The retinoid-RAR complex elicits a variety of biological activities, such as the regulation of cell growth,12 differentiation13 and anti-angiogenesis,14 through the regulation of transcription of various genes either by activating the expression of genes, including RAR-responsive elements (RARE) in the promoter regions,15 or by inhibiting expression of some genes by antagonizing AP-1-mediated gene expression.16

In a previous study, a novel synthetic retinoid, 4-((5,6, 7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl) carbamoyl) benzoic acid (Am-80) showed an inhibitory effect on collagen-induced arthritis in mice, which is an autoimmune disease model, by inhibiting the production of pro-inflammatory cytokines at the transcriptional level.9 Am-80 has approximately 10-fold more potent biological activity than all-trans-retinoic acid by binding to the RARα and RARβ, but not RARγ. Am-80 has superior properties, including stability to light, heat and oxidation and less toxicity and resistance than all-trans-retinoids.

These findings suggest that Am-80 may inhibit contact dermatitis. Previously, a mouse model generating severe contact hypersensitivity17 was established. In this model, mice receiving repeated paintings of DNFB to their ears showed severe dermatitis, together with marked infiltration of inflammatory cells including neutrophils, mononuclear cells and eosinophils, increased hapten-specific IgE in serum and excess expression of mRNA for IFN-γ and IL-4 in cervical lymph nodes.

In the present study, Am-80 is shown to potently inhibit contact dermatitis induced by repeated applications of DNFB through inhibition of the inflammatory cytokines, especially IFN-γ.

Female C57BL/6 mice of the age of 6 weeks, obtained from Japan SLC Inc. (Hamamatsu, Japan), were housed in plastic cages in an air-conditioned room at 24°C, fed a standard laboratory diet and allowed free access to water. All experiments were carried out following the guidelines for the care and use of experimental animals of the Japanese Association for Laboratory Animal Science of 1987.18

The Am-80 was synthesized as described previously.9 The Am-80 was suspended in 0.5% carboxymethyl cellulose (CMC) solution for oral administration. Monoclonal anti-dinitrophenyl (DNP) IgE antibody (Clone SPE-7; Sigma Chemical Co., St Louis, MO, USA), peroxidase-conjugated streptavidin (Dakopatts a/s, Glostrup, Denmark), bovine serum albumin (BSA for ELISA grade; Sigma Chemical Co.), DNP-BSA (LSL Co. Ltd, Tokyo, Japan), NHS-LC-Biotinylation kit (Pierce Chemical Co., Rockford, IL, USA), substrate kit (Sumitomo Bakelite Co. Ltd, Tokyo, Japan), ISOGEN (Nippon Gene Co. Ltd, Tokyo, Japan), reverse transcriptase polymerase chain reaction (RT-PCR) primer sets (β-actin, IFN-γ, IL-1, IL-4; Stratagene Co. Ltd, La Jolla, CA, USA), 1st-STRANDED TM cDNA Synthesis kit (Clontech Lab. Inc., Palo Alto, CA, USA) and PCR Reagents Kit (Perkin Elmer Japan Co. Ltd, Urayasu, Japan) were purchased from listed suppliers.

Ears of mice were painted with 25 µL 0.15% DNFB in vehicle (acetone:olive oil = 3:1) or vehicle alone applied to each side of the ear once a week. Ear thickness was measured at various time points after applying DNFB using an engineering micrometer (R1-A, Ozaki MFG Co. Ltd, Tokyo, Japan) and is expressed as the increase in thickness from that at time 0. The Am-80 suspended in CMC was orally administered once per day throughout the experiment from the first sensitization with DNFB.

Mice ears were removed 24 h after the fifth painting with vehicle or DNFB and fixed with 10% neutral formalin. Ears were then cut into parasagittal slices, dehydrated and embedded in paraffin according to the standard procedures. Paraffin sections were stained with hematoxylin and eosin, then assessed under a light microscope.

To measure anti-DNP IgE levels in serum, sera were obtained from the mice 24 h after the fifth painting with DNFB. The anti-DNP IgE in mice sera were measured using the enzyme-linked immunosorbent assay (ELISA) described below.

The immunoplate (Maxisorp Nunc-Immuno Plate, Nalge Nunc International, Tokyo, Japan) was coated with monoclonal antimouse IgE antibody and incubated at 4°C overnight. To decrease the non-specific reaction of antibodies, the immunoplate was treated with phosphate-buffered saline (PBS) containing 1% BSA, incubated at room temperature for 1 h and washed three times with PBS containing 0.2% Tween 20 (T-PBS). Monoclonal anti-DNP IgE antibody was sequentially diluted as the standard. Diluted serum samples at a volume of 100 µL were added to each well and the plates were incubated at room temperature for 1 h. After washing with T-PBS, 100 µL diluted biotinylated DNP- BSA was added to each well and incubated at room temperature for 1 h. After extensive washing with T-PBS, 100 µL diluted peroxidase-conjugated streptavidin was added to each well. The enzymatic reaction was stopped by adding 100 µL stop solution after incubation at room temperature for 1 h. The optical density of the reaction mixture was read using an automatic ELISA plate reader (Titertek Multiscan MCC/340, Flow Laboratories Inc.) at 450 nm.

The anti-DNP IgE titers were expressed in ng/mL, based on laboratory generated standards and appropriate commercial standards.

Expression levels of mRNA for cytokines in cervical lymph nodes of mice were assessed by the RT-PCR technique. Using ISOGEN, total RNA was extracted from cervical lymph nodes in mice at 4 h after the fifth treatment with DNFB.

The total RNA (500 ng) was reverse-transcribed with the 1st-STRANDED TM cDNA Synthesis Kit at 42°C for 60 min, 94°C for 5 min and then soaked at 5°C for 5 min. The PCR was performed under standard conditions as follows: (i) 5 min at 94°C for 1 cycle; (ii) 1 min 30 s at 94°C , 1 min 30 s at 60°C and 1 min 30 s at 72°C for 30 or 35 cycles; (iii) 10 min at 72°C for 1 cycle; and (iv) 5 min at 5°C.

Primer sets used for the amplification of first-strand cDNA were as follows: IFN-γ, 5'-TACTGCCACGGCACAGTCATTGAA-3', 5'-GCAGCGACTCCTTTTCC- GCTTCCT-3'; IL-1-β, 5'-CAGGATGAGGACATGAGCACC-3', 5'-CTCTGCAGACTCAAACTCCAC-3'; IL-4, 5'-ACGGAGATGGATGTGCCAAACGTC-3', 5'-CGAGTAATCCATTTGCATGATGC-3'; and β-actin, 5'-GTGG- GCCGCTAGGCACCA-3', 5'-CGGTTGGCCTTAGG- GTTCAGGGGGG-3'. The PCR products were resolved by electrophoresis and stained with ethidium bromide to reveal the amplified cDNA.

Results are expressed as the mean ± SEM. One-way ANOVA/Dunnett's post hoc procedure was used to assess the significance of results. P < 0.05 was considered to be statistically significant.

The ears of mice painted with 0.15% DNFB, but not with vehicle alone, provoked severe contact dermatitis in proportion to the number of DNFB applications. Ear swelling was increased at approximately 1 h and reached a peak at 24 h after painting with DNFB (Fig. 1a). The Am-80 was administrated at the dosage of 0.3-3.0 mg/kg per day orally once per day throughout the experiment after the first sensitization with DNFB. The Am-80 significantly inhibited ear swelling 1 h and 24 h after the fifth painting with DNFB, in a dose-dependent manner (Fig. 1b). Throughout this experiment, conspicuous adverse effects, such as loss of weight, were not observed in mice treated with Am-80 (data not shown).

In the histopathologic study, marked infiltration of cells such as neutrophils, lymphocytes and monocytes were elicited in the ears of mice treated with 0.15% DNFB, but not with vehicle alone. In the ears of mice administered Am-80 at the dosage of 3.0 mg/kg per day, infiltration of cells was clearly reduced (Fig. 2).

We investigated the effect of Am-80 on the production of anti-DNP IgE in sera. The sera were obtained from mice 24 h after the fifth painting with DNFB. The amounts of anti-DNP IgE were measured using the ELISA technique. The serum level of anti-DNP IgE was significantly elevated in mice treated with 0.15% DNFB, but not with vehicle alone, in proportion to the number of DNFB applications (data not shown). As shown in Figure 3a, Am-80 did not affect the production of anti-DNP IgE even at the highest dosage (3.0 mg/kg per day, P = 0.563). In addition, the effect of Am-80 on the transcriptional level of productive Cε in cervical lymph nodes was examined. Cervical lymph nodes were isolated from mice 4 h after the fifth treatment with DNFB and total RNA was extracted and purified using ISOGEN. The expression level of productive CεmRNA was assessed using the RT-PCR technique. The Am-80 did not affect the expression level of productive C mRNA in cervical lymph nodes (Fig. 3b). These findings suggested that Am-80 did not affect the production of anti-DNP IgE at both the transcriptional and post-transcriptional levels; and that it in- hibited the contact dermatitis at the post-sensitization phase.

In the preliminary experiment, the expression levels of mRNA for these cytokines in the cervical lymph nodes of DNFB-treated mice reached their peak and plateau 48 h after DNFB-sensitization (data not shown). Therefore, the effect of Am-80 on transcription levels of cytokines, including IFN-γ, IL-1 and IL-4, on the cervical lymph nodes of mice at 4 h after the treatment with DNFB was investigated. Expression levels of mRNA for all cytokines was elevated in cervical lymph nodes of mice treated with DNFB compared with mice treated with vehicle alone. The Am-80 obviously diminished expression levels of mRNA for IFN-γ, but not for IL-1 and IL-4, in a dose- dependent manner (Fig. 4).

Contact dermatitis is an allergic inflammatory skin disease with marked infiltration of inflammatory cells. In the present study, the effect of Am-80 on mouse contact hypersensitivity due to repeated application of DNFB to ears was investigated. In this model, severe elevation of ear swelling and serum anti-DNP IgE levels were observed. Oral administration of Am-80 once per day showed a potent and significant inhibition of the ear swelling and histopathologic changes in the skin lesion. However, Am-80 did not affect the serum level of anti-DNP IgE and the expression level of productive CεmRNA in the cervical lymph node. Immunoglobulin E, following the activation of mast cells, plays a crucial role in the ear swelling response at early time points through the release of a variety of chemical mediators, such as histamine, serotonin and leukotrienes. In the present study, although the serum level of anti-DNP IgE was elevated in parallel with the increase of ear swelling, Am-80 inhibited an increase in ear swelling at 1 h after treatment with DNFB, despite no affect on the anti-DNP IgE level. This finding suggests that Am-80 may inhibit the function of mast cells at the post IgEantigen interaction stage. In addition, Geba et al. have reported the importance of serotonin release from platelets during the early phase reaction of contact hypersensitivity.19 To clarify the mechanism of how Am-80 inhibits the early phase reaction of dermatitis without inhibiting the production of IgE requires further studies.

Much attention has been paid to the role of cytokines in the onset and development of allergic skin disease. Some reports show the roles of IL-4,2 IFN-γ3 and IL-64 in contact dermatitis. Among them, IFN-γ especially contributes to the initiation and progression of contact hypersensitivity. Interferon-γ, which is generated especially by T helper (Th)1 cells, induces various kinds of biologic events, such as the elicitation of adhesion molecule expression, keratinocyte proliferation and down-regulation of Th2-type cytokines.20 Some studies have also shown the functional role of IFN-γ in contact dermatitis. Carroll et al. have reported an augmentation of contact dermatitis in IFN-γ transgenic mice.6 Furthermore, Saulnier et al. have reported an abrogation of dermal infiltration of mononuclear cells and epidermal microabscesses in IFN-γ receptor-deficient mice.7 In the present study, Am-80 clearly decreased the expression level of mRNA for IFN-γ, but not for IL-1 and IL-4, in cervical lymph nodes of DNFB-sensitized mice. Cippitelli et al. have also reported that retinoic acid diminishes the expression level of the IFN-γ gene through down-modulation of the IFN-γ promoter.8 Their findings support those of the present study. In the histopathologic study of the mouse ear, the marked reduction in inflammatory cell infiltration by Am-80 may have been due to the suppression of adhesion molecules by the inhibition of IFN-γ production. However, in contact hypersensitivity many kinds of cytokines are involved in the initiation and development of the reaction and the cytokine network is complicated. Therefore, further studies will be necessary to clarify this point.

In conclusion, it has been demonstrated that Am-80 potently inhibits the contact dermatitis caused by repeated applications of DNFB, probably through the depression of IFN-γ mRNA. Hence, Am-80 may be useful for treatment of contact dermatitis. For the clinical trial, however, further studies, such as investigating the effect of Am-80 in mice chronically inducing contact hypersensitivity, will be necessary.

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