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. 2003 Apr;108(4):562–569. doi: 10.1046/j.1365-2567.2003.01588.x

DS–Nh as an experimental model of atopic dermatitis induced by Staphylococcus aureus producing staphylococcal enterotoxin C

T Yoshioka *, I Hikita *, T Matsutani *, R Yoshida *, M Asakawa *, T Toyosaki-Maeda *, T Hirasawa *, R Suzuki *, A Arimura *, T Horikawa *
PMCID: PMC1782922  PMID: 12667219

Abstract

DS–Nh mice raised under conventional conditions spontaneously develop dermatitis similar to human atopic dermatitis (AD), which is associated with staphylococcal infection. In the present study, we show that Staphylococcus aureus producing staphylococcus exotoxin C (SEC) was recovered from the culture of the skin lesions of DS–Nh mice with AD-like dermatitis and that the serum levels of anti-SEC antibodies from these mice were elevated. We describe here how to promote experimental AD by epicutaneous injection with SEC-producing S. aureus to DS–Nh mice. In order to assess the role of SEC in the pathogenesis of AD, the mitogenic activity, TCRBV repertoire analysis and the production of IL-4 and IFN-γ from spleen mononuclear cells (MNC) from DS–Nh stimulated by SEC were compared with those due to SEA, SEB and TSST. The weakest was the mitogenic activity of SEC, and higher IL-4 responses and lower IFN-γ responses to SEC showed correlation with TCRBV8S2-positive T cells, which were selectively stimulated by SEC. We also demonstrate that SEC-producing S. aureus was able to survive in DS–Nh after intradermal injection. These results suggest a possible role for SEC in the pathogenesis of AD through host–S. aureus relationships.

Introduction

Human atopic dermatitis (AD) is a chronic inflammatory skin disease affecting over 10% of children and is also a major cause of occupation-related disability caused by skin disease,1 which is pathogenically determined by abnormal T cell functions.2 Up to 107 colony-forming units of Staphylococcus aureus can be isolated from tc skin of more than 90% of AD patients.3 In general, only 5% of normal subjects carry S. aureus on their skin, and it is localized mainly in the nose and intertriginous areas. Most AD patients are colonized by S. aureus secreting identical superantigens (SAs), primarily Staphylococcal enterotoxin A (SEA), Staphylococcal enterotoxin B (SEB), Staphylococcal enterotoxin C (SEC) and toxic shock syndrome toxin (TSST).4–6 These SAs, which share the ability to stimulate a large number of some selective TCRBV-positive T cells, may play an important role in the pathogenesis of this disease.

With acute lesions of AD, there is a significant increase in the number of cells expressing IL-4, IL-5 and IL-13 mRNA, suggesting preferential accumulation of Th2 cells. Also, increased expression of IL-4 and IL-5 have been detected in CD4- and CD8-bearing cells in patients with AD.7,8 With chronic AD lesions, expression of Th1 cytokine interferon-γ (IFN-γ) is predominant.9 Thus, both Th1 and Th2 cytokines may contribute to the AD lesions.10 The Th1 cytokines play an important role in cell-mediated immunity and chronic inflammation. In particular, INF-γ-induced expression of MHC Class I and II molecules activates monocytes and macrophages, which play important roles in bacterial clearance. The Th2 cytokines have a critical role in the initiation of the allergic response in S. aureus-associated allergic dermatitis. IL-4 plays an important role as an inducer of IgE production11 and an inhibitor of bacterial clearance.12

In 1974, DS–Nh mice were developed from a colony of an inbred DS strain developed in 1954 from an outbred dd stock of the Central Institute for Experimental Animals, Tokyo, Japan. DS and DS–Nh mice are being maintained at Aburahi Laboratories, Shionogi & Co., Ltd, Shiga, Japan. The Nh non-hair phenotype is inherited in an autosomal dominant fashion. Recently, DS–Nh has been revealed to be applicable as a model mouse for human AD.13 DS–Nh mice showed specific symptoms such as S. aureus-associated dermatitis when kept for a long period under conventional conditions.14 We present the clinical features of DS–Nh kept under conventional conditions in Fig. 1.

Figure 1.

Figure 1

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Clinical features of DS–Nh mice kept under specific pathogen-free (without dermatitis) and conventional (with dermatitis) conditions at 15 weeks of age, and C57BL/6 mice under conventional conditions at 15 weeks of age.

For this study, we used SAs (SEA, SEB, SEC TSST), S. aureus and DS–Nh to clarify the host–bacteria relationship in AD.

Materials and methods

Mice

Male DS–Nh mice used for this study were obtained as F1 (Nh/+) from male DS–Nh (Nh/Nh) × female DS (+/+) kept under specific pathogen-free conditions (free from S. aureus). DS–Nh mice and C57BL/6 were maintained in micro-isolator cages under a 12-hr light/12 hr dark cycle and were provided standard feed and water ad libitum. They were housed in a conventional animal room or in a room under SPF conditions. This study was conducted according to the guidelines for animal experimentation at Kobe University School of Medicine and at Shionogi.

Isolation and identification of bacterial strains on the skin surface

To evaluate the preferential bacterial colonization of the lesions, bacterial cultures were obtained from the facial skin surface of each conventional DS–Nh with a sterile cotton swab-stick, inoculated onto salt egg yolk agar plate (Nissui, Tokyo, Japan), and incubated at 37°. Ten colonies per mouse were picked up at random and identified as subspecies of staphylococci with an AN-ID Test-SP18 kit (Nissui, Tokyo, Japan) according to the manufacturer's instructions.

Detection of S. aureus-derived exotoxins (SAs)

To identify SAs produced by S. aureus, bacteria defined as S. aureus were incubated in 1·5 ml of TSB medium for 36 h for 37°. The washed bacteria were tested for the presence of genes from SAs using polymerase chain reaction (PCR)-based S. aureus-derived exotoxin detection kits (Takara, Kyoto, Japan) according to the manufacturer's instructions.

Mice sensitization

Isolated S. aureus, 4 × 107, in 100 µl of normal saline or placebo (100 µl of normal saline) was placed on a 0·5 × 2 cm patch of serial gauze, which was secured to the skin with a transparent bioocclusive dressing (Johnson and Johnson Medical Inc., Arlington, TX) under S. aureus-free conditions. The patch was kept in place for a day and then removed. Six days later an identical patch was reapplied to the same site. Each mouse had a total of 4 days exposure to the patch separated by a 6-day interval. The patch sites were on the backs of the mice, and the backs of the C57BL/6 mice were shaved at each interval. The symptoms of the patched-exposed skin were assessed and then each mouse was killed after the last patch period.

Immunohistochemical and histochemical staining of skin tissue sections

Frozen and paraffin sections were prepared from the patched back skins for immunohistochemical and histochemical analysis. The frozen sections were pretreated with 2·5 µg/ml purified mouse IgG/1% BSA/PBS. Next, they were immunostained with rat antimouse CD4 (clone H129.19, PharMingen, San Diego, CA) and rat antimouse CD8a (clone 53–6·7, PharMingen, San Diego, CA). After washing in PBS, these slide-embedded sections were treated with biotin-conjugated goat antirat IgG (PharMingen, San Diego, CA). After washing with PBS, antibody-positive cells on these sections were visualized by peroxide–DAB staining and the count of antibody-positive cells for histopathological analysis was performed. Methylgreen was used for the counterstains.

Paraffin sections were stained with haematoxilyn–eosin and acidic toluidine blue for histpathological analysis.

Antibody responses to SEA, SEB, SEC and TSST in serum from DS–Nh

All four SAs used for ELISA, i.e. purified SEA, SEB, SEC and TSST, were purchased from Toxin Technology (Sarasota, FL). The levels of IgG antibodies against the four SAs in the serum samples were assessed by ELISA methods15 using purified SAs as antigens. Purified proteins were diluted to 2 µg/ml in 10 mm phosphate-buffered saline (PBS, pH 7·4), and 100 µl was added to each well of 96-well microplates (Immuno Module F8 Maxisorp). The plates were incubated overnight at 4° to allow binding of the antigens to the wells. Unbound antigens were removed by aspiration, and the wells were washed four times with washing buffer (PBS containing 0·8 g/l of Tween 20). PBS containing 10 g/l of bovine serum albumin (BSA) was added to each well of the microplate and incubated at 25° for 2 hr for blocking. The wells were washed four times with washing buffer and then filled with dilution buffer (PBS containing 1·0 g/l of BSA). The plates coated with SAs were stored at 4° until use.

Additional serum samples were obtained from conventional DS–Nh (aged 5–25 weeks). These serum samples were diluted at 1 : 200 with dilution buffer and 100 µl of diluted serum was added to the SA-coated wells. The plates were incubated at 4° overnight, and then the wells were washed four times with washing buffer. F(ab′)2 rabbit antimurine IgG peroxidase conjugate was diluted at 1000 ng/ml with dilution buffer; 100 µl of this reagent was added to each well, and the plates were incubated at 25° for 2 hr. The wells were again rinsed four times with washing buffer. The reaction was visualized by the subsequent reaction with 100 µl of substrate solution (TMB Peroxidase EIA Substrate Kit; Bio-Rad, Alfred, CA) for 5 min at room temperature. The reaction was terminated by addition of 50 µl of 1 m sulphonic acid and the absorbance of each well was measured with ImmunoReader NJ-2000 (Nihon InterMed Co., Tokyo) at 450 nm.

Analysis of TCR usage

Murine spleen cells (5 × 105) were stimulated with antimurine CD3 antibodies and SAs in the medium at 37° for 4 days. Crude cellular RNAs were extracted from peripheral blood mononuclear cells (PBMC) and stimulated cells by TRIzol™ LS Reagent (BRL, Bethesda, MD) according to the manufacturer's instructions. Adaptor-ligation PCR and microplate hybridization assays were carried out as described previously.16–19 Briefly, 1 µg of total RNA was converted to double-stranded cDNA using a SuperScript cDNA synthesis kit (BRL) according to the manufacturer's instructions, except for priming with BSL-18e primer adaptor containing the NotI site. The P20EA/10EA universal adaptors were ligated to the 5′ end of BSL-18e primed cDNA. Three rounds of Cα- and Cβ-specific PCR were performed using CA and CB sequence-specific oligonucleotide probes (SSOPs) to prepare amplified and biotinylated TCR cDNA pools. Hybridization was carried out between biotinylated PCR products, and VA or VB SSOPs immobilized on carboxylate-modified ELISA plate (Sumitomo Bakelite, Tokyo, Japan). The hybridization was visualized with p-nitrophenylphosphate (Nacalai Tesque, Osaka, Japan). The visualized signals were estimated at 405 nm using Immunoreader NJ-2000 (Nihon Intermed, Tokyo, Japan). Relative usage of the TCRAV or TCRBV region repertoire was calculated by the formula: relative usage of subfamily (%) = corresponding SSOP signal × 100/sum of total TCR V SSOPs signals.

Assay for proliferative responses and measurement of cytokine production after superantigen and antimurine CD3 antibody stimulation

We used 7-week-old mice kept under specific pathogen-free conditions in the following experiments. Murine spleen cells (5 × 105) were incubated in 96-well microplates (Corning Corster Co., Cambridge, MA) in RPMI-1640, 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin with 1 µg/ml of SEA, SEB, SEC and TSST at 37° for 4 days. Murine spleen cells (5 × 105) were incubated in antimurine CD3 antibody-coated 96-well microplate (Corning Corster) in RPMI-1640, 10% FCS, 100 U/ml penicillin and 100 µg/ml streptomycin at 37° for 4 days. Cells were pulsed with 0·5 mCi/well [3H]dThd (Amersham, Little Chalfont, UK) for 8 hr and then harvested. The radioactivity was measured with a liquid scintillation counter. Cell-free supernatants were collected, and IL-4 and IFN-γ concentrations were measured with a Quantikine immunoaasy kit (R&D Systems, Minneapolis, MN).

Bacterial intradermal inoculation and recovery from lymphocytes of DS–Nh

SEC-positive and -negative S. aureus, 4 × 107, in 100 µl of normal saline or a placebo (100 µl of normal saline) was inoculated onto the faces of DS–Nh mice. Three days after bacterial intradermal inoculation the mice were bled and killed, and lymphocytes from parotid and axillary were recovered. Lymphocytes were washed three times with RPMI-1640 and diluted at 107 cells/ml in distilled water (DW). A 100 µl portion of the cell suspension was plated onto tryptic soy agarose (TSA) plate and incubated at 37° for 24 hr, then the number of bacteria counted.

Results

Kinetic percentage of each species of Staphylococci

We examined alterations in the staphylococcus species on the skin of DS–Nh mice housed under conventional conditions. The kinetic percentage of each species of Staphylococci is shown in Table 1. S. aureus was not detectable in the samples obtained prior to and immediately after exposure. At 0 and 2 weeks post-exposure, the common staphylococcal species cultured from the skin of DS–Nh mice were S. cohniiB, S. epidermidis, S. gallinarm, S. sciuri, S. xylosus and S. warneri, with the dominant organisms being S. cohniiB and S. sciuri. After that, S. aureus was predominantly (60–100% frequency) cultured from the skin of DS–Nh mice under conventional conditions at 10–25 weeks post-exposure. At 5 weeks post-exposure, only 3·3% of S. aureus were SEC-positive and the rest were staphylococcal superantigen-negative strains. At 10–25 weeks post-exposure, all S. aureus strains were SEC-positive.

Table 1.

Ratio of staphylococcal species isolated from the skin of DS–Nh mice under conventional conditions and each superantigen produced by isolated S. aureus

Weeks under conventional conditions
0 2 5 10 15 25
Type of Staphylococcus and % frequency
″S. aureus 0 0 60 100 100 100
″S. cohniiB 78 54 0 0 0 0
″S. epidermidis 2 2 0 0 0 0
″S. gallinarum 2 6 0 0 0 0
″S. sciuri 10 30 16 0 0 0
″S. xylosus 4 4 0 0 0 0
″S. warneri 0 0 4 0 0 0
″Others 4 4 20 0 0 0
Type of SAs and their % frequency produced by S. aureus
″SEA – – 0·0 0·0 0·0 0·0
″SEB – – 0·0 0·0 0·0 0·0
″SEC – – 3·3 96·0 100·0 100·0
″SED – – 0·0 0·0 0·0 0·0
″SEE – – 0·0 0·0 0·0 0·0
″TSST – – 0·0 0·0 0·0 0·0
″ND – – 96·7 4·0 0·0 0·0
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Staphylococcus aureus-induced dermatitis in DS–Nh mice

DS–Nh and C57BL/6 mice were patched with 4 × 107 S. aureus, and the clinical course of the disease was followed for 35 days. Seven days after the last bacterial inoculation, erythema and oedema dry skin were observed at the bacterial patched site only in DS–Nh (Fig. 2). To analyse the characteristics of infiltrating T lymphocytes, frozen sections of bacterial- and placebo-patched skin were stained with anti-CD4 and anti-CD8a monoclonal antibodies. The number of antibody-positive cells was counted. Both CD4- and CD8a-positive T lymphocytes increased in bacterial patched skin compared with those exposed to saline. The ratio of CD4 to CD8a T lymphocytes was the highest in the bacterial patched skin (Fig. 2).

Figure 2.

Figure 2

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Clinical features DS–Nh mice patched with S. aureus and placebo, and C57BL/6 mice patched with S. aureus after 7 days from the last patch. Erythema and edema dry skin were observed at the bacterial patched site in DS–Nh mice. CD4-negative, CD8a-positive and TCRBV8S2-bearing T lymphocytes increased in bacterial patched skin compared with those exposed to saline.

Histological changes such as hyperkeratosis and infiltration of inflammatory cells were observed in bacteria-patched skin lesion. The number of total and degranulated mast cells was higher in bacteria patched skin lesions compared with those exposed to saline (Fig. 2).

Antibody titres to SEA, SEB, SEC and TSST in serum samples from DS–Nh mice

To determine the potential pathogenic role of SAs in dermatitis, we examined the serum antibody levels to SEA, SEB, SEC and TSST in the sera from DS–Nh mice under conventional conditions and from DS–Nh mice under SPF conditions. Levels of Ig antibodies to SAs were determined by the ELISA method. Sera from DS–Nh mice under conventional conditions 16–25 weeks post-exposure contained higher levels of Ig antibody against SEC than those from age-matched controls (P < 0·01). There was no significant difference in the levels of serum antibodies to the other SAs between conditions (Fig. 3).

Figure 3.

Figure 3

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Antibody titres against SEA, SEB, SEC and TSST. Sera from DS–Nh mice under conventional conditions contained higher levels of Ig antibody against SEC than those from age-matched controls (P < 0·01).

Selective stimulation of certain Vb-bearing T cells by SEA, SEB, SEC and TSST

In this study, we defined the increase as significant when (i) the percentage was greater than the mean percentage + 2 SD of five controls without stimulation and (ii) the actual percentage obtained in the assay was >10%. Splenocytes from DS–Nh were stimulated with SEA, SEB, SEC and TSST to investigate the TCRBV preference (Fig. 4). The TCRBV repertoire in spleen cells from DS–Nh was analysed at day 0 (before stimulation) and day 4 after stimulation with SAs. The frequency of T cells bearing TCRBV1, 10, 11 and 12 in all DS–Nh increased significantly at day 4 after SEA. The frequency of T cells bearing TCRBV3, 7 and 12 in all DS–Nh increased significantly at day 4 after SEB. The frequency of T cells bearing TCRBV1, 8·2 and 10 in all DS–Nh increased significantly at day 4 after SEC. The frequency of TCRBV18-bearing T cells in all DS–Nh increased significantly at day 4 after TSST stimulation.

Figure 4.

Figure 4

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TCRBV repertoires in spleen cells from 7-week-old DS–Nh kept under SPF condition which were stimulated in vitro with SAs. The bar indicates mean + 2 SD of the TCRBV repertories in PBMC from five DS–Nh mice without in vitro stimulation. Each dot indicates the percentage frequency of TCRBV-bearing T cells in the spleen cells stimulated in vitro with SAs.

Proliferative responses and cytokine production of splenocytes from DS–Nh after SA stimulation

We compared the mitogenic activities of SEA, SEB, SEC and TSST (Fig. 5). All toxins were active on T cells from DS–Nh in the following order: (SEB, TSST) > SEA > SEC.

Figure 5.

Figure 5

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Proliferative responses of spleen cells from DS–Nh mice to CD3 antibodies and different SAs.

IL-4 and IFN-γ production by splenocytes stimulated with SEA, SEB, SEC and TSST was examined to find possible pathogenic differences among these SAs. Culture supernatants were collected at 4 days after stimulation with SAs. IL-4 responses to SAs varied in the order of SEC > (SEB, TSST) > SEA. IFN-γ responses to SAs were in the order of SEA > (SEB, TSST) > SEC. The highest IL-4 responses and lowest IFN-γ responses were observed for SEC compared to the other three Sas (Fig. 6).

Figure 6.

Figure 6

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IL-4 and IFN-γ produced in culture of spleen cells from DS–Nh mice stimulated with SAs.

Bacterial inoculation and detection of bacteria from lymph node

We examined survival ratio of SEC-positive and -negative S. aureus in DS–Nh to understand the host–bacteria relation through superantigens. Three days after inoculation, we examined the live bacteria in parotid and axillary lymph nodes of infected DS–Nh mice. In Table 2, SEC-positive S. aureus could be detected from parotid lymph nodes but not axillary lymph nodes, and SEC-negative type was not detected from either lymph node. The number of detectable bacteria was 10, 19 and 9 per 106 lymphocyte. SEC-positive bacteria were difficult to kill in skin lesions of DS–Nh mice.

Table 2.

Bacterial inoculation and their recovery from DS-Nh

S. aureus no./106 cells
Lymph node Murine no. SEC-positive SEC-negative
Parotid DS–Nh no. 01 10 0
no. 02 19 0
no. 03 9 0
Axillary DS–Nh no. 01 0 0
no. 02 0 0
no. 03 0 0
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Discussion

DS–Nh mice develop dermatitis spontaneously only when raised under conventional conditions, and not under specific pathogen-free (S. aureus-free) conditions.13,14 This spontaneous dermatitis in DS–Nh is comparable to a certain type of human AD.13 In this study, we found that only SEC-producing S. aureus is dominant in skin lesions from aged DS–Nh with AD-like dermatitis. Increased amount of secreted SEC and anti-SEC antibody titre in serum were observed for DS–Nh kept under conventional condition. Figure 3 shows some anti-SEB antibody titres in spite of no SEB-producing S. aureus colonizing the skin in DS–Nh mice. This may have been partially due to cross-reactivity of anti-SEC antibody against SEB, because amino acid sequences of those superantigens are approximate.

Spergel et al. reported that a mouse model of EC sensitization with protein antigen leads to local allergic dermatitis in BALB/c mice.20 In humans, approximately 80% of patients with AD have elevated levels of serum IgE and evidence of IgE antibodies to a variety of allergens.21 These results suggest that protein allergen plays an important role in murine models of allergic dermatitis and human AD. In this study, we could produce experimental dermatitis by EC sensitized with S. aureus from aged DS–Nh under conventional conditions on the backs of S. aureus-free DS–Nh mice. This experiment is the same as in spontaneously developed dermatitis in DS–Nh mice with respect to the following symptoms: erythema, oedema, dry skin and infiltration of CD4-bearing T cells. In DS–Nh mice used as an AD model, SEC-producing S. aureus is a major cause of AD-like dermatitis.

S. aureus is found in the skin lesions of human AD patients, and Staphylococcal enterotoxins, such as SEA and SEB, had been believed to exacerbate AD symptoms through immunological mechanisms. For example, IgE antibodies against SEA and SEB are found in the sera of patients with AD22 suggesting that allergic responses to the SAs play a role in the exacerbation. These SAs also can induce total in vitro IgE synthesis after cross-linking T and B cells23 and allergen-specific IgE are synthesized in AD.24 Total IgE13 and IgG antibodies against SEC were elevated in sera from DS–Nh with AD-like dermatitis. What is puzzling is that these IgE induced by SAs do not play an important role in the pathogenesis of AD in NC mice.25 Although the clinical evidence is persuasive, the mechanisms of the disease-promoting effects of superantigens produced by S. aureus remain to be elucidated. In DS–Nh mice with dermatitis, SEC produced by S. aureus can act as superantigens and/or classic allergens.

Utilizing SEC and splenocytes from DS–Nh, we studied the importance of SEC in DS–Nh mice with spontaneously developed dermatitis. We show that SEA, SEB, SEC and TSST stimulate T cells from DS–Nh. The mitogeneic activity of SEC was the weakest and the highest IL-4 responses and lowest IFN-γ responses were also observed. Although these SAs preferentially stimulated some TCRBVs-positive T cells, only TCRBV8S2-bearing T cells were stimulated selectively by the SEC. This cytokine profile from splenocytes after stimulation with SEC may be caused by TCRBV8S2. Furthermore, it is interesting that these TCRBV8S2-bearing T cells infiltrate the skin lesion in experimental dermatitis. Many of the T cells bearing TCRBV8S2 were NKT cells, which are known to be the major source of IL-4.26–28. This is of interest because TCRBV8S2 plays an important role in the pathogenesis of some allergic and dermal diseases, such as asthma,29,30 contact sensitivity reaction to 2,4,6-trinitro-1-Chlorobenzene(TNCB31) and experimental allergic encephalomyelitis.32

The existence of Th1/Th2 subsets in Th lymphocyte that differ in cytokine secretion patterns and effector functions provides a framework for understanding normal and pathological immune responses.33 Allergic responses at the site of inflammation are considered to be due to development and activation of Th2 cells.34 One of the representative cytokines of Th2 cells is IL-4, and that of Th1 is IFN-γ. IL-4 is known to play many roles in the pathogenesis of AD and is highly expressed in acute AD skin lesions.35 This is important for IgE isotype switching, developing Th2 cells and induction of adhesion molecules on endothelial cells that recruit eosinophils.36 IL-4 also plays a detrimental role in the immune response to S. aureus infection by enhancement of bacterial growth and/or decrease of clearance.37 Furthermore, incubation of normal mouse skin with IL-4 induced increased S. aureus skin binding. Otherwise, IFN-γ plays an important role for host defence against bacteria.38 In our study, we detected only SEC-producing S. aureus in the skin lesion of dermatitis from aged DS–Nh and the dermatitis could be promote by inoculation with the extracted bacteria. T cells from DS–Nh stimulated by SEC secreted much IL-4 and a small amount of IFN-γ. Considering these reports and our findings, SEC led DS–Nh to change the Th1/Th2 cytokine valance. This eventual change may result in S. aureus being able to survive in DS–Nh and able to bind easily to the skin. These long-lived bacteria in the skin may play a pathogenic role in AD as an allergen.

In this study, we proved the importance of SEC in DS–Nh mice with spontaneously developed dermatitis but not in humans. There is published evidence pointing to a more important role of SEB compared to SEC in human atopic dermatitis. We believed that SEB (it may be SEA) may be important in humans with AD, although there are differences among sequences of human and mice TCR V beta chain (specificity of superantigens depends on amino acid sequence of TCR). SEB for human may have as strong an effect in humans as SEC in DS–Nh.

In conclusion, we propose that SECs play a key role in immune responses to S. aureus infection causing AD-like dermatitis in DS–Nh. DS–Nh may be serve as a useful tool for analysing the host–bacteria relationship in certain types of human AD.

Abbreviations

AD

atopic dermatitis

PCR

polymerase chain reaction

PBMC

peripheral blood mononuclear cells

SE

Staphylococcus exotoxin

SEA

Staphylococcal enterotoxin A

SEB

Staphylococcal enterotoxin B

SEC

Staphylococcal enterotoxin C

SSOPs

sequence-specific oligonucleotide probes

TSST

toxic shock syndrome toxin

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