Ige-Mediated Hypersensitivity Against Human Sweat Antigen in Patients with Atopic Dermatitis
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Acta Derm Venereol 2002; 82: 335± 340 INVESTIGATIVE REPORT IgE-mediated Hypersensitivity Against Human Sweat Antigen in Patients with Atopic Dermatitis MICHIHIRO HIDE, TOSHIHIKO TANAKA, YUMI YAMAMURA, OSAMU KORO and SHOSO YAMAMOTO Department of Dermatology, Programs for Biomedical Research, Division of Molecular Medical Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan Sweating aggravates itch in atopic dermatitis, but the a bath, is largely bene® cial to patients (1, 5), and mechanism is unclear. In this study, we examined the suggests the presence of aggravating factors on the skin involvement of type I hypersensitivity in the aggravation surface. On the other hand, immediate-type skin of atopic dermatitis by sweating. Skin tests with auto- reactions to human dander extracts (6, 7) and human logous sweat were positive in 56 of 66 patients (84.4%) sweat (8, 9) have been reported in patients with atopic with atopic dermatitis, but only in 3 of 27 healthy volun- dermatitis. Adachi & Aoki (9) showed the binding teers (11.1%). Sweat samples from both patients and activity of IgE antibody against sweat concentrate healthy volunteers induced varying degrees of histamine collected from a healthy volunteer in patients with release from basophils of patients with atopic dermatitis. atopic dermatitis. More recently, Valenta et al. (10) and However, the histamine release was impaired by removal Natter et al. (11) have identi® ed several intracellular of IgE on the basophils. Incubation of basophils with autoantigens for IgE antibodies in the sera of patients myeloma IgE before sensitization with serum of patients with severe skin manifestations of atopic dermatitis. blocked the ability to release histamine-induced sweat. However, a recombinant protein antigen, Hom s 1, IgE antibody against antigen(s) in sweat may be present reported as the most frequently recognized autoantigen in serum of patients with atopic dermatitis. Key words: by serum IgE of patients with atopic dermatitis, merely histamine; basophils; skin test; autoallergy. induced mild erythematous skin reactions in patients with Hom s 1-speci® c IgE antibodies. Moreover, no signi® cant correlation was observed between the bind- ing activity of serum IgE to the sweat antigen and skin Acta Derm Venereol 2002; 82: 335± 340. reactions to autologous sweat samples in the study by Michihiro Hide, Department of Dermatology, Pro- Adachi & Aoki (9). It is therefore unclear whether such grams for Biomedical Research, Division of Molecu- IgE and autoantigens are involved in the pathogenesis lar Medical Science, Graduate School of Biomedical of atopic dermatitis or not, especially via the mechan- Sciences, Hiroshima University, 1-2-3, Kasumi, ism of type I hypersensitivity. In this study, we show a Minami-ku, Hiroshima 734-8551, Japan. E-mail: high incidence of positive skin reactions to autologous [email protected] sweat in patients with atopic dermatitis regardless of their total serum IgE concentrations, and demonstrate that basophils of the patients release histamine in Atopic dermatitis is a chronic in¯ ammatory skin response to sweat antigen(s) by a mechanism mediated disease that frequently occurs in patients with a per- by speci® c IgE. sonal or family history of atopy (1). Histological analysis of the lesions shows an increase of the mast cell population accompanied by an in® ltration of in¯ am- MATERIALS AND METHODS matory cells, coupled with the dominant expression of Subjects Th2-type cytokines by the lymphocytes, particularly in The following subjects were included in the study: 66 patients acute-phase lesions (1, 2). Challenge by the house dust- (34 men and 32 women; 13 ± 37 years of age, mean+SD mite antigen on the skin of patients with atopic derma- 24.7+5.2) with atopic dermatitis; 7 patients (5 men and 2 titis in vivo and human skin slices sensitized by sera women; 20 ± 35 years of age, mean+SD 24.6+4.8) with of the patients in vitro induces the release of pro- allergic rhinitis without atopic dermatitis, urticaria or asthma; and 27 healthy volunteers (15 men and 12 women; in¯ ammatory mediators and cytokines, such as hista- 20 ± 42 years of age, mean+SD 27.8+6.7) without allergic mine, arachidonic acid metabolites and tumour necrosis symptoms. The protocol of the study was approved by factor-a (3, 4). Elimination and/or avoidance of anti- the ethics committee of Hiroshima University, Faculty of gens for IgE from the environment of patients has Medicine. All patients and volunteers were informed of the aim and protocol and agreed to participate in the study. therefore been suggested as one of the important treat- Twenty-® ve of the 66 patients with atopic dermatitis ments of patients with atopic dermatitis (1). Moreover, had complications of allergic rhinitis and another 15 had cleaning the skin surface after a shower, or after taking complications of asthma. Twelve of these suffered from both # 2002 Taylor & Francis. ISSN 0001-555 5 Acta Derm Venereol 82 336 M. Hide et al. asthma and allergic rhinitis. Histamine H1-receptor antago- with 0.03% human serum albumin (Sigma, A1653, Tokyo, nists were discontinued for at least 3 days prior to the study. Japan). For passive sensitization, the acid-treated basophils Disease severity of each patient was evaluated according were pre-incubated in 2 ml Ca2+ -free, 4 mM EDTA buffer to the grading of atopic dermatitis by Rajka & Langeland solution (13, 15) in the presence or absence of 10 mg/ml (12). Total serum IgE concentrations of the patients were myeloma IgE (Chemicon, Temecola, CA) at 37³C for 30 min, measured using a ¯ uoroenzyme immunoassay kit (UniCap1, followed by the addition of 2 ml serum of patients with atopic Pharmacia-Upjohn, Tokyo, Japan). dermatitis (AD1, see below) at 37³C for 60 min (13, 15). After sensitization, the cells were washed three times with buffer and exposed to the challenge stimuli, as described above. Sweat collection Sweat was collected using the methods described by Adachi & Aoki (9), with a slight modi® cation. The skin of the subject’s Statistics trunk was wiped twice with a cotton swab immersed in 50% Data were summarized as mean+SEM. To calculate isopropyl alcohol. The trunk of the subject was covered with signi® cance levels between groups, two-way ANOVA with 2 ± 3 layers of wrapping ® lm (Saran Wrap1, Asahi-Kasei, Bonferroni’s post test was performed using GraphPad InStat Tokyo, Japan), the edges sealed with adhesive tape. After version 3.01 for Windows 95/NT (GraphPad Software, San running for 15 to 30 min, the ® lm was removed from the Diego, CA). Analyses of a contingency table using the Fisher subjects’ skin and the sweat on the area that was covered was exact test and of linear regression were also performed using collected using an injection syringe (Terumo, SS01T2613S, the same program. Tokyo Japan). The sweat samples were ® ltered (0.22 mm ® lter) (Millipore, SLGV013SL, Bedford, MA) and used immediately for skin testing of the patients or stored at ± 20³C until use RESULTS for analysis in vitro. Skin tests for autologous sweat samples and serum IgE Gel chromatography concentrations One and a half millilitres of ® ltered sweat samples of three Of 66 patients with atopic dermatitis, 56 (84.8%) patients with atopic dermatitis was lyophilized, reconstituted showed positive skin reactions to their own sweat, with saline and part or all of the samples were applied to an whereas only 3 out of 27 individuals (11.1%) in healthy HPLC system. The samples were fractionated in 500 ml saline/ controls showed the positive reactions (p 0.0001; fraction by TSK-GEL G2000 column (TOSOH, Tokyo, 5 Japan) at a ¯ ow rate of 1 ml/min. Table I). In patients with allergic rhinitis there was also a strong association with the positive skin reactions. There was no association with disease Skin test severity of atopic dermatitis. Moreover, there was no In skin tests, 20 ml of crude or fractionated autologous sweat statistical difference in serum IgE concentrations samples, prepared as above, was injected intradermally between patients showing positive skin tests and into the volar forearm skin (crude samples) or back skin (fractionated samples) of the subjects. After 15 min, the those with negative reactions, while serum IgE diameters of the erythema and wheal were measured. The concentration was positively correlated with disease reactions were assessed as ``positive’’ if the diameter of sweat- severity (p50.01, data not shown). Wheal sizes in the induced wheal was equal to or larger than 8 mm, or that of skin tests were not apparently correlated with the the ¯ are equal to or larger than 20 mm. disease severity (data not shown), but marginally correlated with serum IgE concentrations (p50.05) Histamine release assay (Fig. 1). Peripheral blood leucocytes containing about 1% basophils were obtained from patients with atopic dermatitis and from healthy volunteers, as described by Grattan et al. (13), as well Histamine release from basophils of patients with as histamine release assay, except that the ® nal reaction atopic dermatitis by sweat samples of various subjects volume was reduced to 100 ml/tube. Sweat samples and goat anti-human IgE antibody (Seikagaku Co. Tokyo, Japan) Histamine release activity in the sweat samples of were diluted 10 times and 3,000 times, respectively, if not various subjects was studied using the leucocytes of a indicated. A mouse IgG2b monoclonal antibody (CRA-1), which recognizes an epitope on the high af® nity IgE receptor Table I. Skin reactions to autologous sweat (FceRI) without the interference of IgE binding (Kyokuto Pharmaceutical Indust. Co. Ltd, Ibaraki, Japan) (14), was Disease n Positive skin reaction n (%) used as a positive control for the assays at 50 ng/ml. The amounts of histamine were measured by the HPLC system, as Atopic dermatitis 66 56 (84.8)** described by Koro et al.