过敏性湿疹病人体内金黄色葡萄球菌内毒素对组胺和白三烯释放的研究

过敏性湿疹病人体内金黄色葡萄球菌内毒素对组胺和白三烯释放的研究

（国外学术刊物发表）

Staphylococcus aureus Enterotoxins Induce Histamine and Leukotriene Release in Patients with Atopic Eczema

K. Neuber, J. Wehner, K. Hakansson, and J. Ring

Abstract

Peripheral blood basophils from patients with atopic eczema stimulated with the staphylococcal enterotoxins (SE) A, B, D, E and toxic shock syndrome toxin 1 (TSST-1) secreted significantly higher amounts of histamine and leukotriene C4 (LTC4 ) than healthy controls. Furthermore, priming experiments with IL-3, IL-8 and GM -CSF followed by stimulation with enterotoxins showed additional histamine and LTC4 release in the group of atopic eczema patients. Histamine and leukotriene generation from atopic basophils stimulated with staphylococcal enterotoxins may indicate a role of these toxins as possible allergens in at least a subgroup of patients with atopic eczema.

Introduction

Normal as well as diseased skin of patients with atopic eczema (AE) is severely colonized with Staphylococcus aureus [1]. Recently, it has been shown that the majority of S. aureus strains isolated from the skin of patients with AE produce staphylococcal enterotoxins (SEA, B, C, D, E) or toxic shock syndrome toxin-1 (TSST-1). About 50% of the patients have specific IgE directed to staphylococcal toxins [2]. The activation of T-cells by staphylococcal superantigens results in the release of a TH2-like cytokine pattern (IL-4, IL-5) in vitro [3] which, via induction of several effector cells, may be the cause of increased IgE-synthesis and eosinophilia. In this study the influence of staphylococcal enterotoxins (SEs) on histamine and leukotriene release of basophils was studied.

Material and Methods

Patients and Controls. Basophils were obtained from healthy volunteers (n = 9) and from patients with AE (n = 17). The AE diagnosis was performed according to the criteria of Hanifin and Rajka [4]. All patients had a skin colonization with S. aureus as was determined by skin smears. The donors did not receive systemic steroid or antihistamine treatment.

Blood Collection and Cell Preparation. Peripheral blood leukocytes (PBL) were obtained by dextrane-sedimentation for 60-90 min. After three washing steps, the pellet was resuspended in 5 ml HEPES-ACM buffer (pH 7.4). Stimulation Conditions. As stimuli served S. aureus enterotoxins (SEA, SEB, SED, SEE and TSST-1) obtained from Toxin Technology (Sarasota, USA) diluted in carbonate buffer (pH 8.0). For priming experiments interleukins-3, 8 and granulocyte-macrophage colony-stimulating factor (GM -CSF), all obtained from Biermann DPC (Bad Nauheim, Germany), were used. As controls served spontaneous as well as anti-IgE (10-2 M) induced histamine and leukotriene release. Total leukocyte histamine content was determined by sonication of cells for 10 s. Aliquots (500 μl) of leukocyte suspension (1.0-1.5 x107/ml) were stimulated with SEs (100 ng/500 μl) for 30 min at 37 °C in duplicates. For priming experiments leukocytes were prestimulated (10 min) with interleukins in the following concentrations: IL-3 (500 ng/500 μl), IL-8 (40 ng/500 μl) and GM-CSF (20ng / 500 μl). Afterwards, toxins were added for 30 min at 37 °C. After incubation period reactions were stopped by adding 500 μl 0.01 N TFA (C2HF3O2).Supernatants were stored at –20 °C.

Histamine and LTC4 ELISA. Histamine and LTC4 contents of supernatants were determined by ELISA obtained from IBL (Hamburg, Germany) according to the prescription of the manufacturer. The release of histamine, either spontaneously or as induced by stimuli, was recorded as a percentage of the total net leukocyte histamine release.

Analysis of Data. All calculations were performed using the SPSS Statistics package (SPSS, Vers. 5.02). Results were analysed using the nonparametric Wilcoxon signedranks test for paired data or the Wilcoxon non-paired rank-sum test for nonpaired data. Only p-values below 0.05 were accepted as significant.