Motojima S, Adachi T, Manaka K, Arima M, Fukuda T, Makino S
Department of Medicine and Clinical Immunology, Dokkyo University School of Medicine, Tochigi, Japan.
J Allergy Clin Immunol. 1996 Dec;98(6 Pt 2):S216-23. doi: 10.1016/s0091-6749(96)70069-6.
Asthma is characterized by an accumulation of activated eosinophils in the airway. Eosinophil viability-enhancing activity is present in the sputum of patients with asthma, largely because of granulocyte-macrophage colony-stimulating factor (GM-CSF). Bronchial epithelial cells have been shown to release cytokines including GM-CSF when stimulated with IL-1 beta or tumor necrosis factor-alpha.
The study was designed to determine whether eosinophil peroxidase (EPO) stimulates the release of GM-CSF from bronchial epithelial cells.
Epithelial cells (BEAS-2B) were cultured in serum free HD-F12 medium in a 24-well tissue culture plate until they became confluent. The cells were then exposed to EPO (5.9 x 10(-8) to 5.9 x 10(-7) mol/L) for 15 minutes, washed twice, and cultured in 1 ml of HD-F12. The supernatants were harvested at 3, 6, or 24 hours, and GM-CSF concentration was measured by ELISA. BEAS-2B cells were also treated with a system comprising EPO (1.9 x 10(-9) to 5.9 x 10(-8) mol/L) + 10(-5) mol/L H2O2 + 10(-4) mol/L Br for 24 hours.
The GM-CSF concentration in the supernatant pretreated with EPO increased in a time- and concentration-dependent manner compared with control. The release of GM-CSF was not inhibited by catalase but was inhibited by cyclohexamide and by mixing of EPO with heparin, suggesting that the action is due to the cationic property of EPO. When EPO was combined with H2O2 and Br, 5.9 x 10(-9) mol/L EPO + 10(-5) mol/L H2O2 released two times more GM-CSF into the supernatants compared with control.
These results suggest that EPO stimulates epithelial cells to release GM-CSF and forms a self-stimulatory cycle.
哮喘的特征是气道中活化嗜酸性粒细胞的积聚。哮喘患者痰液中存在增强嗜酸性粒细胞活力的活性，这主要归因于粒细胞-巨噬细胞集落刺激因子（GM-CSF）。研究表明，支气管上皮细胞在受到白细胞介素-1β或肿瘤坏死因子-α刺激时会释放包括GM-CSF在内的细胞因子。
本研究旨在确定嗜酸性粒细胞过氧化物酶（EPO）是否刺激支气管上皮细胞释放GM-CSF。
将上皮细胞（BEAS-2B）在无血清的HD-F12培养基中培养于24孔组织培养板中，直至细胞汇合。然后将细胞暴露于EPO（5.9×10⁻⁸至5.9×10⁻⁷mol/L）15分钟，洗涤两次，并在1ml HD-F12中培养。在3、6或24小时收集上清液，通过酶联免疫吸附测定法测量GM-CSF浓度。BEAS-2B细胞也用包含EPO（1.9×10⁻⁹至5.9×10⁻⁸mol/L）+10⁻⁵mol/L过氧化氢+10⁻⁴mol/L溴的体系处理24小时。
与对照组相比，用EPO预处理的上清液中GM-CSF浓度呈时间和浓度依赖性增加。GM-CSF的释放不受过氧化氢酶抑制，但受环己酰亚胺抑制，并且EPO与肝素混合也可抑制，这表明该作用归因于EPO的阳离子特性。当EPO与过氧化氢和溴结合时，与对照组相比，5.9×10⁻⁹mol/L EPO + 10⁻⁵mol/L过氧化氢使上清液中GM-CSF的释放量增加两倍。
这些结果表明EPO刺激上皮细胞释放GM-CSF并形成自我刺激循环。
