Mochizuki Akinori, McEuen Alan R, Buckley Mark G, Walls Andrew F
Immunopharmacology Group, Southampton General Hospital.
J Allergy Clin Immunol. 2003 Jul;112(1):102-8. doi: 10.1067/mai.2003.1511.
Basogranulin, the novel basophil granule protein recognized by the monoclonal antibody BB1, can be released by stimulation with anti-IgE antibody or calcium ionophore. However, the kinetics and regulation of its secretion are unknown.
We quantified basogranulin and histamine release in response to a range of stimuli to assess whether basogranulin secretion is a reliable marker of basophil activation.
Isolated peripheral blood basophils were stimulated with anti-IgE antibody, calcium ionophore, N -formyl-Met-Leu-Phe, and complement C5a. The released basogranulin and histamine were quantified by dot blotting with BB1 and a fluorometric method, respectively. Basogranulin localization was confirmed by flow cytometry.
Both basogranulin and histamine displayed a bell-shaped response curve when basophils were challenged with anti-IgE. Half-maximal release occurred within 30 seconds. Basogranulin levels were maximal by 15 minutes, whereas those for histamine continued increasing to 30 minutes. Wortmannin, a PI3-K inhibitor, suppressed the release of both mediators. Basophils from donors with the "nonreleaser" phenotype secreted neither mediator in response to anti-IgE. Non-IgE-dependent stimuli released both mediators in parallel in a concentration-dependent manner. The correlation between the relative amounts of each mediator released was highly significant (r =.901, P <.0001, n = 87). Flow cytometry revealed that some of the secreted basogranulin adhered to the cell surface.
Basogranulin is secreted along with histamine in response to both FcepsilonR I-related and unrelated stimuli. It is therefore a valid marker of basophil activation and could provide the basis for an immunoassay that distinguishes between basophil and mast cell activation.
嗜碱性粒细胞颗粒蛋白是一种由单克隆抗体BB1识别的新型嗜碱性粒细胞颗粒蛋白，可通过抗IgE抗体或钙离子载体刺激释放。然而，其分泌的动力学和调节机制尚不清楚。
我们对一系列刺激下嗜碱性粒细胞颗粒蛋白和组胺的释放进行定量，以评估嗜碱性粒细胞颗粒蛋白分泌是否为嗜碱性粒细胞活化的可靠标志物。
用抗IgE抗体、钙离子载体、N-甲酰甲硫氨酰亮氨酰苯丙氨酸和补体C5a刺激分离的外周血嗜碱性粒细胞。分别用BB1斑点印迹法和荧光法对释放的嗜碱性粒细胞颗粒蛋白和组胺进行定量。通过流式细胞术确认嗜碱性粒细胞颗粒蛋白的定位。
用抗IgE刺激嗜碱性粒细胞时，嗜碱性粒细胞颗粒蛋白和组胺均呈现钟形反应曲线。半数最大释放发生在30秒内。嗜碱性粒细胞颗粒蛋白水平在15分钟时达到最大值，而组胺水平持续增加至30分钟。PI3-K抑制剂渥曼青霉素可抑制两种介质的释放。具有“非释放者”表型的供体的嗜碱性粒细胞在抗IgE刺激下不分泌任何一种介质。非IgE依赖性刺激以浓度依赖性方式平行释放两种介质。每种介质释放的相对量之间的相关性非常显著（r = 0.901，P < 0.0001，n = 87）。流式细胞术显示，一些分泌的嗜碱性粒细胞颗粒蛋白粘附在细胞表面。
嗜碱性粒细胞颗粒蛋白在FcepsilonR I相关和不相关刺激下均与组胺一起分泌。因此，它是嗜碱性粒细胞活化的有效标志物，可为区分嗜碱性粒细胞和肥大细胞活化的免疫测定提供基础。
