Reimert C M, Skov P S, Poulsen L K
Laboratory of Medical Allergology, National University Hospital, Copenhagen, Denmark.
Allergy. 1998 Feb;53(2):129-38. doi: 10.1111/j.1398-9995.1998.tb03860.x.
A simple microtiter assay for eosinophil activation is described. The assay used 1000-4000 eosinophils/microtiter well, and the design allows for a separate or simultaneous quantitative assessment of eosinophil adhesion to protein-coated microtiter wells and degranulation after stimulation with eosinophil-activating factors. The number of adherent eosinophils is quantified indirectly by expressing the amount of eosinophil cationic protein (ECP) extracted from the adherent fraction of cells in percentage of the total amount of ECP extracted from the cells added to the wells. Degranulation is quantified in the same way by expressing the amount of ECP or eosinophil protein X (EPX) released to the supernatant in percentage of the total amount of ECP or EPX. Known eosinophil-activating agents such as PMA, interleukin (IL)-3, IL-5, GM-CSF, and platelet-activating factor (PAF) all induced a time- and dose-dependent adhesion to albumin-coated wells, whereas L-PAF did not. Kinetic experiments showed that most adhesion occurred within the first 15-30 min, reaching a plateau around 60 min. After prolonged incubation, a decline in adhesion was detected. GM-CSF-induced adhesion was completely inhibited by incubation with monoclonal antibodies directed against the common beta 2-chain (CD18) of the LFA-1, Mac-1, p150,95 complexes. Monoclonal antibodies against CD11a, CD11b, CD11c, VLA-4 ALFA, ICAM-1, VCAM-1, Sialyl-Le(x), ELAM-1, and LECAM had no inhibitory effect. Simultaneous monitoring of adhesion and degranulation after stimulation of eosinophils in albumin-coated wells with either PMA or GM-CSF showed that adhesion always preceded degranulation. Replacing the albumin coating of the microtiter wells with IgG or secretory IgA augmented both the spontaneous and the PMA- or GM-CSF-stimulated responses. In conclusion, the assay allows dynamic evaluation of eosinophil activation and can be used to assess soluble eosinophil-activating factors as well as to study eosinophil activation by solid-phase-bound proteins.
本文描述了一种用于嗜酸性粒细胞活化的简单微量滴定法。该方法在每个微量滴定孔中使用1000 - 4000个嗜酸性粒细胞，其设计允许对嗜酸性粒细胞与蛋白包被的微量滴定孔的黏附以及用嗜酸性粒细胞活化因子刺激后的脱颗粒进行单独或同时的定量评估。通过将从黏附细胞部分提取的嗜酸性粒细胞阳离子蛋白（ECP）量表示为添加到孔中的细胞提取的ECP总量的百分比，间接定量黏附嗜酸性粒细胞的数量。脱颗粒也以相同方式定量，即将释放到上清液中的ECP或嗜酸性粒细胞蛋白X（EPX）量表示为ECP或EPX总量的百分比。已知的嗜酸性粒细胞活化剂，如佛波酯（PMA）、白细胞介素（IL）-3、IL-5、粒细胞-巨噬细胞集落刺激因子（GM-CSF）和血小板活化因子（PAF），均诱导对白蛋白包被孔的时间和剂量依赖性黏附，而L-PAF则不然。动力学实验表明，大多数黏附发生在最初的15 - 30分钟内，在60分钟左右达到平台期。长时间孵育后，检测到黏附下降。GM-CSF诱导的黏附通过与针对LFA-1、Mac-1、p150,95复合物的共同β2链（CD18）的单克隆抗体孵育而被完全抑制。针对CD11a、CD11b、CD11c、VLA-4 ALFA、细胞间黏附分子-1（ICAM-1）、血管细胞黏附分子-1（VCAM-1）、唾液酸化路易斯寡糖（Sialyl-Le(x)）、内皮细胞白细胞黏附分子-1（ELAM-1）和淋巴细胞选择素（LECAM）的单克隆抗体没有抑制作用。在用PMA或GM-CSF刺激白蛋白包被孔中的嗜酸性粒细胞后，同时监测黏附和脱颗粒表明，黏附总是先于脱颗粒。用免疫球蛋白G（IgG）或分泌型免疫球蛋白A（sIgA）替代微量滴定孔的白蛋白包被可增强自发的以及PMA或GM-CSF刺激的反应。总之，该方法允许对嗜酸性粒细胞活化进行动态评估，可用于评估可溶性嗜酸性粒细胞活化因子以及研究固相结合蛋白对嗜酸性粒细胞的活化作用。
