A microtiter assay for activation of eosinophils. Simultaneous monitoring of eosinophil adhesion and degranulation.

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.