Hydrogen peroxide release from human eosinophils on fibronectin: scopoletin enhances eosinophil activation

We have examined the release of H2O2 from PAF or TNFα-stimulated human eosinophils on fibronectin (FN)-coated polystyrene plates. H2O2 release was measured by the standard scopoletin-horseradish peroxidase (SCOP-HRP) method and compared with that measured by a new microplate fluorescent assay for H2O2 using a novel HRP substrate A6550. We observed that the SCOP-HRP method gave a 25-fold higher estimate of H2O2 release from eosinophils than did the A6550-HRP method. Microscopic examination of PAF or TNFα-stimulated eosinophils in buffer alone or A6550-HRP reaction mixture showed that the cells remained generally round, while eosinophils in SCOP-HRP reaction mixture were spread on the fibronectin-coated surface. Measurement of the cellular ATP content after PAF-stimulation showed that only eosinophils activated in SCOP-HRP had a 50% fall in ATP content. This supported our conclusion that measurement of H2O2 release from eosinophils in SCOP-HRP reaction mixture is problematic since the SCOP-HRP system activates eosinophils. However, we also found that A6550-HRP, when present throughout the incubation, resulted in a lower estimate of H2O2 release than expected. The method used to detect eosinophil H2O2 release greatly influences the absolute amount of H2O2 detected.