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

• Published in: Free radical biology & medicine 2000-06, Vol.28 (11), p.1652-1660
• Main Authors: Raible, Donald G, Mohanty, Joy G, Jaffe, Jonathan S, Stella, Hector J, Sprenkle, Boyd E, Glaum, Mark C, Schulman, Edward S
• Format: Article
• Language: English
• Subjects: 7-dihydroxyphenoxazine; Adenosine Triphosphate - metabolism; Amplex Red; Cell Adhesion; Chromogenic Compounds; Eosinophil; Eosinophils - cytology; Eosinophils - drug effects; Eosinophils - metabolism; Fibronectins - metabolism; Free radicals; Horseradish Peroxidase; Humans; Hydrogen peroxide; Hydrogen Peroxide - metabolism; In Vitro Techniques; N-acetyl-3; Oxazines; Peroxidase; Platelet Activating Factor - pharmacology; Scopoletin; Scopoletin - pharmacology; Tumor Necrosis Factor-alpha - pharmacology
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/S0891-5849(00)00279-3

We have examined the release of H 2O 2 from PAF or TNFα-stimulated human eosinophils on fibronectin (FN)-coated polystyrene plates. H 2O 2 release was measured by the standard scopoletin-horseradish peroxidase (SCOP-HRP) method and compared with that measured by a new microplate fluorescent assay for H 2O 2 using a novel HRP substrate A6550. We observed that the SCOP-HRP method gave a 25-fold higher estimate of H 2O 2 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 H 2O 2 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 H 2O 2 release than expected. The method used to detect eosinophil H 2O 2 release greatly influences the absolute amount of H 2O 2 detected.