Hydrogen peroxide release from human eosinophils on fibronectin: scopoletin enhances eosinophil activation.
We have examined the release of H(2)O(2) from PAF or TNFalpha-stimulated human eosinophils on fibronectin (FN)-coated polystyrene plates. H(2)O(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(2)O(2) using a novel HRP substrate A6550. We observed that the SCOP-HRP method gave a 25-fold higher estimate of H(2)O(2) release from eosinophils than did the A6550-HRP method. Microscopic examination of PAF or TNFalpha-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(2)O(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(2)O(2) release than expected. The method used to detect eosinophil H(2)O(2) release greatly influences the absolute amount of H(2)O(2) detected.
MCP Hahnemann School of Medicine, Divisions of Pulmonary and Critical Care Medicine, Philadelphia, PA 19102-1192, USA.
D G
DG
Raible
J G
JG
Mohanty
J S
JS
Jaffe
H J
HJ
Stella
B E
BE
Sprenkle
M C
MC
Glaum
E S
ES
Schulman
D000255:Adenosine Triphosphate / Q000378:metabolism; D002448:Cell Adhesion; D002863:Chromogenic Compounds; D004804:Eosinophils / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism; D006735:Horseradish Peroxidase; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D066298:In Vitro Techniques; D010078:Oxazines; D010972:Platelet Activating Factor / Q000494:pharmacology; D012603:Scopoletin / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D002863:Chromogenic Compounds; D005353:Fibronectins; C105363:N-acetyl-3,7-dihydroxyphenoxazine; D010078:Oxazines; D010972:Platelet Activating Factor; D014409:Tumor Necrosis Factor-alpha; D000255:Adenosine Triphosphate; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D012603:Scopoletin