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.
Raible
D G
DG
MCP Hahnemann School of Medicine, Divisions of Pulmonary and Critical Care Medicine, Philadelphia, PA 19102-1192, USA.
Mohanty
J G
JG
Jaffe
J S
JS
Stella
H J
HJ
Sprenkle
B E
BE
Glaum
M C
MC
Schulman
E S
ES
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