Do human neutrophils make hydroxyl radical? Determination of free radicals generated by human neutrophils activated with a soluble or particulate stimulus using electron paramagnetic resonance spectrometry.

B E Britigan,G M Rosen,Y Chai,M S Cohen

doi:10.1016/s0021-9258(17)38517-4

Abstract

Using electron paramagnetic resonance spectrometry and the spin trap 5,5-dimethyl-1-oxide (DMPO), neutrophil free radical production in response to phorbol myristate acetate and opsonized zymosan was investigated. Using phorbol myristate acetate and zymosan (3 mg/ml), the superoxide spin-trapped adduct 2-2-dimethyl-5-hydroperoxy-1-pyrrolidinyloxyl (DMPO-OOH) and the hydroxyl spin-trapped adduct 2-2-dimethyl-5-hydroxy-1-pyrrolidinyloxyl (DMPO-OH) were detected. Only DMPO-OH was observed with zymosan (0.5 mg/ml). Hydroxyl radical production in the presence of dimethylsulfoxide (Me2SO) and DMPO yields 2,2,5-trimethyl-1-pyrrolidinyloxyl. The only 2,2-trimethyl-1-pyrrolidinyloxyl detected following neutrophil stimulation was that expected from DMPO-OOH degradation. Superoxide dismutase but not catalase inhibited generation of all three spin-trapped adducts. These data indicate that DMPO-OH arose from DMPO-OOH degradation and does not represent hydroxyl radical production. Under certain conditions DMPO-OH is the predominant spin-trapped adduct resulting from neutrophil superoxide production, perhaps due to cellular bioreduction of DMPO-OOH to DMPO-OH. Cytochalasin B, which prevents phagosome closure, inhibited zymosan-stimulated neutrophil oxygen consumption and electron paramagnetic resonance superoxide detection. No hydroxyl radical was detected. Spin trapping with DMPO appears to detect intraphagosomal free-radical formation.

Highlights

Usingelectronparamagneticresonancespectrom- systems, superoxide and hydrogen peroxidereact in an ironetry and the spin trap5,5-dimethyl-l-pyrroline-l-o~-catalyzed processgenerating hydroxyl radical (OH’) (10, 11)
The resulting spectrum was a composite of three distinct products: the superoxide adduct (DMPO-OOH, peak 3 ), hydroxyl adduct (DMPO-OH, peak 2), and the methyl radical adduct (DMPO-CH3,peak 1).The size of the DMPO-OH and DMPO-OOH peaks increased with sequential scans indicating progressive free radical generation (Fig. 3, A and B)
DMPO-CH, formation should occur with hydroxyl radical formation in the presence of Me2S0, which was used as the solvent for Phorbol myristate acetate (PMA)

Summary

Introduction

Usingelectronparamagneticresonancespectrom- systems, superoxide and hydrogen peroxidereact in an ironetry and the spin trap5,5-dimethyl-l-pyrroline-l-o~-catalyzed processgenerating hydroxyl radical (OH’) (10, 11). The only 2,2-trimethyl-l-pyrrolidinyloxyl Spin trapping has been used for detection of free radicals detectedfollowing neutrophil stimulationwas that ex- in many cellular systems (17-22). Superoxidedis- radicals with nitrones or nitroso compounds (spin traps) mutasebutnotcatalaseinhibitedgenerationof all results in theproduction of “long-lived”nitroxide free radicals three spin-trapped adducts. These data indicate that which can be detected using conventional electron paramag-. Sincestable free radicalsaccumulate,spin trapped adduct resulting from neutrophil superoxide trapping is an integrative method of measurement and is production,perhapsdue to cellularbioreductionof inherently more sensitive than procedures measuringinstan-

Methods

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Conclusion