Stimulated human neutrophils limit iron-catalyzed hydroxyl radical formation as detected by spin-trapping techniques

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

Research output: Contribution to journalArticle

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Abstract

Neutrophils stimulated with phorbol myristate acetate (PMA) in the presence of the spin trap 5,5-dimethyl-1-pyrroline 1-oxide (DMPO), dimethyl sulfoxide, and diethylenetriaminepentaacetic acid (DETAPAC) fail to generate hydroxyl radical (.OH), detected as the methyl spin-trapped adduct of DMPO (2,2,5-trimethyl-1-pyrrolidinyloxyl, DMPO-CH3), unless ferric salts (Fe3+) are also added (Britigan, B.E., Rosen, G.M., Chai, Y., and Cohen, M.S. (1986) J. Biol. Chem. 261, 4426-4431). Even then, .OH formation wanes in spite of ongoing superoxide (O2̇-) production. In contrast, ferric salt supplementation of a hypoxanthine/xanthine oxidase O2̇- generating system containing DETAPAC produces continual .OH, suggesting that neutrophils limit the formation of this free radical. To evaluate this hypothesis, neutrophil cytoplasts (largely devoid of granules but able to generate O2̇-) were stimulated with PMA in the presence of Fe3+, DETAPAC, dimethyl sulfoxide, and DMPO. This resulted in continual production of DMPO-CH3. In the presence of dimethyl sulfoxide, HL-60 (promyelocytic) cells differentiate into cells similar in morphology and O2̇- generating capacity to neutrophils. However, their granules lack the iron-binding protein lactoferrin (LF). Ferric salt supplementation of HL-60 cells stimulated with PMA yielded an EPR spectrum similar to cytoplasts. Supernatant obtained following PMA-induced neutrophil degranulation (which releases LF extracellularly) suppressed DMPO-CH3 formation by the hypoxanthine/xanthine oxidase/Fe3+/DETAPAC system. Anti-LF antibody, but not anti-transferrin antibody, prevented stimulated neutrophil supernatant inhibition of hypoxanthine/xanthine oxidase/Fe3+/DETAPAC-mediated .OH formation. Similarly, neutrophils stimulated with PMA in the presence of Fe3+, DETAPAC, and anti-LF antibody (but not anti-transferrin antibody) demonstrated continual formation of .OH. Neutrophil degranulation of LF limits Fe3+-catalyzed .OH formation which in vivo could protect tissue from possible .OH-mediated injury.

Original languageEnglish (US)
Pages (from-to)17026-17032
Number of pages7
JournalJournal of Biological Chemistry
Volume261
Issue number36
StatePublished - Dec 1 1986

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Spin Trapping
Lactoferrin
Hydroxyl Radical
Tetradecanoylphorbol Acetate
Neutrophils
Iron
Oxides
Acids
Xanthine Oxidase
Dimethyl Sulfoxide
Anti-Idiotypic Antibodies
Antibodies
Salts
Transferrin
HL-60 Cells
Iron-Binding Proteins
Superoxides
Free Radicals
Paramagnetic resonance
pyrroline

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Stimulated human neutrophils limit iron-catalyzed hydroxyl radical formation as detected by spin-trapping techniques. / Britigan, B. E.; Rosen, G. M.; Thompson, B. Y.; Chai, Y.; Cohen, M. S.

In: Journal of Biological Chemistry, Vol. 261, No. 36, 01.12.1986, p. 17026-17032.

Research output: Contribution to journalArticle

Britigan, B. E. ; Rosen, G. M. ; Thompson, B. Y. ; Chai, Y. ; Cohen, M. S. / Stimulated human neutrophils limit iron-catalyzed hydroxyl radical formation as detected by spin-trapping techniques. In: Journal of Biological Chemistry. 1986 ; Vol. 261, No. 36. pp. 17026-17032.
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abstract = "Neutrophils stimulated with phorbol myristate acetate (PMA) in the presence of the spin trap 5,5-dimethyl-1-pyrroline 1-oxide (DMPO), dimethyl sulfoxide, and diethylenetriaminepentaacetic acid (DETAPAC) fail to generate hydroxyl radical (.OH), detected as the methyl spin-trapped adduct of DMPO (2,2,5-trimethyl-1-pyrrolidinyloxyl, DMPO-CH3), unless ferric salts (Fe3+) are also added (Britigan, B.E., Rosen, G.M., Chai, Y., and Cohen, M.S. (1986) J. Biol. Chem. 261, 4426-4431). Even then, .OH formation wanes in spite of ongoing superoxide (O2̇-) production. In contrast, ferric salt supplementation of a hypoxanthine/xanthine oxidase O2̇- generating system containing DETAPAC produces continual .OH, suggesting that neutrophils limit the formation of this free radical. To evaluate this hypothesis, neutrophil cytoplasts (largely devoid of granules but able to generate O2̇-) were stimulated with PMA in the presence of Fe3+, DETAPAC, dimethyl sulfoxide, and DMPO. This resulted in continual production of DMPO-CH3. In the presence of dimethyl sulfoxide, HL-60 (promyelocytic) cells differentiate into cells similar in morphology and O2̇- generating capacity to neutrophils. However, their granules lack the iron-binding protein lactoferrin (LF). Ferric salt supplementation of HL-60 cells stimulated with PMA yielded an EPR spectrum similar to cytoplasts. Supernatant obtained following PMA-induced neutrophil degranulation (which releases LF extracellularly) suppressed DMPO-CH3 formation by the hypoxanthine/xanthine oxidase/Fe3+/DETAPAC system. Anti-LF antibody, but not anti-transferrin antibody, prevented stimulated neutrophil supernatant inhibition of hypoxanthine/xanthine oxidase/Fe3+/DETAPAC-mediated .OH formation. Similarly, neutrophils stimulated with PMA in the presence of Fe3+, DETAPAC, and anti-LF antibody (but not anti-transferrin antibody) demonstrated continual formation of .OH. Neutrophil degranulation of LF limits Fe3+-catalyzed .OH formation which in vivo could protect tissue from possible .OH-mediated injury.",
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AU - Rosen, G. M.

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AU - Chai, Y.

AU - Cohen, M. S.

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