title: Granulocyte colony-stimulating factor primes NADPH oxidase in neutrophils through translocation of cytochrome b(558) by gelatinase-granule release.
abstract: Granulocyte colony-stimulating factor (GCSF) primes reduced neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in response to formyl peptide but does not increase oxidase activity when used alone. Both oxidase activity and degranulation require phospholipase D (PLD) activation, and exogenous C(2)-ceramide inhibits both functions through inhibition of PLD activity. We extended these observations to investigate neutrophil responses to GCSF. GCSF at a dosage of 30 to 100 ng/mL, a concentration range that primes superoxide release, stimulated a 60% to 100% increase in gelatinase release from tertiary granules but did not stimulate lactoferrin release from secondary granules. A 75% to 100% dose-dependent increase in PLD activity in GCSF-treated neutrophils was also observed. Gelatinase release and PLD activity were inhibited by 10 micromol/L C(2)-ceramide. The increase in gelatinase release in response to priming concentrations of GCSF suggests that tertiary granules contribute a component of the NADPH oxidase to the plasma membrane. Neutrophils treated with 50 ng/mL GCSF were found to contain 20% more cytochrome b(558) in the plasma membrane fraction than unstimulated cells, consistent with degranulation of only tertiary granules. Correspondingly, in the presence of 10 micromol/L C(2)-ceramide, cytochrome b(558) content in the plasma membrane did not increase after neutrophil activation. In contrast, GCSF did not lead to p47phox translocation to the plasma membrane or phosphorylation. Because phosphorylation and translocation of p47phox are required for oxidase activity, these findings account for the inability of GCSF alone to generate the respiratory burst. We conclude that translocation of cytochrome b(558) was responsible for GCSF priming of NADPH oxidase in neutrophils.
authors.affiliation: Department of Pediatrics, Division of Hematology/Oncology, University of Michigan Ann Arbor, 48109, USA.
authors.forename: Pamela J
authors.initials: PJ
authors.lastname: Mansfield
authors.forename: Vania
authors.initials: V
authors.lastname: Hinkovska-Galcheva
authors.forename: James A
authors.initials: JA
authors.lastname: Shayman
authors.forename: Laurence A
authors.initials: LA
authors.lastname: Boxer
mesh_terms: D002462:Cell Membrane / Q000201:enzymology; D003573:Cytochrome b Group / Q000097:blood* / Q000187:drug effects; D003600:Cytosol / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D000069585:Filgrastim; D018093:Gelatinases / Q000097:blood*; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D019255:NADPH Oxidases / Q000097:blood*; D009504:Neutrophils / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D010739:Phospholipase D / Q000097:blood*; D021381:Protein Transport; D011994:Recombinant Proteins; D022142:Secretory Vesicles / Q000187:drug effects / Q000502:physiology*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D013481:Superoxides / Q000097:blood
chemical_list: D003573:Cytochrome b Group; C064769:N-acetylsphingosine; D011994:Recombinant Proteins; D013481:Superoxides; D016179:Granulocyte Colony-Stimulating Factor; D009240:N-Formylmethionine Leucyl-Phenylalanine; C036888:cytochrome b558; D019255:NADPH Oxidases; D010739:Phospholipase D; D018093:Gelatinases; D013110:Sphingosine; D000069585:Filgrastim