title: Tumor necrosis factor-alpha causes release of cytosolic interleukin-18 from human neutrophils.
abstract: Neutrophils (PMNs) are a vital part of host defense and are the principal leukocyte in innate immunity. Interleukin (IL)-18 is a proinflammatory cytokine with roles in both innate and adaptive immunity. We hypothesize that PMNs contain preformed IL-18, which is released in response to specific inflammatory stimuli. Isolated PMNs were stimulated with a battery of chemoattractants (5 min to 24 h), and IL-18 release was measured. PMNs were also separated into subcellular fractions and immunoblotted with antibodies against IL-18 or were fixed and probed with antibodies to IL-18 as well as to the contents of granules, intracellular organelles, and filamentous actin (F-actin), incubated with fluorescent secondary antibodies, and examined by digital microscopy. Quiescent PMNs contained IL-18 in the cytoplasm, associated with F-actin, as determined by positive fluorescence resonance energy transfer (FRET+). In turn, TNF-alpha stimulation disrupted the association of IL-18 with F-actin, induced a FRET+ interaction of IL-18 with lipid rafts, and elicited IL-18 release. Manipulation of F-actin status confirmed the relationship between IL-18 and F-actin in resting PMNs. Consequently, incubation with monomeric IL-18 binding protein inhibited TNF-alpha-mediated priming of the PMN oxidase. We conclude that human PMNs contain IL-18 associated with F-actin in the cytoplasm and TNF-alpha stimulation causes dissociation of IL-18 from F-actin, association with lipid rafts, and extracellular release. Extracellular IL-18 participates in TNF-alpha priming of the PMN oxidase as demonstrated by inhibition with the IL-18 binding protein.
authors.affiliation: Research Dept., Bonfils Blood Center, 717 Yosemite St., Denver, CO 80230, USA. christopher.silliman@ucdenver.edu
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Silliman
authors.forename: Marguerite R
authors.initials: MR
authors.lastname: Kelher
authors.forename: Fabia
authors.initials: F
authors.lastname: Gamboni-Robertson
authors.forename: Christine
authors.initials: C
authors.lastname: Hamiel
authors.forename: Kelly M
authors.initials: KM
authors.lastname: England
authors.forename: Charles A
authors.initials: CA
authors.lastname: Dinarello
authors.forename: Travis H
authors.initials: TH
authors.lastname: Wyman
authors.forename: Samina Y
authors.initials: SY
authors.lastname: Khan
authors.forename: Nathan J D
authors.initials: NJ
authors.lastname: McLaughlin
authors.forename: Rachel S
authors.initials: RS
authors.lastname: Bercovitz
authors.forename: Anirban
authors.initials: A
authors.lastname: Banerjee
mesh_terms: D000199:Actins / Q000378:metabolism; D056704:Adaptive Immunity; D003600:Cytosol / Q000276:immunology*; D031541:Fluorescence Resonance Energy Transfer; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007113:Immunity, Innate; D015151:Immunoblotting; D018836:Inflammation Mediators / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D020382:Interleukin-18 / Q000378:metabolism*; D021962:Membrane Microdomains / Q000276:immunology; D008856:Microscopy, Fluorescence; D009504:Neutrophils / Q000276:immunology*; D010750:Phosphoproteins / Q000378:metabolism; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: D000199:Actins; D018836:Inflammation Mediators; D036341:Intercellular Signaling Peptides and Proteins; D020382:Interleukin-18; D010750:Phosphoproteins; D014409:Tumor Necrosis Factor-alpha; C118370:interleukin-18 binding protein; C079789:neutrophil cytosol factor 67K