Tumor necrosis factor-alpha causes release of cytosolic interleukin-18 from human neutrophils

• Published in: American Journal of Physiology: Cell Physiology Vol. 298; no. 3; p. C714
• Main Authors: Silliman, Christopher C, Kelher, Marguerite R, Gamboni-Robertson, Fabia, Hamiel, Christine, England, Kelly M, Dinarello, Charles A, Wyman, Travis H, Khan, Samina Y, McLaughlin, Nathan J D, Bercovitz, Rachel S, Banerjee, Anirban
• Format: Journal Article
• Language: English
• Published: United States 01-03-2010
• Subjects: Actins - metabolism; Adaptive Immunity; Cytosol - immunology; Fluorescence Resonance Energy Transfer; Fluorescent Antibody Technique, Indirect; Humans; Immunity, Innate; Immunoblotting; Inflammation Mediators - metabolism; Intercellular Signaling Peptides and Proteins - metabolism; Interleukin-18 - metabolism; Membrane Microdomains - immunology; Microscopy, Fluorescence; Neutrophils - immunology; Phosphoproteins - metabolism; Time Factors; Tumor Necrosis Factor-alpha - metabolism
• ISSN: 1522-1563
• DOI: 10.1152/ajpcell.00011.2009

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.