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

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.

A two-insult in vitro model of PMN-mediated pulmonary endothelial damage: requirements for adherence and chemokine release.

Lysophosphatidylcholines (lyso-PCs), generated during blood storage, are etiologic in a two-insult, sepsis-based model of transfusion-related acute lung injury (TRALI). Individually, endotoxin (LPS) and lyso-PCs prime but do not activate neutrophils (PMNs). We hypothesized that priming of PMNs alters their reactivity such that a second priming agent causes PMN activation and endothelial cell damage. PMNs were primed or not with LPS and then treated with lyso-PCs, and oxidase activation and elastase release were measured. For coculture experiments, activation of human pulmonary microvascular endothelial cells (HMVECs) was assessed by ICAM-1 expression and chemokine release. HMVECs were stimulated or not with LPS, PMNs were added, cells were incubated with lyso-PCs, and the number of viable HMVECs was counted. Lyso-PCs activated LPS-primed PMNs. HMVEC activation resulted in increased ICAM-1 and release of ENA-78, GRO alpha, and IL-8. PMN-mediated HMVEC damage was dependent on LPS activation of HMVECs, chemokine release, PMN adhesion, and lyso-PC activation of the oxidase. In conclusion, sequential exposure of PMNs to priming agents activates the microbicidal arsenal, and PMN-mediated HMVEC damage was the result of two insults: HMVEC activation and PMN oxidase assembly.