IL-1β Converting Enzyme Is a Target for Nitric Oxide-Releasing Aspirin: New Insights in the Antiinflammatory Mechanism of Nitric Oxide-Releasing Nonsteroidal Antiinflammatory Drugs

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Abstract
Caspase-1, the IL-1β converting enzyme (ICE), is required for intracellular processing/maturation of IL-1β and IL-18. NO releasing nonsteroidal antiinflammatory drugs (NSAIDs) are a new class of NSAID derivatives that spare the gastric mucosa. Here, we tested the hypothesis that NCX-4016, a NO-aspirin derivative, inhibits proinflammatory cytokine release from endotoxin (LPS)-challenged monocytes. Our results demonstrated that exposing LPS-stimulated human monocytes to NCX-4016 resulted in a 40–80% inhibition of IL-1β, IL-8, IL-12, IL-18, IFN-γ, and TNF-α release with an EC50 of 10–20 μM for IL-1β and IL-18. Incubating LPS-primed monocytes with NCX-4016 resulted in intracellular NO formation as assessed by measuring nitrite/nitrate, intracellular cGMP concentration, and intracellular NO formation. Exposing LPS-stimulated monocytes to aspirin or celecoxib caused a 90% inhibition of prostaglandin E2 generation but had no effect on cytokine release. NCX-4016, similar to the NO donor S-nitroso-N-acetyl-d-l-penicillamine, inhibited caspase-1 activity with an EC50 of ≈20 μM. The inhibition of caspase-1 by NCX-4016 was reversible by the addition of DTT, which is consistent with S-nitrosylation as the mechanism of caspase-1 inhibition. NCX-4016, but not aspirin, prevented ICE activation as measured by assessing the release of ICE p20 subunit. IL-18 immunoneutralization resulted in a 60–80% reduction of IL-1β, IL-8, IFN-γ, and TNF-α release from LPS-stimulated monocytes. Taken together, these data indicate that incubating human monocytes with NCX-4016 causes intracellular NO formation and suppresses IL-1β and IL-18 processing by inhibiting caspase-1 activity. Caspase-1 inhibition is a new, cycloxygenase-independent antiinflammatory mechanism of NO-aspirin.