Inflammatory events in hippocampal slice cultures prime neuronal susceptibility to excitotoxic injury: a crucial role of P2X7 receptor-mediated IL-1beta release.

We investigated the consequences of transient application of specific stimuli mimicking inflammation to hippocampal tissue on microglia activation and neuronal cell vulnerability to a subsequent excitotoxic insult. Two-week-old organotypic hippocampal slice cultures, from 7-day-old C57BL/6 donor mice, were exposed for 3 h to lipopolysaccharide (LPS; 10 ng/mL) followed by 3 h co-incubation with 1 mM ATP, or 100 microM 2'3'-O-(4-benzoyl-benzoyl) adenosine 5'-triphosphate triethylammonium, a selective P2X(7) receptor agonist. These treatments in combination, but not individually, induced a pronounced activation and apoptotic-like death of macrophage antigen-1 (MAC-1)-positive microglia associated with a massive release of interleukin (IL)-1beta exceeding that induced by LPS alone. Antagonists of P2X(7) receptors prevented these effects. Transient pre-exposure of slice cultures to a combination of LPS and P2X(7) receptor agonists, but not either one or the other alone, significantly exacerbated CA3 pyramidal cell loss induced by subsequent 12 h exposure to 8 microM alpha-amino-3-hydroxy-5-methyl-4-isoxazole propinate (AMPA). Potentiation of AMPA toxicity was prevented when IL-1beta production or its receptor signaling were blocked by an inhibitor of interleukin-converting-enzyme or IL-1 receptor antagonist during application of LPS + ATP. The same treatments did not prevent microglia apoptosis-like death. These findings show that transient exposure to specific pro-inflammatory stimuli in brain tissue can prime neuronal susceptibility to a subsequent excitotoxic insult. P2X(7) receptor stimulation, and the consequent IL-1beta release, is mandatory for exacerbation of neuronal loss. These mechanisms may contribute to determine cell death/survival in acute and chronic neurodegenerative conditions associated with inflammatory events.

(S)-1-((S)-2-{[1-(4-amino-3-chloro-phenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidine-2-carboxylic acid ((2R,3S)-2-ethoxy-5-oxo-tetrahydro-furan-3-yl)-amide (VX-765), an orally available selective interleukin (IL)-converting enzyme/caspase-1 inhibitor, exhibits potent anti-inflammatory activities by inhibiting the release of IL-1beta and IL-18.

(S)-1-((S)-2-{[1-(4-amino-3-chloro-phenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidine-2-carboxylic acid ((2R,3S)-2-ethoxy-5-oxo-tetrahydro-furan-3-yl)-amide (VX-765) is an orally absorbed prodrug of (S)-3-({1-[(S)-1-((S)-2-{[1-(4-amino-3-chlorophenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidin-2yl]-methanoyl}-amino)-4-oxo-butyric acid (VRT-043198), a potent and selective inhibitor of interleukin-converting enzyme/caspase-1 subfamily caspases. VRT-043198 exhibits 100- to 10,000-fold selectivity against other caspase-3 and -6 to -9. The therapeutic potential of VX-765 was assessed by determining the effects of VRT-043198 on cytokine release by monocytes in vitro and of orally administered VX-765 in several animal models in vivo. In cultures of peripheral blood mononuclear cells and whole blood from healthy subjects stimulated with bacterial products, VRT-043198 inhibited the release of interleukin (IL)-1beta and IL-18, but it had little effect on the release of several other cytokines, including IL-1alpha, tumor necrosis factor-alpha, IL-6 and IL-8. In contrast, VRT-043198 had little or no demonstrable activity in cellular models of apoptosis, and it did not affect the proliferation of activated primary T cells or T-cell lines. VX-765 was efficiently converted to VRT-043198 when administered orally to mice, and it inhibited lipopolysaccharide-induced cytokine secretion. In addition, VX-765 reduced disease severity and the expression of inflammatory mediators in models of rheumatoid arthritis and skin inflammation. These data suggest that VX-765 is a novel cytokine inhibitor useful for treatment of inflammatory diseases.

Inactivation of caspase-1 in rodent brain: a novel anticonvulsive strategy.

PURPOSE: Cytokines and related inflammatory mediators are rapidly synthesized in the brain during seizures. We previously found that intracerebral administration of interleukin-1 (IL-1)-beta has proconvulsant effects, whereas its endogenous receptor antagonist (IL-1Ra) mediates potent anticonvulsant actions in various models of limbic seizures. In this study, we investigated whether seizures can be effectively inhibited by blocking the brain production of IL-1beta, by using selective inhibitors of interleukin-converting enzyme (ICE/caspase-1) or through caspase-1 gene deletion. METHODS: Caspase-1 was selectively blocked by using pralnacasan or VX-765. IL-1beta release was induced in mouse organotypic hippocampal slice cultures by proinflammatory stimuli [lipopolysaccharide (LPS) + adenosine triphosphate (ATP)] and measured with enzyme-linked immunosorbent assay (ELISA). IL-1beta production during seizures was measured in the rat hippocampus by Western blot. Seizures were induced in freely moving mice and rats by intrahippocampal injection of kainic acid and recorded by EEG analysis. RESULTS: Caspase-1 inhibition reduced the release of IL-1beta in organotypic slices exposed to LPS+ATP. Administration of pralnacasan (intracerebroventricular, 50 microg) or VX-765 (intraperitoneal, 25-200 mg/kg) to rats blocked seizure-induced production of IL-1beta in the hippocampus, and resulted in a twofold delay in seizure onset and 50% reduction in seizure duration. Mice with caspase-1 gene deletion showed a 70% reduction in seizures and an approximate fourfold delay in their onset. CONCLUSIONS: Inhibition of caspase-1 represents an effective and novel anticonvulsive strategy, which acts by selectively reducing the brain availability of IL-1beta.

IL-18 translational inhibition restricts IFN-gamma expression in crescentic glomerulonephritis.

BACKGROUND: Interleukin-18 (IL-18), a potent inducer of interferon gamma (IFN-gamma) production, is a cytokine involved in the cell-mediated immune response that is expressed during inflammatory and pathologic conditions. IFN-gamma plays a role in the development of some models of glomerulonephritis (GN); however, the role of IL-18 in the production of IFN-gamma during these pathologies has not been studied. METHODS: Rat IL-18 cDNA was isolated and the regulation of IL-18 gene expression was studied. IFN-gamma and IL-18 expression were determined in anti-glomerular basement membrane (GBM) antibody (Ab)-induced GN. Recombinant active IL-18 (rIL-18) was used to further identify its effect on IFN-gamma production during this GN. Glomerular injury and levels of IFN-gamma were assayed in Wistar Kyoto (WKY) rats with anti-GBM GN in the presence or absence of rIL-18. RESULTS: Rat IL-18, similar to the mouse clone, requires processing by the IL-1beta converting enzyme to become activated. A rat IL-18 5'-untranslated region (UTR) translational inhibitor was identified that strongly inhibited the synthesis of IL-18. This translational inhibitor with different lengths (180 and 130 bp) was highly expressed during GN and correlated with minimal IFN-gamma mRNA expression. Injection of recombinant active IL-18 in WKY rats with anti-GBM GN was associated with an increase of glomerular IFN-gamma levels, proliferating cell nuclear antigen (PCNA)-ED1+ cells, and PCNA-CD8+ cells, with worsening of glomerular injury. CONCLUSION: These data suggest that the translational control of IL-18 expression by its 5'-UTR limits the production of IL-18, resulting in restricted expression of mRNA and protein IFN-gamma in this model of GN. Furthermore, it was suggested that possible IL-18/IFN-gamma induction of local proliferation of macrophages and CD8+ cells might be an important mechanism for amplifying CD8+-mediated macrophage-dependent GN.