Redox Signaling-Mediated Regulation of Lipopolysaccharide-Induced Proinflammatory Cytokine Biosynthesis in Alveolar Epithelial Cells

Abstract

The regulation of cytokine gene transcription and biosynthesis involves the reduction-oxidation (redox)-sensitive nuclear factor-κB (NF-κB), whose activation is mediated by an upstream kinase that regulates the phosphorylation of inhibitory-κB (IκB). It was hypothesized that lipopolysaccharide (LPS)-induced biosynthesis of interleukin-1β, interleukin-6, and tumor necrosis factor-αin vitro is regulated by redox equilibrium. In alveolar epithelial cells, we investigated the role of L-buthionine-(S,R)-sulfoximine (BSO), an irreversible inhibitor of γ-glutamylcysteine synthetase, the rate-limiting enzyme in GSH biosynthesis, 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), which inhibits glutathione oxidized disulfide reductase, pyrrolidine dithiocarbamate (PDTC), an antioxidant/prooxidant thiuram, and N-acetyl-L-cysteine (NAC), an antioxidant and GSH precursor, in regulating LPS-induced cytokine biosynthesis and IκB-α/NF-κB signaling. BSO blockaded the phosphorylation of IκB-α, reduced its degradation, and inhibited NF-κB activation, besides augmenting LPS-mediated biosynthesis of cytokines. BCNU up-regulated LPS-induced release of cytokines, an effect associated with partial phosphorylation/degradation of IκB-α and inhibition of the DNA binding activity. PDTC, which partially affected LPS-induced IκB-α phosphorylation/degradation, otherwise blockading NF-κB activation, reduced LPS-dependent up-regulation of cytokine release. Pretreatment with BSO did not abolish the NAC-dependent reduction of LPS-induced cytokine release, despite the fact that NAC marginally amplified IκB-α phosphorylation/degradation and suppressed NF-κB activation. These results indicate that cytokines are redox-sensitive mediators and that the IκB-α/NF-κB pathway is redox-sensitive and differentially implicated in mediating redox-dependent regulation of LPS-induced release of proinflammatory cytokines.