Inhibition of glutathione-related enzymes augments LPS-mediated cytokine biosynthesis: involvement of an IkappaB/NF-kappaB-sensitive pathway in the alveolar epithelium

Abstract

The regulation of lipopolysaccharide (LPS)-mediated pro-inflammatory cytokine biosynthesis by reduction-oxidation (redox)-sensitive enzymes involved in maintaining intracellular glutathione homeostasis was investigated in fetal alveolar type II epithelial cells (fATII). Inhibition of glutathione-oxidized disulfide reductase, which recycles GSSG --> 2GSH, by the action of 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) augmented LPS-dependent secretion of interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF)-alpha. BCNU increased [GSSG] concentration at the expense of [GSH], thereby favoring oxidation equilibrium. Inhibition of gamma-glutamylcysteine synthetase, the rate-limiting enzyme in the biosynthesis of GSH, by the action of L-buthionine-(S,R)-sulfoximine (BSO), potentiated LPS-induced IL-1beta, IL-6 and TNF-alpha production. Similar to BCNU, BSO depleted [GSH] and induced the accumulation of [GSSG]. BCNU and BSO reduced LPS-mediated phosphorylation of inhibitory-kappaB (IkappaB-alpha), allowing its cytosolic accumulation. This effect was associated with the inhibition of the nuclear translocation of selective nuclear factor (NF)-kappaB subunits: NF-kappaB1 (p50), RelA (p65), RelB (p68) and c-Rel (p75), but not NF-kappaB2 (p52). BCNU and BSO reduced LPS-induced NF-kappaB activation as determined by the electrophoretic mobility shift DNA-binding assay. Analytical analysis of the effect of modulating the dynamic redox ratio ([GSH]+[GSSG])/[GSSG] revealed a novel role for GSSG as a disulfhydryl compound which mediates an inhibitory effect on NF-kappaB activation. It is concluded that selective modulation of redox-sensitive enzymes has an immunopharmacological potential in regulating pro-inflammatory cytokines and that the TkappaB-alpha/NF-kappaB pathway is redox-sensitive and differentially involved in mediating redox-dependent regulation of cytokine signaling.