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

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)-1h, IL-6 and tumor necrosis factor (TNF)-a. BCNU increased [GSSG] concentration at the expense of [GSH], thereby favoring oxidation equilibrium. Inhibition of g-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-1h, IL-6 and TNF-a production. Similar to BCNU, BSO depleted [GSH] and induced the accumulation of [GSSG]. BCNU and BSO reduced LPS-mediated phosphorylation of inhibitory-nB (InB-a), allowing its cytosolic accumulation. This effect was associated with the inhibition of the nuclear translocation of selective nuclear factor (NF)-nB subunits: NF-nB1 (p50), RelA (p65), RelB (p68) and c-Rel (p75), but not NFnB2 (p52). BCNU and BSO reduced LPS-induced NF-nB activation as determined by the electrophoretic mobility shift DNAbinding assay. Analytical analysis of the effect of modulating the dynamic redox ratio ([GSH]+[GSSG])/[GSSG] revealed a 1567-5769/02/$ see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S1567 -5769 (02 )00117 -0 Abbreviations: NAC, N-acetyl-L-cysteine; AEBSF, 4-2-aminoethyl-benzene sulfonyl fluoride–HCl; BSA, bovine serum albumin; BSO, Lbuthionine-(S,R)-sulfoximine; BCNU, 1,3-bis-(2-chloroethyl)-1-nitrosourea; DTT, dithiothreitol; DMEM, Dulbecco’s modified Eagle medium; EMSA, electrophoretic mobility shift assay; ELISA, enzyme-linked immunosorbent assay; fATII, fetal alveolar type II epithelial cells; FCS, fetal calf serum; GSSG, glutathione oxidized; GSSG-RD, glutathione oxidized disulfide reductase; GSH, L-g-glutamyl-L-cysteinyl-glycine; gGCS, g-glutamylcysteine synthetase; HBSS, Hanks’ balanced salt solution; InB, inhibitory-nB; IL, interleukin; LPS, lipopolysaccharideendotoxin; MAPK, mitogen-activated protein kinase; NF-nB, nuclear factor-nB; PBS, phosphate-buffered saline; PDTC, pyrrolidine dithiocarbamate; RNS, reactive nitrogen species; ROS, reactive oxygen species; redox, reduction–oxidation; TMB, tetramethyl-benzidine dihydrochloride; Rf, transepithelial monolayer resistance; TNF-a, tumor necrosis factor-a. * Corresponding author. Tel.: +1-415-476-8984; fax: +1-415-476-8841. E-mail address: [email protected] (J.J. Haddad). www.elsevier.com/locate/intimp International Immunopharmacology 2 (2002) 1567–1583 novel role for GSSG as a disulfhydryl compound which mediates an inhibitory effect on NF-nB activation. It is concluded that selective modulation of redox-sensitive enzymes has an immunopharmacological potential in regulating pro-inflammatory cytokines and that the InB-a/NF-nB pathway is redox-sensitive and differentially involved in mediating redox-dependent regulation of cytokine signaling. D 2002 Elsevier Science B.V. All rights reserved.