Oxidant-mediated lung epithelial cell tolerance: the role of intracellular glutathione and nuclear factor-kappaB 1 1Abbreviations: A549 cells, human alveolar epithelial type II cell line; AP-1, activator protein-1; BSO; dl-buthionine (SR)-sulfoximine; COPD, chronic obstructive pulmonary disease; DMEM, Dulbecco’s modified Eagle’s medium; DTT, dithiothreitol; GSHMEE, glutathione monoethyl ester; H2O2, hydrogen peroxide; IkB, inhibitory binding protein kB; LDH, lactic dehydrogenase; NAC, N-acetyl-l-cysteine; NF-kB, nuclear factor-kappa b; NPSH, non-protein sulphydryls; PKC, protein kinase C; and ROS, reactive oxygen species.

The airway epithelium is injured by oxidants inhaled as atmospheric pollutants or produced during inflammatory responses. We studied the effect of modulating the antioxidant intracellular glutathione, both using thiol compounds and by the adaptive effect of hyperoxia, on oxidant-induced injury and activation of the nuclear factor- kappa B (NF-κB) in two cell lines: the human bronchial (16HBE) and type II alveolar epithelial cells (A549). The thiol antioxidants glutathione (GSH) and glutathione monoethyl ester (GSH-MEE) [2 mM] increased GSH levels (nmol/mg protein) in A549 cells (GSH 383 ± 26 and GSH-MEE 336 ± 23 vs control 171 ± 13, P < 0.001) and in 16HBE cells (GSH 405 ± 33, GSH-MEE 362 ± 37 vs control 198 ± 12, P < 0.001, N = 3). Treatment of hyperoxia (95% oxygen) also increased GSH levels between 4 and 24 hr exposure compared with control ( P < 0.01). Hydrogen peroxide (H 2 O 2 ) (0.01 mM) induced NF-κB activation, whereas hyperoxia exposure did not affect NF-κB activation in either cell line. Pretreatment with dl-buthionine ( SR )-sulfoximine, which decreased intracellular glutathione, increased NF-κB binding induced by H 2 O 2 and increased lactate dehydrogenase (LDH) release ( P < 0.001). Pretreatment with the thiol compounds and hyperoxia totally inhibited H 2 O 2 -induced NF-κB binding and cell injury as measured by LDH release. These data indicate the importance of intracellular glutathione and inhibition of NF-κB in both protection/tolerance against oxidant-induced epithelial cell injury, and NF-κB activation in response to oxidative stress which may be important in lung inflammation. Thus, increasing intracellular glutathione may be of therapeutic relevance if able to modulate NF-κB activation and hence attenuate inflammation.