Introduction
The therapeutic potential of glutathione (GSH), a ubiquitous thiol, and its immunopharmacological properties in the alveolar epithelium are not well characterized. GSH plays a major role in maintaining intracellular reduction-oxidation (redox) balance and in regulating pathways augmented by oxidative stress (Haddad & Land, 2000a). The chemioxyexcitation regulation of redox homeostasis is dependent on flux kinetics and duration of reactive oxygen species (ROS) exposure. Furthermore, GSH depleting/repleting agents manipulate the cytokine network, an effect antagonistically reversed by restoring equilibrium.
Methods
We elaborated in vitro using fetal alveolar type II epithelial cells pathways linked to redox disequilibrium and subsequent effects on chemioxyexcitation-induced up-regulation of the cytokine network. Measurement of cytokines in cell-free supernatants was performed by a two-site, solid-phase, enzyme-linked immunosorbent assay (ELISA).
Results
L-Buthionine-(S,R)-sulfoximine, a selective and irreversible inhibitor of g-glutamylcysteine synthetase, the rate-limiting enzyme in the biosynthesis of GSH (Griffith & Meister, 1979), induced the formation of intracellular ROS and up-regulated the release of pro-inflammatory cytokines IL-1b, IL-6 and TNF-a. Pretreatment with 1,3-bis-(2-chloroethyl)-1-nitrosourea, which blocks the NADPH-dependent recycling of oxidized disulfide glutathione (GSSG), reduced ROS-induced cytokine production, in a manner similar to pyrrolidine dithiocarbamate, an antioxidant/pro-oxidant agent which elevates GSSG (Haddad & Land, 2000b; Schreck et al., 1992). The antioxidant and GSH precursor, N-acetyl-L-cysteine (Bernard, 1991), abrogated cytokine production concomitant with suppression of ROS formation, an effect mimicked by g-glutamylcysteinyl-ethyl ester, a permeating GSH. The administration of cysteine, the rate-limiting essential amino acid in the de novo biosynthesis of GSH, in the form of pro-drugs 2-oxothiazolidine-4-carboxylate and S-adenosyl-L-methionine, selectively mitigated the chemioxyexcitation-dependent cytokine release. Mimicking the effect of glutathione peroxidase by using its cell-permeating mimetic, 2-phenyl-1,2-benzisoselenazol-3(2H)-one, an anti-inflammatory antioxidant, neutralized the formation of ROS through the GSH-peroxidase coupled reaction, thereby blocking the pathway to cytokine enhancement.
Conclusions
Our results indicate that modulating redox equilibrium by pharmacological thiols bears clinical consequences for the therapeutic treatment of pediatric distresses and oxidative stress mediated exacerbation of lung injury in which pro-inflammatory cytokines act as major participants in their pathophysiology.
References
Bernard, G.R. 1991. Am J Med 91(3C), 55S-59S.
Griffith, O.W. & Meister, A. 1979. J Biol Chem 254, 7558-7560.
Haddad, J.J.E. & Land, S.C. 2000a. Am J Physiol 278, L492-L503.
Haddad, J.J.E. & Land, S.C. 2000b. Biochem Biophys Res Commun 271, 257-267.
Schreck, R., Meier, B., Mannel, D.N., Droge, W. & Baeuerle, P.A. 1992. J Exp Med 175, 1181-1194.