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We have investigated the role that the nuclear factor (NF)-kappaB plays in regulating the biosynthesis of tumor necrosis factor (TNF)-alpha, an inflammatory cytokine. Irreversible inhibition of the proteasome complex by carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (MG-132; 1-50 microM) had no inhibitory effect on LPS-mediated TNF-alpha biosynthesis. Furthermore, selective inhibition of NF-kappaB by the action of caffeic acid phenylethyl ester (CAPE; 1-100 microM) and sulfasalazine (SSA; 0.1-10 mM), a potent and irreversible inhibitor of NF-kappaB, partially attenuated, but did not abolish, LPS-dependent TNF-alpha secretion. Incorporation of a selectively permeant inhibitor of NF-kappaB, SN-50 (1-20 microM), a peptide which contains the nuclear localization sequence (NLS) for the p50 NF-kappaB subunit, and the amino-terminal sequence of Kaposi fibroblast growth factor to promote cell permeability, attenuated in a dose-dependent manner LPS-mediated release of TNF-alpha. It is concluded that the NF-kappaB pathway is partially implicated and that its blockade attenuates, but does not abrogate, LPS-dependent TNF-alpha biosynthesis in alveolar epithelial cells., (Copyright 2001 Academic Press.)

1. Pyrimidylpiperazine (Y-40138), a synthetic derivative of N-[1-(4-([4-(pyrimidin-2-yl)piperazin-1-yl]methyl)phenyl)cyclopropyl] acetamide, is a novel dual regulator of pro- and anti-inflammatory cytokines in vivo. The aim of the present study was to determine the signal transduction mechanisms implicated in vitro. 2. In alveolar epithelial cells, pre-treatment (30 min) with Y-40138 reduced LPS-induced biosynthesis of IL-1 beta, IL-6 and TNF-alpha, an effect paralleled by up-regulating an anti-inflammatory counter-loop mediated through IL-10. 3. This differential regulation of pro- and anti-inflammatory signals was accompanied by an inhibition of the nuclear localization of selective NF-kappa B subunits, particularly NF-kappa B(1) (p50), RelA (p65), the major transactivating member of the Rel family, RelB (p68) and c-Rel (p75). In addition, Y-40138 blockaded, in a dose-dependent manner, the LPS-induced nuclear activation of NF-kappa B. 4. Analysis of the upstream pathway involved in Y-40138-dependent retardation of LPS-induced NF-kappa B translocation/activation revealed the involvement of an I kappa B-alpha sensitive pathway. Pre-treatment with Y-40138 ameliorated LPS-induced degradation of I kappa B-alpha in the cytosolic compartment and retarded its phosphorylation, suggesting the involvement of an upstream kinase. 5. Recombinant IL-10 (0 -- 10 ng ml(-1)) blockaded, in a dose-dependent manner, LPS-induced biosynthesis of IL-1 beta, IL-6 and TNF-alpha. Furthermore, rhIL-10 reduced the DNA binding activity of NF-kappa B. Immunoneutralization of endogenous IL-10 by a polyclonal alpha IL-10 (5 microg ml(-1)) reversed the inhibitory effect of Y-40138 on pro-inflammatory cytokines and partially restored the DNA binding activity of NF-kappa B. 6. These results indicate that Y-40138 mediated dual immunoregulation of pro- and anti-inflammatory cytokines is IL-10 sensitive and mediated through the I kappa B-alpha/NF-kappa B signal transduction pathway.

The potential anti-inflammatory role of alpha-melanocyte-stimulating hormone (alpha-MSH)-related tripeptide, lysine(11)-D-proline-valine(13) (KDPV), an analogue of interleukin (IL)-1beta(193-195) and an antagonist of IL-1beta/prostaglandin E(2), is not well characterized in the alveolar epithelium. In a model of foetal alveolar type II epithelial cells in vitro, we showed that lipopolysaccharide endotoxin (LPS) differentially, but selectively, induced the nuclear subunit composition of nuclear factor kappaB(1) (NF-kappaB(1)) (p50), RelA (p65) and c-Rel (p75), in parallel to up-regulating the DNA-binding activity (supershift indicating the presence of the p50-p65 complex). LPS accelerated the degradation of inhibitory kappaB-alpha (IkappaB-alpha), accompanied by enhancing its phosphorylation in the cytosolic compartment but not in the nucleus. KDPV suppressed, in a dose-dependent manner, the nuclear localization of p50, p65 and p75, an effect that led to the subsequent inhibition of NF-kappaB activation. Interleukin-1 receptor antagonist (IL-1ra) decreased the nuclear abundance of p50, p65 and p75, and subsequently depressed the DNA-binding activity induced by LPS. Analysis of the mechanism involved in the KDPV- and IL-1ra-mediated inhibition of NF-kappaB nuclear localization revealed a reversal in IkappaB-alpha phosphorylation and degradation, followed by cytosolic accumulation. LPS induced endogenous IL-1beta biosynthesis in a time-dependent manner; the administration of exogenous recombinant human interleukin 1 (rhIL-1) resulted in a dose-dependent activation of NF-kappaB. KDPV and IL-1ra abrogated the effect of rhIL-1. Pretreatment with the non-steroidal anti-inflammatory drug (NSAID) indomethacin, an inhibitor of cyclo-oxygenase, blocked the LPS-induced activation of NF-kappaB. These results indicate the involvement of prostanoid-dependent (NSAID-sensitive) and IL-1-dependent (IL-1ra-sensitive) mechanisms mediating LPS-induced NF-kappaB translocation and activation, a pathway that is regulated, in part, by a negative feedback mechanism transduced through IkappaB-alpha, the major cytosolic inhibitor of NF-kappaB.

Electrophoretic mobility shift assays revealed minimal levels of NF-kappaB activity in rat distal lung epithelial cells cultured at fetal (23 mmHg) or adult alveolar (100 mmHg) P(O2), but revealed significant activation of this transcription factor in cells exposed to a rise in P(O2) mimicking that experienced at birth. This response was entirely abolished by pretreating cells with 5 mM sulfasalazine (SSA). This shift in P(O2) also evoked a rise in apical Na+ conductance (G(Na+)) that may underlie the O2-evoked stimulation of Na+ transport seen in these cells. Pretreatment with SSA had no effect upon G(Na+) in cells cultured continually at adult or fetal P(O2) but did inhibit the increase in G(Na+) seen in cells that had experienced the rise in P(O2). O2-evoked activation of NF-kappaB may thus mediate the increased Na+ transport that occurs when the distal lung epithelial cells are exposed to a physiologically-relevant increase in P(O2)., (Copyright 2001 Academic Press.)

The O(2) and redox-sensitive transcription factors hypoxia inducible factor-1alpha (HIF-1alpha) and nuclear factor-kappaB (NF-kappaB) are differentially regulated in the alveolar epithelium over fetal to neonatal oxygen tensions. We have used fetal alveolar type II epithelial cells to monitor their regulation in association with redox responsiveness to antioxidant pretreatment in vitro. N-Acetyl-l-cysteine, a glutathione (GSH) precursor and a potent scavenger of reactive oxygen species, induced HIF-1alpha and ameliorated NF-kappaB nuclear abundance and DNA binding activity, respectively, in a dose-dependent manner. Analysis of variations in glutathione homeostasis at ascending DeltapO(2) regimen with N-acetyl-(L)-cysteine reveals increased GSH at the expense of the oxidized form of glutathione (GSSG), thereby shifting GSH/GSSG into reduction equilibrium. Pyrrolidine dithiocarbamate (PDTC), which exerts both antioxidant and pro-oxidant effects, provoked a substantial increase in HIF-1alpha nuclear abundance, with no apparent effect on its activation. PDTC reduced NF-kappaB nuclear abundance and its inhibitory effects on binding activity are dose-dependent. Assessment of glutathione homeostasis with PDTC shows increasing levels of GSSG at the expense of GSH, lowering GSH/GSSG in favor of an oxidative equilibrium. Our results indicate the hypoxic activation of HIF-1alpha and the hyperoxic induction of NF-kappaB in the fetal epithelium is redox-sensitive and, thus, tightly regulated by the GSH/GSSG equilibrium. This highlights glutathione as a key regulatory component for determining genetic responsiveness to oxidant/antioxidant imbalance in normal lung development and pathophysiological conditions.

Fetal alveolar type II (fATII) epithelial cells were used to evaluate the role of signaling factors involved in oxidative stress-induced programmed cell death (PCD; apoptosis). Bcl-2, an antiapoptotic proto-oncogene, showed maximum abundance in hypoxia and mild reoxygenation, but declined thereafter. The Bcl-2 counterpart, Bax, which promotes PCD, displayed an increasing logarithmic profile with ascending DeltapO(2) regimen, such that the ratio of Bcl-2/Bax decreased as pO(2) increased. The expression of p53, a cell cycle regulator, paralleled Bax abundance. Pretreatment of fATII cells with l-buthionine-(S,R)-sulfoximine, an irreversible inhibitor of gamma-glutamylcysteine synthetase, the rate-limiting enzyme in the biosynthesis of glutathione (GSH), enhanced Bax and p53 expression over Bcl-2. The GSH analogue, gamma-glutamylcysteinyl-ethyl ester, down-regulated Bax/p53 abundance but restored that of Bcl-2, thereby increasing Bcl-2/Bax. The antioxidant and GSH precursor N-acetyl-l-cysteine favored Bcl-2 at the expense of Bax/p53, whereas pyrrolidine dithiocarbamate induced Bax against Bcl-2, with mild effect on p53. Sulfasalazine, a potent and specific inhibitor of NF-kappaB, induced Bax at the expense of Bcl-2, in a p53-dependent manner. We conclude that the differential expression of signaling factors involved in PCD in the alveolar epithelium is redox-sensitive and mediated, at least in part, by a negative feedback mechanism transduced by NF-kappaB., (Copyright 2000 Academic Press.)

To test the genetic capacity of the perinatal lung to respond to O(2) shifts that coincide with the first respiratory movements, rat fetal alveolar type II (fATII) epithelial cells were cultured at fetal distal lung PO(2) (23 Torr) and then exposed to postnatal (23 --> 76 Torr; mild hyperoxic shift), moderate (23 --> 152 Torr; moderate hyperoxic shift), or severe (23 --> 722 Torr; severe hyperoxic shift) oxygenation. Nuclear abundance and consensus binding characteristics of hypoxia-inducible factor (HIF)-1alpha and nuclear factor (NF)-kappaB (Rel A/p65) plus glutathione biosynthetic capacity were determined. Maximal HIF-1alpha activation at 23 Torr was sustained over the postnatal shift in (Delta) PO(2) and was elevated in vivo throughout late gestation. NF-kappaB was activated by the acute postnatal DeltaPO(2) in fATII cells, becoming maximal with moderate and severe oxygenation in vitro and within 6 h of birth in vivo, declining thereafter. fATII cell and whole lung glutathione and GSH-to-GSSG ratio increased fourfold with a postnatal DeltaPO(2) and were matched by threefold activity increases in gamma-glutamylcysteine synthetase and glutathione synthase. GSH concentration depletion by L-buthionine-(S, R)-sulfoximine abrogated both HIF-1alpha and NF-kappaB activation, with HIF-1alpha showing a heightened sensitivity to GSH concentration. We conclude that O(2)-linked genetic regulation in perinatal lung epithelium is responsive to developmental changes in glutathione biosynthetic capacity.