During beneficial inflammation, potentially tissuedamaging granulocytes undergo apoptosis before being cleared by phagocytes in a non-phlogistic manner. Here we show that the rate of constitutive apoptosis in human neutrophils and eosinophils is greatly accelerated in both a rapid and concentration-dependent manner by the fungal metabolite gliotoxin, but not by its inactive analog methylthiogliotoxin. This induction of apoptosis was abolished by the caspase inhibitor zVAD-fmk, correlated with the inhibition of nuclear factor-kappa B (NF-B), and was mimicked by a cell permeable inhibitory peptide of NF-B, SN-50; other NF-B inhibitors, curcumin and pyrrolidine dithiocarbamate; and the proteasome inhibitor, MG-132. Gliotoxin also augmented dramatically the early (2-6 h) pro-apoptotic effects of tumor necrosis factor-␣ (TNF-␣) in neutrophils and unmasked the ability of TNF-␣ to induce eosinophil apoptosis. In neutrophils, TNF-␣ caused a gliotoxin-inhibitable activation of an inducible form of NF-B, a response that may underlie the ability of TNF-␣ to delay apoptosis at later times (12-24 h) and limit its early killing effect. Furthermore, cycloheximide displayed a similar capacity to enhance TNF-␣ induced neutrophil apoptosis even at time points when cycloheximide alone had no proapoptotic effect, suggesting that NF-B may regulate the production of protein(s) which protect neutrophils from the cytotoxic effects of TNF-␣. These data shed light on the biochemical and molecular mechanisms regulating human granulocyte apoptosis and, in particular, indicate that the transcription factor NF-B plays a crucial role in regulating the physiological cell death pathway in granulocytes.

Increased levels of glutathione (GSH) occur in the epithelial lining fluid (ELF) of chronic cigarette smokers. Therefore we investigated the effect of cigarette smoke condensate solution (CSC) on GSH synthesis and the regulation of T-glutamylcysteine synthetase (~CS) in human type II alveolar epithelial cells (A549). CSC exposure increased GSH levels, TGCS activity and ~CS heavy subunit (HS) mRNA, as well as increasing DNA binding of the activator protein-1 (AP-1) and the human antioxidant response element (hARE). Transfection of deletion constructs of the ~GCS-HS promoter in a chloramphenicol acetyl transferase (CAT) reporter system revealed that an hARE, present within promoter, is not required tot the CSC mediated induction. We conclude that CSC induction of ~GCS-HS expression is associated with AP-1/AP-I-like responsive elements.

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