Reactive oxygen species can function as intracellular messengers, but linking these signaling events with specific enzymes has been difficult.

It has been reported that interleukin-8 (IL-8) and cyclooxygenase-2 (COX-2) expression is regulated by peroxisome proliferator-activated receptor (PPAR)-␥ synthetic ligands. We have shown previously that cytosolic phospholipase A 2 (cPLA 2 ) is able to activate gene expression through PPAR-␥ response elements (Pawlic-

-Interferon gamma (IFN-␥) plays a role in a variety of lung inflammatory responses, and corticosteroids are frequently employed as a treatment in these conditions. Therefore, the effect of IFN-␥, of the corticosteroid dexamethasone (Dex), or of both on gene expression was studied in normal human bronchial epithelial (NHBE) cells. NHBE cells were exposed to medium alone, IFN-␥ (300 U/ml), Dex (10 Ϫ7 M), or both IFN-␥ and Dex for 8 or 24 h. Gene expression was examined using oligonucleotide microarrays. A principal components analysis demonstrated that the IFN-␥ treatment effect was the primary source of differences in the data. With a 5% false discovery rate, of the 66 genes upregulated by IFN-␥ by twofold or greater at 8 h and 287 genes upregulated at 24 h, coincubation with Dex inhibited the expression of 2 genes at 8 h and 45 genes at 24 h. Prominent among these were cytokines and secreted proteins. Dex cotreatment increased expression of 65 of the 376 genes that were inhibited by IFN-␥ by 50% at 24 h. The majority of these genes encode cell cycle or nuclear proteins. Dex alone increased the expression of only 22 genes and inhibited the expression of 7 genes compared with controls at 24 h. The effect of Dex on IFN-␥-induced changes suggests a specific, targeted effect on IFN-␥ responses that is substantially greater than the effect of Dex alone. Dex had little effect on the immediate early response to IFN-␥ but a significant effect on the late responses.

Oxidative stress is thought to be a factor influencing many inflammatory responses, including arachidonic acid (AA) release. We have studied the effect of hydrogen peroxide on AA and prostaglandin E(2) release, cytosolic phospholipase (cPLA(2)) steady-state mRNA, cPLA(2) protein levels, cPLA(2) enzyme activity, and cPLA(2) phosphorylation in a human lung epithelial cell line: A549 cells. Hydrogen peroxide caused a dose-dependent increase of A23187-stimulated AA and prostaglandin E(2) release, with a maximum effect at 1 h. This effect is associated with a maximum specific cPLA(2) activity at 1 h, and with a significant increase in cPLA(2) Serine 505 phosphorylation. All these effects were abolished, in a dose-related manner, by the epithelial growth factor receptor kinase inhibitor, AG 1478. To further investigate the pathway leading to the increase cPLA(2) phosphorylation, we used cells transfected with a Ras dominant negative vector and mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) and p38 kinase inhibitors. Cells transfected with the Ras dominant negative vector exhibited diminished hydrogen peroxide-induced AA release and cPLA(2) phosphorylation as compared with cells transfected with the Ras expression vector. Both MEK and p38 kinase inhibitors inhibited the hydrogen peroxide effect on AA release and specific cPLA(2) activity. Finally, cells stably transfected with an antisense cPLA(2) vector exhibited diminished A23187-stimulated AA release in response to hydrogen peroxide as compared with cells stably transfected with empty expression vector. Collectively, these data show that hydrogen peroxide increases cPLA(2) activity through its phosphorylation utilizing an epithelial growth factor/Ras/extracellular signal-regulated kinase and p38 pathway.

The effects of nitric oxide (NO) on human neutrophil chemotactic responses and release of interleukin (IL)-8 was studied. Neutrophils exposed to chemoattractants (IL-8, FMLP, leukotriene B 4 , and C5a) failed to show increases in intracellular guanosine 3,5-cyclic monophosphate (cGMP), an indicator of NO production. Although NO increased cGMP in neutrophils, neither of two NO donors (sodium nitroprusside and 3-morpholino-sydonimine) nor a NO synthase inhibitor (N v -nitro-L-arginine) altered FMLP-or IL-8 -elicited neutrophil chemotaxis (P ú .25 for all). However, lipopolysaccharide-induced IL-8 production was increased in a dose-dependent manner by a combination of sodium nitroprusside and N-acetylcysteine (P Å .03) or by S-nitrosoglutathione (P Å .004). NO-augmented IL-8 release was not reproduced by treating neutrophils with dibutyryl-cGMP. Upregulation of IL-8 release by NO was associated with increased IL-8 mRNA levels (P Å .009). These data suggest that NO does not directly affect neutrophil chemotaxis but may indirectly alter chemotactic responses by increasing IL-8 production via a cGMP-independent pathway.