CALL FOR PAPERS Oxidant Signaling in Lung Cells Differential roles for NF- (cid:1) B in endotoxin and oxygen induction of interleukin-8 in the macrophage

Differential roles for NF- (cid:1) B in endotoxin and oxygen induction of interleukin-8 in the macrophage. alveolar macrophage is an important source of interleukin (IL)-8 during pulmonary injury. The IL-8 gene promoter sequence contains nuclear factor (NF)- (cid:1) B, NF-IL6, and activator protein (AP)-1 binding sequences. These sites may have differing regulatory roles in hyperoxia-exposed macrophages than in those stimulated by bacterial lipopoly-saccharide (LPS). U-937 and THP-1 macrophage-like cells were exposed to air-5% CO 2 or 95% O 2 -5% CO 2 , with or without 1.0 (cid:2) g/ml of LPS, and transfected with an IL-8 promoter-reporter containing NF- (cid:1) B, NF-IL6, or AP-1 mutations. Hyperoxia and LPS caused additive increases in IL-8 production by U-937 cells, whereas THP-1 cells responded only to LPS. An NF- (cid:1) B mutation ablated baseline and O 2 - and LPS-stimulated reporter activity in both cell lines, whereas NF-IL6 mutations had little effect. An AP-1 mutation had an intermediate effect. LPS, but not hyperoxia, stimulated nuclear translocation of NF- (cid:1) B in both cell lines. Pharmacological blockade of NF- (cid:1) B nuclear translocation ablated LPS-, but not hyperoxia-, stimulated IL-8 production. Although an intact promoter NF- (cid:1) B site is crucial to macrophage IL-8 production, only LPS-stimulated production appears to require additional nuclear translocation of NF- (cid:1) B. INTERLEUKIN Pharmacological inhibition of NF- (cid:1) B nuclear translocation. The proteasome inhibitor MG-132 (Peptides International, Louisville, KY) was used to block proteasomal degradation of the inhibitor of NF- (cid:1) B, which prevents the nuclear translocation of NF- (cid:1) B (22). MG-132 (50 (cid:2) M) was added to the cell cultures 1 h before the onset of experimental conditions, and cells were exposed for 5 h to the conditions described previously. Statistical methods. The effects of potentially interacting variables (e.g., LPS and hyperoxia) on outcomes were assessed using two-way ANOVA. Univariate comparisons were made using the Student’s t -test. Log transformation was used for stabilization of variance, as required. Alterations in luciferase output were described qualitatively. Error bars in graphs represent the standard error of the mean. using densitometry. The intensity of the speciﬁc NF- (cid:1) B bands in air was used to deﬁne baseline NF- (cid:1) B levels. Relative band intensity was compared using 2-way ANOVA. LPS caused an increase in NF- (cid:1) B translocation (* P (cid:9) 0.0005), whereas hyperoxia alone exhibited no effect. N (cid:9) 5 experiments. C : representative gel electrophoresis shows that neither AP-1 nor NF-IL6 translocation into the nucleus was affected by hyperoxia or LPS exposures over a 6-h period. Stimulation with phorbol 12-myristate 13-acetate (PMA; 1 (cid:2) g/ml) caused increased nuclear translocation of AP-1 but did not affect NF-IL6 nuclear translocation. Nonspeciﬁc, nonradioactive oligonucleotides (AP-1 for NF-IL6, Sp1 for AP-1; N) did not prevent association of nuclear protein to the 32 P-labeled oligonucleotide, whereas a nonradioactive speciﬁc oligonucleotide (S) did do so. Depicted results are typical of 3–5 experiments.