Increased Transcription of IL-8 in Endothelial Cells Is Differentially Regulated by TNF-a and Oxidized Phospholipids

Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (Ox-PAPC) upregulates a spectrum of inflammatory cytokines and adhesion molecules different from those induced by classic inflammatory mediators such as tumor necrosis factor-a (TNF-a) or lipopolysaccharide. Interestingly, Ox-PAPC also induces the expression of a set of proteins similar to those induced by TNF-a or lipopolysaccharide, which include the chemokines monocyte chemotactic protein-1 (MCP-1) and interleukin (IL)-8. To elucidate the molecular mechanisms of Ox-PAPC–induced gene expression and to determine whether Ox-PAPC and other inflammatory mediators such as TNF-a utilize common signaling pathways, we examined the transcriptional regulation of IL-8 by Ox-PAPC and TNF-a in human aortic endothelial cells. Both Ox-PAPC and TNF-a induced the expression of IL-8 mRNA in a dose-dependent fashion; however, the kinetics of IL-8 mRNA accumulation between the 2 ligands differed. Ox-PAPC–induced IL-8 mRNA was seen as early as 30 minutes, peaked between 4 and 8 hours, and decreased substantially by 24 hours. In contrast, TNF-a–induced IL-8 mRNA synthesis was elevated at 30 minutes, peaked at 2 hours, and reached basal/undetectable levels by 6 hours. Actinomycin D experiments suggested that both Ox-PAPC and TNF-a regulate the expression of IL-8 at the transcriptional level. Furthermore, the half-life of IL-8 mRNA for both ligands was similar (,30 minutes), suggesting that mRNA stability was not responsible for the differences in the kinetics of IL-8 accumulation between the 2 ligands. Transient transfection studies with reporter constructs containing 1.48 kb of the IL-8 promoter identified an Ox-PAPC–specific response region between 2133 and 21481 bp of the IL-8 promoter. In contrast, TNF-a activation of the IL-8 promoter was mediated almost entirely through the nuclear factor-kB and activation protein-1 response elements present between 270 and 2133 bp of the IL-8 promoter. Thus, although Ox-PAPC and TNF-a both induced IL-8 synthesis, our data suggest that the 2 ligands utilize different mechanisms in the regulation of IL-8 transcription. (Arterioscler Thromb Vasc Biol. 2001;21:1585-1591.) We have previously shown that oxidized 1-palmitoyl-2arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC), a major bioactive component of minimally modified LDL (MM-LDL), is present in atherosclerotic lesions and other sites of chronic inflammation.1,2 Similar to other inflammatory cytokines such as tumor necrosis factor-a (TNFa), Ox-PAPC activates endothelial cells to enhance monocyte-endothelial interactions partly through the induction of chemokines such as monocyte chemotactic protein-1 (MCP-1) and interleukin (IL)-8.3,4 Interestingly, Ox-PAPC (or MM-LDL) but not TNF-a enhances monocyte-endothelial binding by mediating the activation of b1-integrins, resulting in deposition of the connecting segment-1 domain of fibronectin on the apical surface of endothelial cells.5 On the other hand, TNF-a enhances endothelial-monocyte interactions through the induction of E-selectin and vascular cell adhesion molecule-1, which are not affected by Ox-PAPC.6 Thus, Ox-PAPC and TNF-a utilize both similar and distinct induction patterns of gene expression to initiate endothelialmonocyte interactions. Although the molecular mechanisms of gene induction by TNF-a are well understood, the target receptors, signaling pathways, and molecular mechanisms by which Ox-PAPC (1) initiates functional changes in endothelial cells and (2) enhances endothelial-monocyte interactions