CysLT1 receptor engagement induces activator protein-1-and NF-κB-dependent IL-8 expression

Because cysteinyl-leukotrienes (cysLTs) are major protagonists in the pathophysiology of human asthma, and because neutrophils are involved in the more severe form of asthma, we studied the potential for leukotriene (LT) D-4 to induce synthesis of the chemokine IL-8 through activation of the CysLT1 receptor. We found LTD4 to induce IL-8 gene expression in monocytic THP-1 cells and human dendritic cells with complete abrogation by selective CysLT1 antagonists. Human embryonic kidney-293 cells stably transfected with CysLT1 were used to better study the transcriptional regulation of the IL-8 promoter. Stimulation of the cells with graded concentrations of LTD4 resulted in a time- and concentration-dependent induction of IL-8 transcription and protein synthesis. Use of IL-8 promoter mutants with substitutions in their NF-kappa B, activator protein (AP)-1, and NF-IL-6 binding elements revealed a requirement for NF-kappa B and AP-1, but not NF-IL-6, in LTD4-induced activation of the IL-8 promoter. Overexpression of dominant-negative I kappa B alpha inhibited the IL-8 transactivation induced by LTD4. NF-kappa B DNA binding activity was induced by LTD4, as determined by electrophoretic mobility shift assays, and could be supershifted by antibodies against p50 and p65. Supershift assays after LTD4 stimulation also indicated the formation of a c-Jun/c-Fos complex. Moreover, our results demonstrate that LTD4 upregulates the expression of c-fos and c-jun at the mRNA level. Our data show for the first time that LTD4, via the CysLT1 receptor, can transcriptionally activate IL-8 production, with involvement of the transcription factors p50, p65, Fos, and Jun. These findings provide mechanistic and potentially therapeutic elements for modulation of the inflammatory component of asthma.