Transcriptional Regulation of NGAL by the Pro-Inflammatory Cytokines IL-17 and TNFα.

IL-17 is a proinflammatory cytokine synthesized by a newly discovered subset of helper T-cells called T_H17. In our search for targets that are regulated by IL-17, we have found that the gene for the bacteriostatic protein neutrophil gelatinase associated lipocalin (NGAL) is strongly induced in epithelial cells by this cytokine. NGAL is a siderophore-binding protein that binds bacterial catecholate-type ferric siderophores and exerts a bacteriostatic effect under iron limiting conditions. It has been observed that NGAL synthesis is induced in inflamed epithelium of the lungs and colon. Using the type II pneumocyt-derived cell line A549 as a model, we found that NGAL was up-regulated 27-fold at RNA level, and 14-fold at protein level, when co-stimulated for 24 hours with IL-17 (200ng/ml) and TNFα (20ng/ml), whereas no induction was seen with either cytokine alone compared to unstimulated cells. The expression of NGAL in A549 cells, when stimulated with IL-17 and TNFα, was dependent on de novo protein synthesis as demonstrated by inhibition of NGAL mRNA production by cycloheximide. Deletion and substitution analysis of the NGAL promoter and subsequent stimulation of transfected cells with IL-17 and TNFα showed that a functional NF-κB-binding site was essential for promoter activation whereas AP-1 and C/EBP-sites were dispensable. Quantitative RT-PCR showed a 13-fold induction of IkB-ζ mRNA in A549 cells when stimulated with IL-17 and TNFα with peak level at 1½ hours and a slow decline for up to 24 hours. Cells stimulated with IL-17 alone resulted in 8-fold mRNA induction at 1½ hours followed by a decline to a 3-fold induction level of mRNA, which lasted for 24 hours. In contrast, cells stimulated with TNFα showed a 9-fold mRNA induction at 1½ hours but then rapidly returned to back-ground level at 3 hours post-induction. These data, and previous studies of the NGAL promoter (Cowland et al. (2006), 176, 5559), demonstrates that the NGAL promoter requires binding of the transcription factor NF-κB as well as the NF-κB-binding co-factor IkB-ζ for transcriptional activation. Our data indicate that TNFα stimulates transcription of the IkB-ζ gene and NF-κB binding to the NGAL promoter whereas IL-17 stabilizes IkB-ζ mRNA and thus enables translation of this co-factor. Only when acting together the two cytokines induce the formation of an NF-κB:IκB-ζ-complex that can stimulate NGAL transcription. We have found that human b-defensin 2 (hBD2) is regulated in the same manner. The functions of NGAL and hBD2 in the innate immune response are well characterized, and the present data demonstrates that IL-17, which is synthesized only by the newly discovered T_H-17 cell population, is involved in transcriptional regulation of these effector molecules. The data furthermore demonstrate that cells of the adaptive immune system is able to activate genes of the innate immune system.