Wild-type and mutated plasmids were transfected into Jurkat cells. The transfected cells were infected without or with

Corby. The activities are expressed relative to that of cells transfected with -133-luc followed by mock-infection, which was defined as 1. Luciferase activities were DNA Damage inhibitor normalized based on the Renilla luciferase activity from phRL-TK. The numbers on the bars depict fold induction relative to the basal level measured in uninfected cells. LUC, luciferase. Graph data are mean ± SD values of three experiments. To identify the cis-acting element(s) in the -133 to -50 bp region of the IL-8 promoter, which served as a L. pneumophila-responsive regulatory element, we prepared and tested selleck compound site-directed mutant constructs (Fig. 5C). Mutation in the NF-κB site (NF-κB mut-luc) and AP-1 site (AP-1 mut-luc) suppressed selleck products L. pneumophila-induced IL-8 expression. However, mutation of the NF-IL-6 site (NF-IL-6 mut-luc) had no such effect. These results indicate that activation of the IL-8 promoter in Jurkat cells in response to L. pneumophila infection requires an intact binding site for the NF-κB and AP-1 elements. Flagellin-dependent activation of NF-κB Because the internal mutational analysis of IL-8 promoter indicated that L. pneumophila infection activated

transcription through the NF-κB site, it was important to identify the nuclear factor(s) that binds to this site. The NF-κB sequence derived from the IL-8 promoter was used as a probe in electrophoretic mobility shift assay (EMSA). Jurkat cells were infected with Corby strain at different times after challenge, and nuclear protein extracts were prepared and analyzed to determine NF-κB DNA binding activity. As shown in Fig. 6A, a complex was induced in these cells within 30 min after infection with Corby and increased in a time-dependent manner. This NF-κB binding activity

to IL-8 promoter was reduced by the addition of either cold probe or a typical NF-κB sequence derived from the IL-2 receptor (IL-2R) α-chain (IL-2Rα) enhancer but not by an oligonucleotide containing the AP-1 binding site (Fig. 6B, lanes 3 to 5). Next, we characterized the L. pneumophila-induced Bumetanide complexes identified by the IL-8 NF-κB probe. These complexes were diminished and supershifted by the addition of anti-p50 or anti-p65 antibody (Fig. 6A, lanes 6 to 10), suggesting that L. pneumophila-induced IL-8 NF-κB complexes are composed of p50 and p65. Based on these results, one can conclude that L. pneumophila infection seems to induce IL-8 gene expression at least in part through induced binding of p50 and p65 to the NF-κB site in the IL-8 promoter region. Figure 6 NF-κB signal is essential for flagellin-dependent activation of the IL-8 promoter by L. pneumophila. (A) Flagellin is required for induction of NF-κB binding activity. Nuclear extracts from Jurkat cells infected with Corby or flaA mutant were mixed with IL-8 NF-κB probe (MOI, 100:1).