(A) Total RNA was extracted from Jurkat cells infected with AA100jm, dotO mutant, Corby, or flaA mutant (MOI of 100) for the indicated ITF2357 cell line time intervals and used for RT-PCR. (B) Jurkat cells were infected with the indicated concentrations of L. pneumophila for 4 h. Total RNA was extracted and used for RT-PCR. (C) Total RNA was extracted from CD4+ T cells infected with Corby (MOI of 50) for 3 h and used for RT-PCR. (D) Jurkat cells were infected with live L. pneumophila Corby or flaA mutant (MOI of 100) for 4 h or incubated with L. pneumophila under the indicated treatment for 4 h. PFA, paraformaldehyde.
Total RNA was extracted and used for RT-PCR. Representative examples of three experiments with similar results. To determine the GDC-0449 correlation between IL-8 VX-689 expression level and L. pneumophila bacterial proteins, heat-killed Corby was used to infect Jurkat
cells at a multiplicity of infection (MOI) of 100. At 4 h, IL-8 was not expressed in Jurkat cells infected with the heat-killed strain (Fig. 2D). Furthermore, IL-8 gene expression was not induced when paraformaldehyde-fixed L. pneumophila was used (Fig. 2D). However, bacteria heated at 56°C for 30 min induced IL-8 expression. These results suggest that the surface proteins of bacteria but not lipopolysaccharide are required for IL-8 induction. Considered together, it seems that Legionella flagellin is involved in IL-8 expression in T cells. Flagellin is recognized by toll-like receptor 5 (TLR5) . Thus, we also examined the expression of TLR2, TLR3, TLR4, and TLR5 mRNAs in Jurkat and CD4+ T cells. All TLR mRNAs examined were expressed in Jurkat and CD4+ T cells (Fig. 3A and 3B). Furthermore, their expression levels did not change by L. pneumophila infection in CD4+ T cells
(Fig. 3B) and Jurkat cells (data not shown). nearly Figure 3 TLR mRNA expression in T cells. (A) Expression of TLR mRNA in Jurkat cells. Total RNA was extracted from Jurkat cells and used for RT-PCR. (B) CD4+ T cells were infected without or with Corby (MOI of 50) for 3 h. Total RNA was extracted from CD4+ T cells and used for RT-PCR. Representative examples of three experiments with similar results. IL-8 production from Jurkat cells during infection with L. pneumophila We used enzyme-linked immunosorbent assay (ELISA) to determine IL-8 protein levels in culture supernatants of Jurkat cells at 8, 12, or 24 h after infection with either the parental strain Corby or flaA mutant strain at an MOI of 100. IL-8 was induced by Corby in a time-dependent manner. On the other hand, the amount of IL-8 produced by Jurkat cells infected with the flaA mutant strain was significantly less than that by cells infected with the wild-type strain (Fig. 4A). Corby-induced IL-8 production by Jurkat cells was MOI-dependent (Fig. 4B). Corby also induced a significant amount of IL-8 from CD4+ T cells (Fig. 4C). Figure 4 IL-8 production from Jurkat cells during infection with L. pneumophila strains.