FIGURE 5.

PLA K. pneumoniae CPS induces TNF-α and IL-6 secretion through ROS. A–C and F, data are expressed as the mean ± S.D. for three separate experiments, although in D and E, the results presented are representative of those obtained in three different experiments. A, J774A.1 macrophages were incubated with PLA K. pneumoniae CPS (3 μg/ml) in the presence or absence of NAC (10 mm) for the indicated time, and then ROS levels were measured by detection of the fluorescence intensity of the fluorophore carboxyl-2′,7′-dichlorofluorescein and expressed relative to those at time 0. * indicates a significant difference at the level of p < 0.05. B, HEK293-mTLR4/MD2/CD14 and HEK293-null cells were incubated for the indicated times with PLA K. pneumoniae CPS (CPS) or TFA-treated PLA K. pneumoniae CPS (CPS-TFA) (both 3 μg/ml), and then ROS levels were measured as in A. C, J774A.1 macrophages (1 × 10⁶/ml) were incubated for 6 h with PLA K. pneumoniae CPS (3 μg/ml) in the presence or absence of NAC, and then TNF-α or IL-6 in the culture medium was measured by ELISA. * indicates a significant difference at the level of p < 0.05 compared with PLA K. pneumoniae CPS alone. D, J774A.1 macrophages were incubated for the indicated time with PLA K. pneumoniae CPS (3 μg/ml) in the presence or absence of NAC (10 mm), and phosphorylation of MAPK was measured by Western blotting. E, J774A.1 macrophages were incubated for the indicated time with PLA K. pneumoniae CPS (3 μg/ml) in the presence or absence of NAC (10 mm), and then phosphorylation of PKC-δ and PKC-α was measured by Western blotting. F, RAW-Blue^TM cells were incubated for 24 h with PLA K. pneumoniae CPS or TFA-treated PLA K. pneumoniae CPS (CPS-TFA) (both 3 μg/ml) in the presence or absence of NAC (10 mm), and then secreted embryonic alkaline phosphatase activity was measured by QUANTI-Blue^TM. * indicates a significant difference at the level of p < 0.05.