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. 2011 Apr 8;286(24):21041–21051. doi: 10.1074/jbc.M111.222091

FIGURE 3.

FIGURE 3.

TLR4 is the major receptor for PLA K. pneumoniae CPS. A, J774A.1 cells (1 × 105/ml) were incubated for 6 h with PLA K. pneumoniae CPS (3 μg/ml; left of panel), E. coli LPS (1 μg/ml; center of panel), or Pam3Cys (1 μg/ml; right of panel) in the presence or absence of an anti-TLR4/MD2 neutralizing antibody or control IgG2a (both 10 μg/ml), and then TNF-α in the culture medium was measured by ELISA. B, PLA K. pneumoniae CPS competes with fluorescent LPS (LPS-Alexa) for binding to the cell surface. Paraformaldehyde-fixed J774A.1 macrophages were preincubated for 30 min at 4 °C with PLA K. pneumoniae CPS, E. coli LPS, or Pam3Cys (all 20 μg/ml) and then incubated for 30 min at 4 °C with LPS-Alexa (2 μg/ml), and cell surface-bound LPS-Alexa was analyzed by flow cytometry. C, impaired cytokine production in PLA K. pneumoniae CPS-stimulated TLR4-deficient macrophages. HeNC2 and GG2EE cells (1 × 106/ml) were stimulated for 6 h with PLA K. pneumoniae CPS, E. coli LPS, or Pam3Cys, and then TNF-α in the culture medium was measured by ELISA. D, HEK293-mTLR4/MD2/CD14 and HEK293-null cells (1 × 106/ml) were stimulated for 24 h with PLA K. pneumoniae CPS (CPS), TFA-treated PLA K. pneumoniae CPS (CPS-TFA), or E. coli LPS, and then IL-8 in the culture medium was measured by ELISA. E, peritoneal macrophages (5 × 105/ml); F, splenocytes (1 × 106/ml) isolated from wild-type and TLR4-deficient mice were stimulated for 24 h with PLA K. pneumoniae CPS (CPS), TFA-treated PLA K. pneumoniae CPS (CPS-TFA), E. coli LPS, or Pam3Cys, and then TNF-α in the culture medium was determined by ELISA. In all figures, the data are expressed as the mean ± S.D. for three separate experiments. * indicates a significant difference at the level of p < 0.05.