Inhibition of C. pneumoniae-mediated NF-κB activation with anti-TLR4 antibodies. (A) Representative CLSM images after incubation of type II cells pre-treated or not with anti-TLR4 antibodies followed by C. pneumoniae incubation for 30 min. Cells were stained with anti-IκBα antibody and anti-p65 antibody both detected by a secondary antibody coupled to Alexa594 (red), F-actin was stained with Alexa488-conjugated phalloidin (green) and nuclear DNA with DAPI (blue). In C. pneumoniae infected cells IκBα is decreased and p65 is localized in the nucleus. In cells that were treated with anti-TLR4 antibodies IκBα is not decreased and p65 is not localized in the nucleus. The effect on IκBα and NF-κB p65 was studied semi-quantitatively by CLSM as described in Materials and Methods. The C. pneumoniae-mediated IκBα degradation resulting in a 5.6-fold decrease versus control cells and NF-κB p65 translocation in the nucleus resulting in a 18-fold increase versus control cells. Areas of overlap between blue and red are pink, and between red and green are yellow, the bar equals 10 μm. (n = 5 experiments). (B and C) The amount of cytoplasmatic IκBα and of nuclear p65 of type II cells in controls (lane 1), incubated with C. pneumoniae for 30 min (lane 2) and pre-incubated with anti-TLR4 (HTA) antibodies prior to C. pneumoniae exposure for 30 min (lane 3) was quantified by Western blot analysis. Values of n = 3 experiments are given as means ± SD in arbitrary units. Open bars, IκBα; solid bars, p65. Asterisk indicates a significant difference to controls (1) and cells pre-incubated with anti-TLR4 antibodies (3) (P < 0.0001).