Fig. 4.

Anti-Toll-like receptor (TLR)-3 antibody reduced poly I:C-stimulated intercellular adhesion molecule-1 (ICAM-1) expression and production. (a) Total RNA was extracted from HT-29 cells and subjected to reverse transcription–polymerase chain reaction. The clear 213 base pairs (bp) band was detected (lane 2). This band was not detected in the absence of TLR-3-specific primers (lane 1). The expression of β-actin was shown in the lower panel. (b) The HT-29 cell lysate was prepared and subjected to immunoprecipitation followed by Western blotting. When the cell lysate was incubated with anti-TLR-3 antibody, a clear 115 kDa band was detected (right), but not in non-specific control antibody (left). (c) HT-29 cells were plated on coverslips and incubated with control (left) or anti-TLR-3 antibody (right) followed by fluorescein isothiocyanate–goat anti-mouse immunoglobulin (H+L). Very weak but significant stainings were detected in the anti-TLR-3 antibody staining. (d) HT-29 cells were pretreated with 1µg/ml of anti-TLR-3 antibody for 2 h and stimulated with poly I:C for 3 h. Total RNA was extracted and subjected to real-time polymerase chain reaction. Anti-TLR-3 antibody treatment reduced the induction of ICAM-1 significantly. Addition of control antibody was not affected on ICAM-1 induction. *P < 0·05 for the comparison of anti-TLR-3 plus and minus conditions. (e) After pretreatment with anti-TLR-3 antibody, as above, the cells were stimulated further with poly I:C for 24 h. The culture supernatants were collected and subjected to enzyme-linked immunosorbent assay for estimation of sICAM-1. The amount of sICAM-1 in poly I:C-stimulated HT-29 cells were set as 100% and compared with antibody-treated cells.