TLR2 is necessary for SAA-induced G-CSF expression. (A) Inhibition of SAA-induced G-CSF secretion in mouse BMDM by an anti-TLR2 mouse antibody but not an anti-TLR4 rat antibody (5 μg/mL each). Antibody treatment was for 1 hour. Isotype-matching IgG controls for the mouse and rat antibodies were included. The secreted G-CSF (591 pg/mL/10⁶ cells) by SAA without antibody pretreatment was set as 100%. (B) The G-CSF mRNA level was determined by real-time PCR using RNA prepared from SAA (1 μM)-stimulated or unstimulated (NS) BMDM from WT C57BL/6 and Tlr2^−/− mice. The relative concentrations of the G-CSF transcript are presented as fold changes over unstimulated sample (mean plus or minus SEM from 4 experiments, each in duplicate). (C) BMDMs from WT C57BL/6, Tlr2^−/−, and Tlr4^lps-del mice were stimulated with 1 μM SAA, and the secreted G-CSF was determined at the indicated time points using ELISA. Data are presented as mean plus or minus SEM of 3 experiments, each performed in duplicate. (D,E) The TLR2-overexpressed HeLa cells (HeLa/TLR2) or mock-transfected HeLa cells (HeLa/vector) were transiently transfected with G-CSF luciferase reporter cDNA and then stimulated with different reagents for 5 hours. The luciferase activity was measured as described in “Luciferase report assay.” Data are mean plus or minus SEM of 2 to 4 experiments, each performed in triplicate. In panel D, cells were stimulated with SAA (0.1 μM), Pam3CSK4 (1 μg/mL), PGN (1 μg/mL), LTA (10 μg/mL), zymosan (10 μg/mL), and LPS (1 μg/mL). Heat-treated (100°C, 25 minutes) and proteinase K- (50 μg/mL, 1 hour) treated SAA were also used in the study. In panel E, cells were stimulated with different concentrations of SAA (0.02 and 0.1 μM), Pam3CSK4 (0.1 and 1 μg/mL), and PGN (0.2 and 1 μg/mL), either alone or in combination.