Signaling through TLR4 enhances B cell chemokine responsiveness. (A) Receptor expression. Splenic B cell was activated with LPS for 18 h or not activated. Stimulated B cell profiles, nonactivated B cell profiles, and isotype controls for indicated receptor are shown. A representative result is depicted from six performed experiments. (B) Chemotaxis assays. Stimulated and nonactivated B cell was subjected to 2 h of chemotaxis in response to CXCL12, CCL19, or CXCL13. The specific migration is shown. Results are mean and SE of sextuplet samples from six experiments (*, P < 0.01 vs. nonactivated B cell). (C) Chemotaxis to CXCL16, CXCL9, or CCL25. Results are mean and SE of sextuplet samples from six experiments. (D) Homing assay. Ten million differentially labeled LPS-activated and nonactivated B cells were intravenously transferred to recipient mice (six experiments each with three pairs of mice; *, P < 0.01 vs. nonactivated B cell). 2 h after transfer, cells of the recipient mice were analyzed by flow cytometry. Shown are the actual number of transferred B cells found in blood (/10 µl), spleen (× 10³ cells), iLN (× 10² cells), and pLN (× 10² cells). (E) LN transit assay. 10 million differentially labeled LPS-treated B cells and nontreated B cells were intravenously transferred (six experiments each with three pairs of mice; *, P < 0.01 vs. nonactivated B cell). 100 µg/mouse of anti–L-selectin antibody was injected intravenously 2 h after transfer. Control mice were injected with PBS (white bars) and analyzed immediately. Cells were isolated from the indicated sites and analyzed by flow cytometer at 12 h (shaded bars) and 24 h (black bars) after antibody injection. (F) Ratios. The ratios between 12 h of antibody, 24 h of antibody, and PBS-treated control groups are shown from the data in E. Error bars indicate means ± SEM each with three pairs of mice in six experiments.