CDPs down-regulate CXCR4 and up-regulate CCR7 on TLR stimulation in vitro. FACS-isolated CDPs were cultured in cytokines alone (control) or stimulated by adding the TLR agonists Pam3csk4, LPS, or CpG. Twelve hours later, mRNA was isolated, and chemokine receptor expression was assessed by quantitative real-time PCR and normalized to Rn18s. Bar graphs show relative expression of mRNA for Cxcr4 (A) and Ccr7 (D) in cultures with cytokines alone (gray) or after addition of TLR agonists (red). Surface protein expression was determined by flow cytometry after 21 hours. Histograms show protein surface expression (blue filled) of CXCR4 (B) and CCR7 (E), overlaid with isotype-matched controls (dotted lines). The relative quantification of protein expression as a ratio between the mean fluorescence intensity of the specific antibody stain and isotype-matched controls is shown for CXCR4 (C) and CCR7 (F). mRNA expression is shown as means and SEM of 4 (A) or 5 (D) independent experiments, including independent cell sorts, cultures, and real-time PCR runs. The fold-changed mRNA expression and the level of statistical significant difference between stimulated cells and control cultures is indicated on the graphs. The histogram overlays (B,E) and the quantification of changes in protein expression (C,F) are shown as 1 representative experiment of 2 independent experiments.
