Figure 5

CCL19 binding, CCL19-induced CCR7 internalization and CCR7-dependent signaling are inhibited by the CCR7 TM4 peptide. (A) Flow cytometric analysis of CCL19-Ig (100 ng/ml) binding in H9 cells with the CCR7 TM4 peptide (red open histogram) or the shuffled peptide (blue open histogram). The gray-filled histogram shows the staining with control immunoglobulin. MFI is indicated on the histograms (left). The relative MFI of CCL19-Ig binding with the CCR7 TM4 peptide or the shuffled peptide is shown (right). Data are mean ± SD of three independent experiments. (B) Flow cytometric analysis of CXCL10-Ig binding with (red open histogram) or without (blue open histogram) the CCR7 TM4 peptide. The gray-filled histogram shows the staining with control immunoglobulin. MFI is indicated on the histogram (left). The relative MFI of CXCL10-Ig binding is shown (right). Data are mean ± SD of three independent experiments. (C) CCR7 expression levels after addition of 1 μg/ml CCL19 were evaluated in the presence (black line) or absence (gray line) of the CCR7 TM4 peptide by flow cytometry using anti-CCR7 antibody. Data are mean ± SD of three independent experiments. (D) CCR7 (left) or CXCR4 (right) expression levels after addition of the indicated chemokine for 30 minutes were evaluated in the presence (black bars) or absence (gray bars) of the CCR7 TM4 peptide by flow cytometry using anti-human CCR7 antibody or anti-human CXCR4 antibody. (E) CCR7 ligand-induced phosphorylation of Akt and p44/42 MAPK (Erk1/2) at the indicated time points after 1 µg/ml CCL19 treatment with or without 15 μg/ml CCR7 TM4 peptide. The levels of phosphorylated and total Akt and Erk1/2 were analyzed by Western blotting using antibodies against phosphorylated Akt at Thr308 (pAkt), phosphorylated Erk1/2 (pErk), total Akt (tAkt), total Erk1/2 (tErk) or GAPDH. A representative experiment from three independent experiments is shown. (F) The ratio of pAkt/tAkt (left) and pErk/tErk (right) were analyzed by densitometric analysis. Data are expressed as percentage of pAkt or pErk levels of control treatment at 0.5 min. Data are mean ± SEM of three independent experiments. *p < 0.05 by Student’s t test; NS, not significant.