Figure 4

Potentiation of CCL19, but not CCL21 induced chemotaxis by CRAM blockage. A. Upper panel: MEC-1 cells (CRAM⁺/CCR7⁺) were used to do a chemotaxis assay toward 200 ng/ml CCL19 or CCL21, 48 h after transfection with negative control siRNA (black) or CCRL2 siRNA (grey). Experiment was carried out 5 times in triplicate, data shown are means + SD and statistical analysis were done by Mann-Whitney U-test, all chemotaxis indexes reached significance when compared with migration toward buffer (p ≤ 0.05): potentiation of CCL19, but not CCL21 induced chemotaxis by CRAM down-regulation. B. Upper panel: Chemotaxis migration toward CCL19 was done adding inert CRAM ligands in the upper part of the well CCL5 (H = 50 ng/ml, L = 5 ng/ml) and chemerin (H = 500 ng/ml, L = 50 ng/ml) or CCL25 as a control of exclusion for CCX-CKR involvement in the effect observed. Experiments were carried out 3 times in triplicate. Data shown are means + SD and analysis using Mann-Whitney U-test, all chemotaxis indexes reach significance when compared with migration toward buffer (P ≤ 0.001). Lower panel: as a CRAM specificity control, similar experiments were done, this time toward CCL21, for the conditions where significant changes were observed with CCL19. Neither CCL5-L nor chem-H altered the chemotaxis induced by CCL21. C. Schematic illustration of molecular mechanisms illustrating the previous results: potentiation of CCL19, but not CCL21 induced chemotaxis by CRAM blockage by competitors or reduced level of expression of the receptor.