Figure 3

CCR7 activation by CCL19 but not CCL21 is increased by anti-CRAM blocking antibody. A. MEC-1 cells were incubated with 10 ng/ml CCL19 (left) or 10 ng/ml CCL21 (right) in the presence of an anti-CRAM antibody or of relevant isotype control for the indicated periods of time. Addition of anti-CRAM antibody increased the phosphorylation of MAPK p44/42. Upper panels show western blot results of phosphorylated or control p44/42, lower panels depict the quantification of five and three independent similar experiments for CCL19 and CCL21, respectively. The intensity of each phosphorylated p44/42 band has been normalised to the corresponding unstimulated control, and the result expressed in function of the time 0 point for isotype control for each experiment. Mean + SD are depicted, unpaired T-test analysis results in significant p values for 10 and 15 minute incubations with CCL19. No significance was observed with CCL21. B. The potentiation of CCL19 induced response by anti-CRAM antibody was confirmed in a chemotaxis experiment, where MEC-1 cells were stimulated to migrate toward CCL19 either in the presence of antibody against CRAM (light grey) or of corresponding isotype control (dark grey). CRAM antibody induced a significant increase of migration toward CCL19 (unpaired T-test, p = 0.03). No changes were observed for similar experiments with CCL21 (data from 3 independent experiments done in triplicate). C. Schematic illustration of molecular mechanisms explaining the previous results: potentiation of CCL19 induced p44/42 phosphorylation by CRAM blockage.