Fig. 1. mBMEC express the CCR2 receptor. (A) Expression of CCR2 mRNA in quiescent and IL-1ß-treated cells examined by RT-PCR. GADPH, glyceraldehyde phosphate dehydrogenase; PC, positive control: a mixture of cDNA provided by the supplier of the chemokine receptors and GADPH primers. Volumes of CCR2 bands were expressed as percentage of control gene (GADPH) bands amplified in same PCR reactions. Each bar represents the mean±s.e.m. of three independent experiments. Asterisks indicated significant differences (P<0.001) from corresponding control levels. (B) Western blot analysis of CCR2 receptors. Cells were either quiescent mBMEC in first passage or were treated with 10 ng/ml IL-1ß for 6, 12 and 24 hours. PC, positive control: lysate of murine peritoneal macrophages. Results are presented as means±s.e.m. of three independent experiments; ^*P<0.01. (C) CCR2^+/+ or CCR2^–/– mBMEC were incubated with rabbit anti-CCR2 antibody followed by fluorescein-conjugated secondary antibody. Scale bar 80 µm. (D) mBMEC chemotaxis. Different concentrations of MCP-1 were placed in the lower wells of chemotaxis chambers and mBMEC were placed in the upper wells. After incubation at 37°C the cells on the Neuroprobes filter were counted. Chemotaxis index represents the ratio of migrating endothelial cells in the presence of MCP-1 and in the absence of MCP-1 (control medium). In a separate set of experiments, MCP-1 was also added to a suspension of mBMEC or anti-MCP-1 antibody (1 µg/ml) was added to the lower chamber.