Effect of NiCl2 on expression of functional MCP-1.
NiCl2 induces expression of functional MCP-1 in a time- and concentration-dependent manner. (A-C) HUVECs (A, C) or HDMECs (B) were cultured for 4 hours in the presence of 0, 0.1, 0.5, 1.0, and 2.0 mM NiCl2 (A- B) or for 0, 1, 2, 4, 8, or 24 hours in the presence of 1.5 mM NiCl2 (C). Supernatants were then harvested and studied for MCP-1 content by ELISA. The SEM of each point did not exceed 5% of the mean value. (D-E) HUVECs (D) or HDMECs (E) were exposed to 1.0 mM NiCl2, 1 ng/mL TNFα, or medium as control for 8 hours. Subsequently, supernatants were harvested and studied for monocyte chemotactic activity by means of chemotaxis assays as described in “Materials and methods.” In panel E, chemotaxis was determined in the absence or presence of neutralizing polyclonal goat IgG against human MCP-1 or MIP-1α as indicated. Migrated cells were counted in 4 high power fields per well; each supernatant was evaluated in triplicate. Results are presented as mean ± SEM. The inset in panel D visualizes MCP-1 mRNA expression by HUVECs exposed to medium, 1.0 mM NiCl2, or 1 ng/mL TNFα as indicated. The mRNA expression was detected at a single cell level by in situ hybridization.
