Title

The role of endothelial chemokine receptor CCR2 in monocyte chemoattractant protein-1-mediated leukocyte migration across the blood-brain barrier

Date of Completion

January 2004

Keywords

Biology, Neuroscience|Health Sciences, Pharmacology|Health Sciences, Immunology

Degree

Ph.D.

Abstract

Previous results from this laboratory revealed the presence of high-affinity, saturable binding sites for monocyte chemoattractant protein-1 (MCP-1) along human brain microvessels (Andjelkovic et al., 1999; Andjelkovic and Pachter, 2000), which suggested that CCR2, the recognized receptor for this chemokine, was expressed by the brain microvascular endothelium. To directly test the role of CCR2 in mediating MCP-1 interactions with the brain microvasculature, MCP-1 binding activity was assessed in murine brain microvessels isolated from wildtype mice and CCR2(−/−) mice engineered to lack this receptor. Results demonstrate that MCP-1 binding is greatly attenuated in microvessels prepared from CCR2(−/−) mice as compared to their wildtype controls. Moreover, microvessels from wildtype mice exhibited MCP-1-induced downmodulation following MCP-1 binding, and that the recovery of this binding activity was not dependent upon de novo protein synthesis. Furthermore, it was shown that MCP-1 was internalized within wild-type microvessels, but not within microvessels obtained from CCR2(−/−) mice. This further validated that CCR2 is obligatory for MCP-1 endocytosis. The internalization of MCP-1, but not transferrin, was inhibited by the disruption of caveolae. Internalized MCP-1 also co-localized at some sites with caveolin-1, a major protein of caveolae, implying that this chemokine is endocytosed, in part, via non-clathrin-coated vesicles. These results prompt consideration that MCP-1 signals may be relayed across the blood-brain barrier by highly specialized interactions of this chemokine with its cognate receptor—CCR2—along brain microvascular endothelial cells. ^ The contribution of CCR2 to MCP-1-induced transendothelial migration was evaluated through ‘mix-and-match’ experiments where endothelial or mononuclear cells from either wildtype or CCR2(−/−) were utilized. Specifically, this study assessed if CCR2 expression by brain microvascular endothelial cells (BMEC) facilitates peritoneal macrophage (pMØ) transendothelial migration. Results were that the expression of CCR2 by pMØ is required for these cells to undergo MCP-1-stimulated transmigration. Moreover, it was also demonstrated that CCR2 expression by BMEC was found to be critical for pMØ transmigration. Furthermore, cellular components from both wildtype and CCR2(−/−) were found to support transendothelial migration in response to another chemokine, MIP-1α. These findings underscore the contribution of CCR2 in MCP-1-stimulated transmigration and highlight a novel role for chemokine receptors on endothelia. ^