Characterization of binding sites for chemokines MCP-1 and MIP-1 alpha on human brain microvessels

The presence of binding sites for the beta chemokines monocyte chemoattract ant protein-1 (MCP-1) and macrophage inflammatory protein-1 alpha (MIP-1 al pha) has recently been identified on human brain microvessels. We extend th ese findings in this report to reveal that such sites exemplify characteris tics of the recognized major receptors for MCP-1 and MIP-1 alpha: CCR2, and CCR1 and CCR5, respectively. Specifically, labeled MCP-1 binding to isolat ed brain microvessels was inhibited by unlabeled MCP-1 and MCP-3, the latte r another CCR2 ligand, but not by MIP-1 alpha. Inhibition of labeled MIP-1 alpha binding was achieved with unlabeled MIP-1 alpha and RANTES, the latte r a beta chemokine that binds to both CCR1 and CCR5, but not by MCP-1. Labe led MIP-1 alpha binding was also antagonized by unlabeled MCP-3, which is a lso recognized by CCR1, and MIP-1 beta, which is a ligand for CCR5. Labeled MCP-1 and MIP-1 alpha were further observed to be internalized within the endothelial cells of brain microvessels, following their binding to the mic rovascular surface at 37 degrees C. Additionally, exposure of microvessels to unlabeled MCP-1 or MIP-1 alpha was accompanied by the initial loss and s ubsequent recovery of surface binding sites for these chemokines, which occ urred on a time scale consistent with ligand-induced endocytosis and recycl ing. These collective features bear striking similarity to those that chara cterize interactions of MCP-1 and MIP-1 alpha with their receptors on leuko cytes and underscore the concept of cognate chemokine receptors on brain mi crovascular endothelium.