SEROLOGIC ANALYSIS OF THE MOUSE-BETA CHEMOKINE-JE MONOCYTE CHEMOATTRACTANT PROTEIN-1

Mouse monocyte chemoattractant protein-1 (MCP-1), previously termed JE , is a member of the beta chemokine gene family and a homologue of the human monocyte chemoattractant protein, MCP-1. Mouse rMCP-1 was used to immunize hamsters for the production of mAb. Seven mouse MCP-1-spec ific mAbs were characterized: two of these mAbs cross-reacted with the human MCP-1, as determined by ELISA. A sensitive and specific capture ELISA for MCP-1 quantitation, which allowed measurement of mouse MCP- 1 levels in supernatants from cells stimulated with inflammatory agent s, was developed. LPS-stimulated astrocytes produce the highest levels of MCP-1 (80 ng/ml); macrophages and mesangial cells produce lower le vels of MCP-1 (2 to 14 ng/ml) after LPS stimulation. IL-1 and TNF-alph a stimulation also can induce low levels of MCP-1 production. Western blot analysis demonstrated that the predominant native form of mouse M CP-1 is a 30-kDa glycoprotein. Two mAbs (2H5 and 6C7) demonstrated dos e-dependent neutralization of mouse MCP-1 chemotactic activity. To loc alize the epitope recognized by one of these neutralizing Abs, the mAb was used to bind a series of genetically engineered truncated variant s of human MCP-1. The C-terminal residues 62 to 67 on human MCP-1 mole cules seem to be critical to express the epitope recognized by the neu tralizing 2H5 anti-MCP-1 mAb. However, multiple sites on the MCP-1 mol ecule seem to be critical for bioactivity. Thus, these Ab reagents pro vide a useful tool to explore the biology of the mouse MCP-1 beta chem okine.