cDNA cloning of guinea pig monocyte chemoattractant protein-1 and expression of the recombinant protein.
Monocyte chemoattractant protein-1 (MCP-1) cDNA was cloned from guinea pig spleen cells stimulated with Con A. The cDNA comprised 647 bp with an open reading frame that encoded a 120 amino acid protein. The sequence similarity of the first 99 amino acids to human MCP-1 is 56%. Recombinant guinea pig MCP-1 was expressed in COS-7 cells, then purified by a three step procedure with orange A-agarose, carboxymethyl-HPLC, and reversed phase-HPLC. The purified protein was found around 25 kDa as a broad band on a polyacrylamide gel under reducing conditions. Guinea pig MCP-1 attracted about 34% of input human monocytes at 5 x 10(-9) M. Guinea pig peritoneal exudate macrophages migrated toward guinea pig MCP-1 dose dependently, but only 1% of input cells responded to guinea pig MCP-1 at its optimal concentration of 5 x 10(-9) M. Human MCP-1 attracted 1% of input guinea pig peritoneal exudate macrophages at its highest concentration of 2.5 x 10(-8) M. Neither human nor guinea pig MCP-1 attracted guinea pig peritoneal resident macrophages. These results suggest that monocytes lose responsiveness to MCP-1 after differentiating to macrophages. Finally, intradermal injection of the recombinant protein into guinea pigs caused marked macrophage infiltration. Cloned and expressed guinea pig MCP-1 will help in studying the role of MCP-1 in vivo.
Yoshimura
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Immunopathology Section, National Cancer Institute-Frederick Cancer Research and Development Center, MD 21702.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D018932:Chemokine CCL2; D002630:Chemotactic Factors / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology; D003001:Cloning, Molecular; D016207:Cytokines / Q000235:genetics*; D004247:DNA / Q000235:genetics; D006168:Guinea Pigs; D006801:Humans; D007271:Injections, Intradermal; D008264:Macrophages; D008969:Molecular Sequence Data; D009000:Monocytes / Q000187:drug effects / Q000276:immunology; D011994:Recombinant Proteins / Q000494:pharmacology
D018932:Chemokine CCL2; D002630:Chemotactic Factors; D016207:Cytokines; D011994:Recombinant Proteins; D004247:DNA