Neutrophil attractant/activation protein-1 and monocyte chemoattractant protein-1 in rabbit. cDNA cloning and their expression in spleen cells

Abstract

Rabbit neutrophil attractant/activation protein-1 (NAP-1) and monocyte chemoattractant protein-1 (MCP-1) were investigated. Rabbit spleen cells stimulated with 5 micrograms/ml of Con A produced both neutrophil and monocyte chemotactic activity. Physicochemical characteristics of those activities obtained by HPLC gel filtration and HPLC chromatofocusing were very similar to those of human NAP-1 and MCP-1, suggesting that rabbit spleen cells produce NAP-1 and MCP-1 after Con A stimulation. A cDNA library was constructed from mRNA purified from Con A-stimulated rabbit spleen cells and screened with oligonucleotide probes. By two rounds of screening, NAP-1 and MCP-1 cDNA were cloned. NAP-1 cDNA comprises 1500 bp with an open reading frame that encodes for a 101-amino acid protein highly similar to human NAP-1. MCP-1 cDNA comprises 607 bp with an open reading frame that encodes for a 124-amino acid protein highly similar to human MCP-1. Expression of NAP-1 and MCP-1 mRNA by rabbit spleen cells was studied. Both Con A- and LPS-stimulated spleen cells expressed NAP-1 and MCP-1 mRNA, but the kinetics of expression were different. Con A rapidly induced high NAP-1 and MCP-1 mRNA expression. LPS also rapidly induced NAP-1 mRNA expression, but high MCP-1 mRNA expression was not observed until 15 h after stimulation. Immunoprecipitation of metabolically labeled NAP-1 and MCP-1 with anti-human NAP-1 or MCP-1 polyclonal antibodies was attempted. Immunoprecipitated rabbit NAP-1 with a molecular mass of about 7 kDa was detected by SDS-PAGE and radioautography, but MCP-1 was not. Cloned rabbit NAP-1 and MCP-1 will give us opportunities to study the role of NAP-1 and MCP-1 in vivo.