Identification and characterization of MIP-1α/CCL3 isoform 2 from bovine serum as a potent monocyte/dendritic cell chemoattractant

Abstract

Bovine serum is a rich source of cytokines and growth factors supporting in vitro cell culture. Here, a novel bovine monocyte chemotactic factor (boMCF-1) was isolated from commercial bovine serum by a four step purification procedure including adsorption to silicic acid, heparin affinity and cation-exchange chromatography and reversed phase HPLC. Homogeneous boMCF-1 was characterized as a 7717Da protein by mass spectrometry and identified by Edman degradation as the predicted product of bovine macrophage inflammatory protein-1α gene (boMIP-1α/CCL3) isoform 2 (lacking three NH2-terminal amino acids), belonging to the MIP subfamily of CC chemokines. Monocyte chemotactic activity of boCCL3 isoform 2 was completely desensitized by human CCL3 and CCL5, partially by CCL7 and not by CCL2. Its activity was better inhibited by CCR1 than by CCR5 blockade. BoCCL3 isoform 2, present in bovine serum at about 10ng/ml, functioned as a most potent chemoattractant for immature (but not mature) dendritic cells with a minimal effective concentration of 0.03ng/ml and a maximal chemotactic index of 30 at 0.3ng/ml. Its chemotactic activity on immature dendritic cells was significantly desensitized by human CCL3, CCL5 and CCL7. Blockade of CCR5 rather than CCR1 partially prevented chemotactic activity, whereas blockade of both further enhanced this inhibition. BoCCL3 isoform 2 was not chemokinetic but, like human CCL3, synergized with CXCL12 in monocytic cell chemotaxis. Since it cannot be deduced which is the exact human homolog of boCCL3 isoform 2, further research is required to study the biology of other boCCL3 family members.