Production and identification of natural monocyte chemotactic protein from virally infected murine fibroblasts

Abstract

Primary cultures of mouse embryonic fibroblasts and confluent monolayers of mouse fibroblastoid cells (L929) were found to secrete a chemotactic factor specific for monocytes. It biological activity was deduced from both the migration distance under agarose and the number of migrated monocytes in the micropore filter method. The monocyte chemotactic protein (MCP) was inducible in these cells by double-stranded RNA and by infection with virus. In embryonic fibroblasts MCP was also produced in response to the cytokines interleukin-1 (IL-1) and tumor necrosis factor (TNF). Under all conditions for induction of MCP tested no production of chemotactic activity for granulocytes could be detected. MCP activity from virally infected L929 cells was concentrated and purified by sequential adsorption to controlled pore glass, heparin-Sepharose chromatography, ion-exchange FPLC and reversed-phase HPLC. Pure MCP was found to occur mainly as a 7-8-kDa protein. Although the mature protein possessed a blocked NH2-terminus, it was identified by enzymatic cleavage and sequence analysis of an internal fragment. The sequence obtained corresponded to a part of the cDNA-derived protein sequence of the murine 'competence' (JE) gene, inducible in fibroblasts by cytokines and virus. In all probability the 7-8-kDa MCP form represents the natural product of the mouse gene JE. Murine MCP can thus be classified in the novel family of small inducible inflammatory proteins.