Molecular cloning, recombinant expression and characterization of GMCSF from the rhesus monkey, Macaca mulatta

Abstract

Recent studies have found that, in addition to hematopoiesis, granulocyte–macrophage colony-stimulating factor (GMCSF) plays pivotal roles in multiple immune disorders. The gene encoding Macaca mulatta GMCSF (mmGMCSF) was cloned from stimulated peripheral blood mononuclear cells (PBMCs). Concanavalin A (Con A) and mismatched allogeneic antigen-stimulation significantly increased the production of mmGMCSF by monkey PBMCs. The gene encoding mature mmGMCSF was first expressed as a soluble fusion protein in Escherichia coli, and native mmGMCSF was further prepared by protease cleavage. The recombinant mmGMCSF induced antigen-presenting dendritic cells from monkey PBMCs, suggesting a central role of mmGMCSF in the immune system of the rhesus monkey. Although the predicted mature mmGMCSF protein differs from human GMCSF (hGMCSF) at six amino acid residues, mmGMCSF showed a strong ability to support human TF-1 cell survival. Additional co-immunoprecipitation experiments revealed that mmGMCSF cross reacts with the hGMCSF receptor (hGMCSFR). In addition, the hGMCSF-neutralizing agents hGMCSFR-Fc and anti-hGMCSF antibody reduced the biological effects of mmGMCSF on TF-1 cells. These results suggest that recombinant mmGMCSF might be used for the in vitro evaluation of novel hGMCSF-neutralizing agents prior to the in vivo preclinical evaluation of these agents in the rhesus monkey model.