Identification and characterization of a differentiation antigen in human neutrophils and monocytes

Abstract

Human promyelocytic leukemia (HL-60) cells were induced to differentiate into macrophage-like cells by treatment with 10(-7) mol/L 1,25-dihydroxyvitamin D3 (VD3). A monoclonal antibody (MoAb, 60B8), reactive with the particulate of the differentiated cells but not of the untreated cells, was isolated. The antigen recognized by the MoAb became apparent two days after VD3 treatment, and its concentration increased and peaked on day 6. Human neutrophils, followed by monocytes and differentiated HL-60 cells, showed the greatest abundance of the antigen. Monocytes cultured for eight days in vitro lost the antigen. No 60B8 antigen was seen in other blood cells. The MoAb precipitated two polypeptides with an apparent molecular weight (mol wt) of 15,000 (15 k) and 13 k in the detergent-solubilized, 35S-methionine-labeled lysate of the differentiated HL-60 cells. Double-sandwich type enzyme- linked immunosorbent assay (ELISA) devised for the quantitative assay of 60B8 antigen indicated that some 2% to 5% of neutrophil protein was 60B8 antigen. This antigen was not exposed on the neutrophil cell surface, since the cells were not stained immunofluorescently with either mono- or polyclonal antibody, unless they had become permeable. The neutrophil membrane and the granules were separated on the Percoll density gradient, and the antigen was found localized in the plasma membrane-rich fraction. These findings suggested that 60B8 antigen is a novel differentiation antigen for phagocytic cells.