Murine macrophage precursor characterization. II. Monoclonal antibodies against macrophage precursor antigens

Abstract

The aim of the present study was the phenotypical analysis of early stages in macrophage (M phi) differentiation. For this purpose, we produced a panel of syngeneic rat hybridomas, which secreted antibodies (mAb) against M phi precursor antigens. As immunogens we used immortalized M phi precursors (P.J. M. Leenen et al., Eur. J. Immunol. 1990, 20: 15). We screened the obtained mAb in the following in vitro models of M phi differentiation: (a) a panel of M phi cell lines ordered in a linear differentiated sequence; (b) immature and mature mononuclear phagocytes obtained from bone marrow (BM) culture; (c) a panel of M phi precursor hybrids, and (d) differentiated and control M phi precursor hybrid cells. Four mAb, ER-MP12, ER-MP20, ER-MP54 and ER-MP58, were selected. These mAb recognize antigens which disappear in the course of M phi differentiation. Next, we investigated whether these mAb also recognized M phi precursors in normal BM. For this purpose, ER-MP-positive and -negative BM fractions were isolated using a fluorescence-activated cell sorter. Fractions were cultured in M phi-colony-stimulating factor-containing conditioned medium, and the resulting mature M phi progeny was quantified using the MTT assay. The present experiments indicate that ER-MP12 and ER-MP20 detect a subpopulation of BM M phi precursors, whereas ER-MP58 stains virtually all M phi precursors. Biochemical analysis of radioiodinated antigens revealed that these mAb recognize different molecules. ER-MP12 and ER-MP20 bound to single-chain (glyco)proteins of 140 kDa and 14 kDa, respectively. ER-MP54 precipitated multiple polypeptides, of which the major chains have an apparent molecular mass of 90, 80-85 and 70-75 kDa. Based on the molecular mass of the recognized antigens and the mAb specificities we conclude that ER-MP12, ER-MP54 and ER-MP58 recognize hitherto unknown antigens of murine M phi precursor cells. The antigen detected by ER-MP20 is most likely identical to Ly-6C.