Leenen P J, Melis M, Slieker W A, Van Ewijk W
Department of Cell Biology II and Immunology, Erasmus University, Rotterdam, The Netherlands.
Eur J Immunol. 1990 Jan;20(1):27-34. doi: 10.1002/eji.1830200105.
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.
本研究的目的是对巨噬细胞（M phi）分化早期阶段进行表型分析。为此，我们制备了一组同基因大鼠杂交瘤，它们分泌针对M phi前体抗原的抗体（单克隆抗体）。作为免疫原，我们使用了永生化的M phi前体（P.J.M. Leenen等人，《欧洲免疫学杂志》，1990年，20卷：15页）。我们在以下M phi分化的体外模型中筛选获得的单克隆抗体：（a）一组按线性分化顺序排列的M phi细胞系；（b）从骨髓（BM）培养物中获得的未成熟和成熟单核吞噬细胞；（c）一组M phi前体杂交瘤，以及（d）分化的和对照的M phi前体杂交细胞。选择了四种单克隆抗体，即ER-MP12、ER-MP20、ER-MP54和ER-MP58。这些单克隆抗体识别在M phi分化过程中消失的抗原。接下来，我们研究这些单克隆抗体是否也能识别正常骨髓中的M phi前体。为此，使用荧光激活细胞分选仪分离出ER-MP阳性和阴性的骨髓组分。将这些组分在含有M phi集落刺激因子的条件培养基中培养，并用MTT法对产生的成熟M phi后代进行定量。目前的实验表明，ER-MP12和ER-MP20检测到骨髓M phi前体的一个亚群，而ER-MP58几乎能标记所有的M phi前体。对放射性碘化抗原的生化分析表明，这些单克隆抗体识别不同的分子。ER-MP12和ER-MP20分别与140 kDa和14 kDa的单链（糖）蛋白结合。ER-MP54沉淀出多种多肽，其中主要链的表观分子量为90、80 - 85和70 - 75 kDa。根据所识别抗原的分子量和单克隆抗体的特异性，我们得出结论，ER-MP12、ER-MP54和ER-MP58识别迄今未知的小鼠M phi前体细胞抗原。ER-MP20检测到的抗原很可能与Ly-6C相同。
