Eur. J. Immunol. 1990.20: 15-25 Murine macrophage precursor hybrids 15 zy Pieter J. M. Leenen, Walentina A. T. Slieker, Marleen Melis and Willem Van Ewijk Department of Cell Biology I1 and Immunology, Erasmus University, Rotterdam Murine macrophage precursor characterization I. Production, phenotype and differentiation of macrophage precursor hybrids* This study reports on the earliest stages of mononuclear phagocyte differentia- tion. A crucial question in this developmental process is whether mature macrophage ( M a ) heterogeneity is already appointed at the precursor cell level. For this purpose, we produced clonal populations of mononuclear phagocytes from bone marrow culture by somatic cell hybridization with two hypoxanthine, aminopterin, thymidine-sensitive myeloid cell lines. A panel of 22 stable hybrids was obtained from these fusions. Differentiation stage analysis of the hybrids indicated that all cell lines had immature mononuclear phagocyte characteristics. The hybrids exhibited typical myeloid morphology and mainly nonadherent growth. Mature MQ, features, such as expression of the cell surface antigens Mac-1, Mac-2 and F4180, phagocytosis of latex beads, and expression of nonspecific esterase and acid phosphatase activity, were virtually absent. The immature MQ, markers Thy-1, MIV25 and MIV52, on the other hand, were readily expressed, although heterogeneity was observed among different hybrid cell lines. We then analyzed the differentiation potential of seven hybrids by culture of the cells in the presence of post-lipopolysaccharide serum supplemented with interferon-y and found that the expression of mature MQ, characteristics was induced. However, the various hybrids showed divergent patterns of mature MQ, marker induction. ROC2 cells, for instance, showed extensive morphological and phenotypical differentiation without concomitant induction of phagocytosis. In contrast, W1C4 cells showed significant induction of phagocytosis without simultaneous increase of phosphatase and esterase activity. RlCl cells were unique in the strong induction of Ia antigen expression. Together, our data indicate that (a) early MQ, differentiation stages can be rescued by somatic cell hybridization, and that (b) the obtained cell lines are able to mature according 1 Introduction Mononuclear phagocytes originate from the BM, where precursors develop from pluripotent hemopoietic stem cells [l]. Monocytes are transported via the peripheral blood and mature to MQ, upon entrance of the various tissues. Mature McP constitute a heterogeneous population with regard to functional, morphological and phenotypic aspects (for reviews see [2-41). However, consensus about arranging mononuclear phagocytes in discrete subsets, as can be done for lymphoid cells, has not been reached. It remains to be conclusively established whether specialized subsets exist, or, alternatively, the mononuclear phagocyte lineage as a whole forms a continuum in which different activation and maturation stages, induced by extrinsic [I 79321 zyxwvu * This work was supported by research grant zyxwvutsrq 80-25 from the Netherlands Asthma Foundation. Correspondence: Pieter J. M. Leenen, Department of Cell Biology zyxwvu I1 and Immunology, Erasmus University, PO. Box 1738, NL~3000 DR Rotterdam, The Netherlands Abbreviations: BMDM: BM-derived mononuclear phago- cyte(s) BMDM-4,(7): BMDM, isolated after 4(7) days of cul- ture HECS: Human endothelium culture SN HGF Hybridoma growth factor (IL6) LCM: L cell-conditioned medium M-CSF Macrophage-CSF PLS: Post-LPS serum to divergent differentiation' programs. factors, perform distinct immunological functions. Argu- ments have been provided for both views [3-71. Further- more, the question may be raised whether mononuclear phagocyte heterogeneity is already appointed at the pre- cursor level, as has been suggested previously [5, 8, 91. To address these questions, MQ, and their precursors have to be analyzed at the clonal level. An attractive approach is immortalization of single cells through hybridization with a suitable tumor cell line. In this way, representatives of different mature MQ, populations were immortalized by using a mature McP cell line as fusion partner [ 10-131. The resulting mature MQ, hybrids showed extensive hetero- geneity with respect to phenotypes and functional capaci- ties, such as phagocytosis, antigen presentation, IL 1 and PG secretion. Even during prolonged culture of the various mature MQ, hybrids, the distinct phenotypes are stably maintained. Possibly, the distinct mature MQ, hybrids represent naturally occurring McP populations. Further, the phenotypical diversity of these hybrids suggests that the different mature MQ, phenotypes are predetermined, rather than induced by extrinsic factors, as the hybrids are kept under identical culture conditions. In the present study, we have investigated whether MQ, heterogeneity is already appointed at the precursor level.To enable clonal analysis of McP precursor stages, we used somatic cell hybridization of BM-derived mononuclear phagocytes with myeloid tumor cell lines. By choosing z 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990 001 4-2980/90/0101-0015$02.50/0