Identification and characterization of the monoblast in mononuclear phagocyte colonies grown in vitro

Abstract

A liquid culture technique for growing mononuclear phagocyte colonies on a glass surface is described. This useful and reliable technique made it possible to study immature mononuclear phagocytes. In the mononuclear phagocyte colonies the cells grow separate from each other in a single layer. Three types of cells are recognized in these colonies, namely nondividing macrophages, and proliferating promonocytes and monoblasts. The macrophage and the promonocyte exhibit the typical characteristics previously demonstrated by the other methods, whereas the monoblast could only be fully characterized by the present liquid culture method. This proliferating cell (labeling index with [3H]thymidine, 92-96%) is almost round (diameters, 10 X 10 mum), has only a small rim of strongly basophilic cytoplasm, almost devoid of granules, and shows a certain degree of ruffling of the cell surface. The monoblast is positive for esterase with alpha-naphthyl butyrate as substrate (91%), for peroxidase (78% in the peroxidase-positive colonies), and lysozyme (43%). The monoblast is able to pinocytize dextran sulphate (15-20%) and to phagocytize opsonized bacteria (20-30%), latex particles (47%), and IgG-coated red cells (96%). IgG receptors (94%) and complement receptors (16%) are present at the cell surface. In these respects the monoblast has the typical characteristics of the mononuclear phagocytes, but its properties show it to be a more immature cell type than the promonocyte. On the basis of these criteria and the sequence of appearance of the different cell types during incubation and during the development of the individual mononuclear phagocyte colony, monoblasts being present before promonocytes appear in the colony, it is concluded that the monoblast is the precursor of the promonocyte. In these cultures granulocyte colonies are also formed, consisting of myeloblasts, (pro)myelocytes, stabs, and polymorphonuclear neutrophils. Besides the typically tight structure of this kind of colony, the granulocytic cells themselves are quite distinct from the mononuclear phagocytes by their morphology, cytochemical characteristics (e.g. all negative for esterase with alpha-naphthyl butyrate, but 96% positive with N-acetyl DL-alanyl 1-naphthylester), functional characteristics (pinocytic index 13-21%; phagocytic index; for opsonized bacteria 15-36%, for latex particles 10%, and for IgG-coated red cells 0%), and their very small number of IgG receptors and lack of complement receptors. On the basis of these criteria, these granulocytic cells are easily distinguished from the immature cells of the mononuclear phagocyte colonies. The present study confirms the conclusion that the mononuclear phagocytes are a separate cell line, quite distinct from the granulocytic series, since even the most immature cells so far identified--the monoblast and the myeloblast--have quite different characteristics.