Flow cytometric analysis of PKH26-labeled goldfish kidney-derived macrophages

We recently demonstrated that a goldfish macrophage cell line (GMCL) and pr imary in vitro-derived kidney macrophage (IVDKM) cultures contain three dis tinct macrophage subpopulations. Morphological, cytochemical, functional, a nd how cytometric characterization of these sub-populations suggested that they may represent cells of the macrophage lineage temporally arrested at d istinct differentiation junctures of fish macrophage development (putative early progenitors, monocytes, and macrophages). In this study, we examined the proliferation and differentiation events leading to the generation of m ature macrophage-like cells from goldfish kidney hematopoietic tissues. The flow cytometric studies were done after labeling macrophages with PKH26 fl uorescent dye and analysis of the data using the MODFIT software. Our resul ts showed that IVDKM cultures proliferated non-synchronously, suggesting th e presence of a temporal control mechanism regulating the number of cells e ntering the paths towards maturation. Such control is most evident during e arly progenitor proliferation and differentiation events. Our results showe d that proliferation may not be a requirement for differentiation of early progenitors to putative monocyte and macrophage subsets. Detailed observati on of the mature macrophage-like subpopulation indicated that: 1) they appe ar to develop from both, the differentiation of monocyte-like cells, and di rect differentiation of early progenitors in the absence of a monocyte-like stage; and (2) mature macrophage-like cells appeared to be capable of self -proliferation. Our results suggest the presence of alternate pathways of f ish macrophage development other than the classical hematopoietic pathway. (C) 2000 Elsevier Science Ltd. All rights reserved.