Flow cytometric isolation and phenotypic characterization of two subsets of ED2+ (CD163) hepatic macrophages in rats

Aims: Macrophages in the liver are well known for their functional heterogeneity. However, subpopulations of the hepatic macrophages are not well defined. Methods: Two subsets of hepatic macrophages isolated from rats via FACS with immunolabeling of ED2 (anti‐CD163) antibody were studied for phenotypic and functional characteristics. Results: A subset showed an ED2high and autofluorescencehigh (ED2high/AFhigh) phenotype, exhibiting characteristics consistent with the description of the Kupffer cells (KC). A second subset, displaying an ED2dim/AFdim phenotype, was smaller in size, monocyte‐like and weak in phagocytosis. Transmission electron microscopy demonstrated that both subsets are phagocytes. Quantitative RT‐PCR revealed that in addition to expression of macrophage‐related surface markers such as CD14, ED1 (CD68), fucose receptor, and CD163, the ED2dim/ AFdim cells expressed mRNA encoding for myeloid lineage differentiation markers ERMP12 (PECAM) and ERMP20 (Ly‐6C). These two subsets exhibited differential in gene expression of selected cytokines, extracellular matrix proteinases, and Toll‐like receptor in normal livers, as well as significantly upregulated expression in cholestatic livers induced by bile duct ligation. Conclusion: The data suggest that the ED2high/AFhigh population of the liver cells represent the conventional Kupffer cells. The ED2dim/AFdim cells, however, are small hepatic resident macrophages characteristically different from the conventional Kupffer cells.