Although mouse liver F4/80(+) Kupffer cells consist of cytokine-producing CD11b(+) cells and phagocytic CD68(+) cells, an undefined CD11b(-) CD68(-) subset (30%) also exists. We herein demonstrate a more fundamental classification by adding CD32 (FcγRII), which covers most liver F4/80(+) cells and the distinct functions of them. Among the F4/80(+) cells, 50%, 40%, and 30% of cells were CD32(+), CD68(+), and CD11b(+), respectively, and one-half of the CD68(+) cells coexpressed CD32. CD68(+) and CD32(+) cells, but not CD11b(+) cells, expressed a phagocytosis-related CRIg. Gy (6) irradiation depleted liver CD11b(+) cells and those in the spleen, bone marrow, and peripheral blood but not liver CD32/CD68(+) cells. Transfer of bone marrow cells into the irradiated mice reconstituted liver CD11b(+) cells. Conversely, clodronate pretreatment depleted only liver CD32/CD68(+) cells but not liver CD11b(+) cells and peripheral blood or spleen CD11b(+) monocytes/macrophages. Moreover, the CD32(+) cells might be precursors of CD68(+) cells, as a large proportion of CD32(+) cells expressed the c-kit (CD117), and CD34 and CD32(+) cells acquired CD68 immediately after bacteria administration. CD32/CD68(+) cells, but not CD11b(+) cells, expressed resident macrophage-specific MerTK and CD64 (FcγRI). Challenge with Staphylococcus aureus or liver metastatic EL-4 tumor cells indicated that the CD68(+) subset is engaged in systemic bactericidal activity, whereas the CD11b(+) subset is pivotal for liver antitumor immunity. Human liver CD14(+) Kupffer cells could also be classified into three similar subsets. These results suggest that liver CD68(+) Kupffer cells and CD11b(+) Kupffer cells/macrophages are developmentally and functionally distinct subsets.

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We previously reported that F4/80+ Kupffer cells are subclassified into CD68+ Kupffer cells with phagocytic and ROS producing capacity, and CD11b+ Kupffer cells with cytokine-producing capacity. Carbon tetrachloride (CCl4)-induced hepatic injury is a well-known chemical-induced hepatocyte injury. In the present study, we investigated the immunological role of Kupffer cells/macrophages in CCl4-induced hepatitis in mice. The immunohistochemical analysis of the liver and the flow cytometry of the liver mononuclear cells showed that clodronate liposome (c-lipo) treatment greatly decreased the spindle-shaped F4/80+ or CD68+ cells, while the oval-shaped F4/80+ CD11b+ cells increased. Notably, severe hepatic injury induced by CCl4 was further aggravated by c-lipo-pretreatment. The population of CD11b+ Kupffer cells/macrophages dramatically increased 24 hour (h) after CCl4 administration, especially in c-lipo-pretreated mice. The CD11b+ Kupffer cells expressed intracellular TNF and surface Fas-ligand (FasL). Furthermore, anti-TNF Ab pretreatment (which decreased the FasL expression of CD11b+ Kupffer cells), anti-FasL Ab pretreatment or gld/gld mice attenuated the liver injury induced by CCl4. CD1d−/− mouse and cell depletion experiments showed that NKT cells and NK cells were not involved in the hepatic injury. The adoptive transfer and cytotoxic assay against primary cultured hepatocytes confirmed the role of CD11b+ Kupffer cells in CCl4-induced hepatitis. Interestingly, the serum MCP-1 level rapidly increased and peaked at six h after c-lipo pretreatment, suggesting that the MCP-1 produced by c-lipo-phagocytized CD68+ Kupffer cells may recruit CD11b+ macrophages from the periphery and bone marrow. The CD11b+ Kupffer cells producing TNF and FasL thus play a pivotal role in CCl4-induced acute hepatic injury.

TNF and Fas/FasL are vital components, not only in hepatocyte injury, but are also required for hepatocyte regeneration. Liver F4/80+Kupffer cells are classified into two subsets; resident radio-resistant CD68+cells with phagocytic and bactericidal activity, and recruited radio-sensitive CD11b+cells with cytokine-producing capacity. The aim of this study was to investigate the role of these Kupffer cells in the liver regeneration after partial hepatectomy (PHx) in mice. The proportion of Kupffer cell subsets in the remnant liver was examined in C57BL/6 mice by flow cytometry after PHx. To examine the role of CD11b+Kupffer cells/Mφ, mice were depleted of these cells before PHx by non-lethal 5 Gy irradiation with or without bone marrow transplantation (BMT) or the injection of a CCR2 (MCP-1 receptor) antagonist, and liver regeneration was evaluated. Although the proportion of CD68+Kupffer cells did not significantly change after PHx, the proportion of CD11b+Kupffer cells/Mφ and their FasL expression was greatly increased at three days after PHx, when the hepatocytes vigorously proliferate. Serum TNF and MCP-1 levels peaked one day after PHx. Irradiation eliminated the CD11b+Kupffer cells/Mφ for approximately two weeks in the liver, while CD68+Kupffer cells, NK cells and NKT cells remained, and hepatocyte regeneration was retarded. However, BMT partially restored CD11b+Kupffer cells/Mφ and recovered the liver regeneration. Furthermore, CCR2 antagonist treatment decreased the CD11b+Kupffer cells/Mφ and significantly inhibited liver regeneration. The CD11b+Kupffer cells/Mφ recruited from bone marrow by the MCP-1 produced by CD68+Kupffer cells play a pivotal role in liver regeneration via the TNF/FasL/Fas pathway after PHx.