No abstract

Although concanavalin A (Con-A)-induced experimental hepatitis is thought to be induced by activated T cells, natural killer T (NKT) cells, and cytokines, precise mechanisms are still unknown. In the current study, we investigated the roles of Kupffer cells, NKT cells, FasL, tumor necrosis factor (TNF), and superoxide in Con-A hepatitis in C57BL/6 mice. Removal of Kupffer cells using gadolinium chloride (GdCl 3 ) from the liver completely inhibited Con-A hepatitis, whereas increased serum TNF and IFN-␥ levels were not inhibited at all. Unexpectedly, anti-FasL antibody pretreatment did not inhibit Con-A hepatitis, whereas it inhibited hepatic injury induced by a synthetic ligand of NKT cells, ␣-galactosylceramide.

We have reported that the mouse hepatic injury induced by either α-galactosylceramide (α-GalCer) or bacterial DNA motifs (CpG-ODN) is mediated by the TNF/NKT cell/Fas-ligand (FasL) pathway. In addition, F4/80+ Kupffer cells can be subclassified into CD68+ subset with a phagocytosing capacity and CD11b+ subset with a TNF-producing capacity. CD11b+ subset increase if mice are fed high-fat and cholesterol diet (HFCD). The present study examined how a HFCD affects the function of NKT cells and F4/80+ CD11b+ subset and these hepatitis models. After the C57BL/6 mice received a HFCD, high-cholesterol diet (HCD), high-fat diet (HFD) and control diet (CD) for four weeks, the HFCD mice increased surface CD1d and intracellular TLR-9 expression by the CD11b+ population compared to CD mice. Hepatic injury induced either by α-GalCer or CpG-ODN was more severe in HCD and HFCD mice compared to CD mice, which was in proportion to the serum TNF levels. In addition, liver cholesterol levels but not serum cholesterol levels nor liver triglyceride levels were involved in the aggravation of hepatitis. The FasL expression of NKT cells induced by both reagents was upregulated in HFCD mice. Furthermore, the liver mononuclear cells and purified F4/80+ CD11b+ subset from HFCD mice stimulated with either reagent in vitro produced a larger amount of TNF than did those from CD mice. Intracellular TNF production in F4/80+ CD11b+ cells was confirmed. The increased number of F4/80+ CD11b+ Kupffer cells/macrophages by HFCD and their enhanced TNF production thus play a pivotal role in TNF/NKT cell/FasL dependent hepatic injury.

Although B cells in vertebrates have been thought to lack phagocytic activity, there has been a recent report of such ability by the B cells of early vertebrates such as fish and frogs. Here, we show for the first time that mouse liver IgM(+) B cells actively phagocytose microsphere beads and Escherichia coli and that they effectively kill bacterial cells. Such phagocytic activity is not observed in other liver MNCs, except for F4/80(+) Kupffer cells. In the presence of fresh mouse serum (but not heat-inactivated serum), the heat-killed E. coli phagocytic activity of liver B cells increased significantly but was inhibited significantly by anticomplement component C3 antibody, suggesting E. coli opsonization by serum factors, including complement components. Upon i.v. injection of FITC-labeled E. coli into mice, a substantial proportion of liver B cells phagocytosed the bacteria, as compared with spleen B cells. Functional phagolysosome formation in liver B cells was supported by several reagents showing an acidic change and lysosomes in the phagocytosed vacuoles. Indeed, mouse liver B cells killed viable E. coli more efficiently than did spleen B cells in vitro. Further, E. coli-phagocytic liver B cells produced a substantial amount of IL-12. These results indicate that liver B cells have phagocytic and bactericidal activities similar to those of dedicated phagocytes and may contribute to bacterial clearance.

Dietary lipids/cholesterol may modulate liver immune function. We have recently found that mouse F4/80 Kupffer cells are classified into phagocytic CD68 Kupffer cells and cytokine-producing CD11b Kupffer cells. We here investigate how a high-fat and/or high-cholesterol diet affects innate immune liver mononuclear cells. For 4 weeks, C57BL/6 mice were fed a high-fat and high-cholesterol diet (HFCD), a high-cholesterol diet (HCD), a high-fat diet (HFD), or a control diet (CD). High-fat and high-cholesterol diet and HCD increased liver cholesterol levels; serum cholesterol levels increased in HFCD and HFD mice but not in HCD mice. The increased proportion of natural killer (NK) cells, downregulated NK1.1 expression of natural killer T cells, and enhanced CD69 and IL-12 receptor β mRNA expression of liver lymphocytes indicate the activation of them by HFCD. IL-12 production from Kupffer cells and interferon γ production from NK/natural killer T cells activated by LPS and/or IL-12 both increased. IL-12 pretreatment more effectively improved the survival of HFCD mice relative to the survival of CD mice upon injections of liver metastatic EL-4 cells. In contrast, HFCD mouse survival decreased after LPS injection and generalized Shwartzman reaction. Consistently in HFCD mice, Toll-like receptor 4 mRNA expression of whole Kupffer cells was upregulated, and CD11b Kupffer cells proportionally increased. Although the proportion of CD68 Kupffer cells decreased in HFCD mice, phagocytic activity of them was enhanced. Mice fed with HCD rather than those fed with HFD showed features closer to HFCD mice. Thus, enhanced function of mouse liver mononuclear cells is likely dependent on the liver cholesterol level, rather than the liver triglyceride level.