Masahiro Nakashima

Characterization of two F4/80-positive Kupffer cell subsets by their function and phenotype in mice

BACKGROUND & AIMS Liver Kupffer cells have been suggested to be heterogeneous macrophage lineage cells. We explored this possibility by classifying the mouse Kupffer cells into subpopulations and characterizing them by their phenotype and function. METHODS Liver mononuclear cells (MNCs) from C57BL/6 mice were isolated and their phenotypes and functions were analyzed. The effects of clodronate liposomes and gadolinium chloride (GdCl(3)) on Kupffer cells were also investigated. RESULTS Approximately 25% of liver MNCs were F4/80(+) Kupffer cells. Of these, 46% were CD11b(-)CD68(+), 22% were CD11b(+)CD68(-), and 6% were CD11b(+)CD68(+). CD68(+) cells showed potent phagocytic activity and reactive oxygen species (ROS) production capacity after lipopolysaccharide (LPS) stimulation, whereas CD11b(+) cells did not. CD11b(+) cells showed a strong capacity for the production of cytokines (TNF and IL-12), which was much less prominent in CD68(+) cells. At 24h after LPS or Escherichia coli injection into mice, the proportions of CD11b(+)CD68(-) and CD11b(+)CD68(+) cells increased but that of CD11b(-)CD68(+) cells decreased. The increase in CD11b(+)CD68(+) cells appeared to be derived from the CD11b(+)CD68(-) subset. Although the CD11b(+) cells augmented phagocytic activity after LPS injection, they did not increase ROS production, suggesting their weak lytic activity. Injection of clodronate or GdCl(3) into mice depleted the CD68(+) cells but increased CD11b(+) cells proportionally because CD68(+) cells may phagocytose these toxic reagents and undergo apoptosis. GdCl(3)-treated mice also consistently increased serum TNF after LPS challenge. CONCLUSIONS Two F4/80(+) Kupffer cell subsets may exist, a CD68(+) subset with phagocytic activity and a CD11b(+) subset with cytokine-producing capacity.

Superoxide produced by Kupffer cells is an essential effector in concanavalin A–induced hepatitis in mice

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 (GdCl3) from the liver completely inhibited Con‐A hepatitis, whereas increased serum TNF and IFN‐γ 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, α‐galactosylceramide. Furthermore, GdCl3 pretreatment changed neither the activation‐induced down‐regulation of NK1.1 antigens as well as T cell receptors of NKT cells nor the increased expression of the CD69 activation antigen of hepatic T cells. CD68+ Kupffer cells greatly increased in proportion in the early phase after Con‐A injection; this increase was abrogated by GdCl3 pretreatment. Anti‐TNF antibody (Ab) pretreatment did not inhibit the increase of Kupffer cells, but it effectively suppressed superoxide/reactive oxygen production from Kupffer cells and the resulting hepatic injury. Conversely, depletion of NKT cells in mice by NK1.1 Ab pretreatment did suppress both the increase of CD68+ Kupffer cells and Con‐A hepatitis. Consistently, the diminution of oxygen radicals produced by Kupffer cells by use of free radical scavengers greatly inhibited Con‐A hepatitis without suppressing cytokine production. However, adoptive transfer experiments also indicate that a close interaction/cooperation of Kupffer cells with NKT cells is essential for Con‐A hepatitis. Conclusion: Superoxide produced by Kupffer cells may be the essential effector in Con‐A hepatitis, and TNF and NKT cells support their activation and superoxide production. (HEPATOLOGY 2008;48:1979‐1988.)

Distinct development and functions of resident and recruited liver Kupffer cells/macrophages

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.

Antitumor immunity produced by the liver Kupffer cells, NK cells, NKT cells, and CD8 CD122 T cells.

Mouse and human livers contain innate immune leukocytes, NK cells, NKT cells, and macrophage-lineage Kupffer cells. Various bacterial components, including Toll-like receptor (TLR) ligands and an NKT cell ligand (α-galactocylceramide), activate liver Kupffer cells, which produce IL-1, IL-6, IL-12, and TNF. IL-12 activates hepatic NK cells and NKT cells to produce IFN-γ, which further activates hepatic T cells, in turn activating phagocytosis and cytokine production by Kupffer cells in a positive feedback loop. These immunological events are essentially evoked to protect the host from bacterial and viral infections; however, these events also contribute to antitumor and antimetastatic immunity in the liver by activated liver NK cells and NKT cells. Bystander CD8+CD122+ T cells, and tumor-specific memory CD8+T cells, are also induced in the liver by α-galactocylceramide. Furthermore, adoptive transfer experiments have revealed that activated liver lymphocytes may migrate to other organs to inhibit tumor growth, such as the lungs and kidneys. The immunological mechanism underlying the development of hepatocellular carcinoma in cirrhotic livers in hepatitis C patients and liver innate immunity as a double-edged sword (hepatocyte injury/regeneration, septic shock, autoimmune disease, etc.) are also discussed.

Pivotal advance: characterization of mouse liver phagocytic B cells in innate immunity.

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.

Involvement of the TNF and FasL produced by CD11b Kupffer cells/macrophages in CCl4-induced acute hepatic injury.

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.

Activation of CD11b+ Kupffer Cells/Macrophages as a Common Cause for Exacerbation of TNF/Fas-Ligand-Dependent Hepatitis in Hypercholesterolemic Mice

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.

Insulin Treatment Directly Restores Neutrophil Phagocytosis and Bactericidal Activity in Diabetic Mice and Thereby Improves Surgical Site Staphylococcus aureus Infection

ABSTRACT Bacterial infections, including surgical site infections (SSI), are a common and serious complication of diabetes. Staphylococcus aureus, which is eliminated mainly by neutrophils, is a major cause of SSI in diabetic patients. However, the precise mechanisms by which diabetes predisposes to staphylococcal infection are not fully elucidated. The effect of insulin on this infection is also not well understood. We therefore investigated the effect of insulin treatment on SSI and neutrophil function in diabetic mice. S. aureus was inoculated into the abdominal muscle in diabetic db/db and high-fat-diet (HFD)-fed mice with or without insulin treatment. Although the diabetic db/db mice developed SSI, insulin treatment ameliorated the infection. db/db mice had neutrophil dysfunction, such as decreased phagocytosis, superoxide production, and killing activity of S. aureus; however, insulin treatment restored these functions. Ex vivo treatment (coincubation) of neutrophils with insulin and euglycemic control by phlorizin suggest that insulin may directly activate neutrophil phagocytic and bactericidal activity independently of its euglycemic effect. However, insulin may indirectly restore superoxide production by neutrophils through its euglycemic effect. HFD-fed mice with mild hyperglycemia also developed more severe SSI by S. aureus than control mice and had impaired neutrophil phagocytic and bactericidal activity, which was improved by insulin treatment. Unlike db/db mice, in HFD mice, superoxide production was increased in neutrophils and subsequently suppressed by insulin treatment. Glycemic control by insulin also normalized the neutrophil superoxide-producing capability in HFD mice. Thus, insulin may restore neutrophil phagocytosis and bactericidal activity, thereby ameliorating SSI.

Effect of Cevimeline on Salivary Components in Patients with Sjögren Syndrome

The aim of this study is to clarify the effects of cevimeline on various components in human saliva, such as immunoglobulin A (IgA), lysozyme, α-amylase and squamous cell carcinoma (SCC) antigen. Twelve female patients with Sjögren syndrome (SS) and 14 healthy women were enrolled. After the first saliva collection, one capsule (30 mg) of cevimeline was administered to each subject. Saliva was collected again after 90 min. The salivary flow rate and concentration of each component were measured. In both groups the salivary flow rate and amylase concentration were significantly increased by cevimeline. The lysozyme and IgA concentrations did not change significantly in both groups. The SCC antigen concentration did not change significantly in the SS group, but it decreased significantly in the control group. The secretion rates of amylase and IgA showed significant increases in both groups. The secretion rate of lysozyme significantly increased only in the control group, while the secretion rate of SCC significantly increased only in the SS group. Cevimeline augments not only the salivary flow rate but also the secretion rate of some digestive and/or defense factors from infections. It may be beneficial for SS patients to continue taking cevimeline to prevent oral infections, and other serious sequelae.

The immunologic outcome of enhanced function of mouse liver lymphocytes and Kupffer cells by high-fat and high-cholesterol diet.

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 &bgr; mRNA expression of liver lymphocytes indicate the activation of them by HFCD. IL-12 production from Kupffer cells and interferon &ggr; 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.