Yoshiko Habu

The liver as a crucial organ in the first line of host defense: the roles of Kupffer cells, natural killer (NK) cells and NK1.1 Ag+ T cells in T helper 1 immune responses

Summary: The liver remains a hematopoietic organ after birth and can produce all leukocyte lineages from resident hematopoietic stem cells. Hepatocytes produce acute phase proteins and complement in bacterial infections. Liver Kupffer cells are activated by various bacterial stimuli, including bacterial lipopolysaccharide (LPS) and bacterial superantigens, and produce interleukin (IL)‐12. IL‐12 and other monokines (IL‐18 etc.) produced by Kupffer cells activate liver natural killer (NK) cells and NK1.1 Ag+ T cells to produce interferon‐g and thereby acquire cytotoxicity against tumors and microbe‐infected cells. These liver leukocytes and the T helper 1 immune responses induced by them thus play a crucial role in the first line of defense against bacterial infections and hematogenous tumor metastases. However, if this defense system is inadequately activated, shock associated with multiple organ failure takes place. Activated liver NK1.1 Ag+ T cells and NK cells also cause hepatocyte injury. NK1.1 Ag+ T cells and another T‐cell subset with an intermediate T‐cell receptor, CD122+CD8+ T cells, can develop independently of thymic epithelial cells. Liver NK cells and NK1.1 Ag+ T cells physiologically develop in situ from their precursors, presumably due to bacterial antigens brought from the intestine via the portal vein. NK cells activated by bacterial superantigens or LPS are also probably involved in the vascular endothelial injury in Kawasaki disease.

Decrease of CD56+T cells and natural killer cells in cirrhotic livers with hepatitis C may be involved in their susceptibility to hepatocellular carcinoma

CD56+T cells and CD56+natural killer (NK) cells are abundant in the human liver. The aim of this study was the further characterization of these cells in the liver with or without hepatitis C virus (HCV) infection. Liver mononuclear cells (MNC) were isolated from liver specimens obtained from the patients during abdominal surgery. In addition to a flow cytometric analysis, liver MNC and PBMC were cultured with the immobilized anti‐CD3 Ab, IL‐2, or a combination of IL‐2 and IL‐12 and their IFN‐γ production and the antitumor cytotoxicity were assessed. The liver MNC of HCV (−) patients contained 20% CD56+T cells whereas the same proportions decreased to 11% in chronic hepatitis livers and to 5% in cirrhotic livers. The proportion of NK cells also decreased in the cirrhotic livers. On the other hand, the populations of these cells in PBMC did not significantly differ among patient groups. The IFN‐γ production and the cytotoxicity against K562 cells, Raji cells, and a hepatocellular carcinoma, HuH‐7 cells, greatly decreased in the cirrhotic liver MNC. In contrast, the cytotoxicity in PBMC did not significantly differ among the patient groups and was lower than that in the liver MNC of HCV (−) patients. CD56+T cells and NK cells but not regular T cells purified from liver MNC cultured with cytokines showed potent cytotoxicities against HuH‐7 cells. These results suggest that a decreased number of CD56+T cells and NK cells in cirrhotic livers may be related to their susceptibility to hepatocellular carcinoma.

Combined spinal and general anesthesia attenuates liver metastasis by preserving Th1/Th2 cytokine balance

Background: Many studies have shown that regional anesthesia improves postoperative outcome and particularly lessens infection by attenuating perioperative immunosuppression related to the stress response to surgery and general anesthesia. However, it remains to be determined whether regional anesthesia improves oncologic outcome after surgery. Methods: C57BL/6 mice were subjected to laparotomy during sevoflurane general anesthesia alone or combined with spinal block achieved with bupivacaine (5 &mgr;g) and morphine (1.25 &mgr;g). Control groups were anesthetized only or were untreated. Liver was removed 5 h after surgery to assess antitumor killer cell activity and production of interferon γ and interleukin 4 by liver mononuclear cells, or mice were inoculated intravenously with liver-metastatic EL4 cells and hepatic metastases were counted 12 days later. Results: Laparotomy during sevoflurane anesthesia significantly increased the number (± SD) of liver metastases from 15.5 ± 8.7 (control) and 19.4 ± 5.4 (sevoflurane alone) to 33.7 ± 8.9. Sevoflurane anesthesia plus spinal block significantly reduced this increase to 19.8 ± 9. The in vitro killer activity of liver mononuclear cells against EL4 cells decreased from 32.7% (control) and 29.4% (sevoflurane alone) to 18.5% after sevoflurane plus laparotomy, and the addition of spinal block increased activity to 26.6%. The interferon-γ/interleukin-4 ratio decreased from 89.3 (control) and 95.7 (anesthesia alone) to 15.7 after sevoflurane plus laparotomy, and the addition of spinal block increased the ratio to 46.5. Conclusions: The addition of spinal block to sevoflurane general anesthesia accompanying surgery attenuates the suppression of tumoricidal function of liver mononuclear cells, presumably by preserving the T helper 1/T helper 2 (Th1/Th2) balance, and thereby reduces the promotion of tumor metastasis.

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.

Systematic characterization of human CD8+ T cells with natural killer cell markers in comparison with natural killer cells and normal CD8+ T cells

We investigated the function of CD56+ CD8+ T cells (CD56+ T cells) and CD56− CD57+ CD8+ T cells (CD57+ T cells; natural killer (NK)‐type T cells) and compared them with those of normal CD56− CD57− CD8+ T cells (CD8+ T cells) and CD56+ NK cells from healthy volunteers. After the stimulation with immobilized anti‐CD3 antibodies, both NK‐type T cells produced much larger amounts of interferon‐γ (IFN‐γ) than CD8+ T cells. Both NK‐type T cells also acquired a more potent cytotoxicity against NK‐sensitive K562 cells than CD8+ T cells while only CD56+ T cells showed a potent cytotoxicity against NK‐resistant Raji cells. After the stimulation with a combination of interleukin (IL)‐2, IL‐12 and IL‐15, the IFN‐γ amounts produced were NK cells ≥ CD56+ T cells ≥ CD57+ T cells > CD8+ T cells. The cytotoxicities against K562 cells were NK cells > CD56+ T cells ≥ CD57+ T cells > CD8+ T cells while cytotoxicities against Raji cells were CD56+ T cells > CD57+ T cells ≥ CD8+ T cells ≥ NK cells. However, the CD3‐stimulated proliferation of both NK‐type T cells was smaller than that of CD8+ T cells partly because NK‐type T cells were susceptible to apoptosis. In addition to NK cells, NK‐type T cells but not CD8+ T cells stimulated with cytokines, expressed cytoplasmic perforin and granzyme B. Furthermore, CD3‐stimulated IFN‐γ production from peripheral blood mononuclear cells (PBMC) correlated with the proportions of CD57+ T cells in PBMC from donors. Our findings suggest that NK‐type T cells play an important role in the T helper 1 responses and the immunological changes associated with ageing.

The Mechanism of a Defective IFN-γ Response to Bacterial Toxins in an Atopic Dermatitis Model, NC/Nga Mice, and the Therapeutic Effect of IFN-γ, IL-12, or IL-18 on Dermatitis

NC/Nga (NC) mice raised under conventional conditions (Conv. NC mice) spontaneously develop dermatitis similar to human atopic dermatitis, whereas NC mice raised under the specific pathogen-free conditions do not develop dermatitis. In the present study, we show that the representative Th1 cytokine, IFN-γ levels in the sera of NC mice, injected with either staphylococcal enterotoxin B or endotoxin (LPS), to be severalfold lower than those of normal mice. The low IFN-γ response to staphylococcal enterotoxin B was correlated to the lack of regular Vβ8+ T cells and Vβ8+ NK T cells, and the low IFN-γ response to LPS was correlated to an impaired IL-18 production of macrophages. The CD3-stimulated IL-4 production from liver and spleen T cells from Conv. NC mice in vitro was greatly augmented. The serum IL-4 levels of untreated Conv. NC mice also were higher than those of normal mice and specific pathogen-free NC mice. Treatment of Conv. NC mice either with IFN-γ, IL-12, or IL-18 twice a week from 4 wk of age substantially inhibited the elevation of the serum IgE levels, serum IL-4 levels, and dermatitis, and IL-12 or IL-18 treatment also reduced the in vitro IL-4 production from CD3-stimulated liver T cells. The systemic deficiency in the Th1 response to bacterial stimulation thus leads to a Th2-dominant state and may induce an abnormal cellular immune response in the skin accompanied with an overproduction of IgE and a susceptibility to dermatitis in NC mice.

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.)

Induction of CD16+ CD56bright NK Cells with Antitumour Cytotoxicity not only from CD16− CD56bright NK Cells but also from CD16− CD56dim NK Cells

The aim of this study was to examine the effect of cytokines on different subsets of NK cells, while especially focusing on CD16− CD56dim cells and CD16− CD56bright cells. When human peripheral blood mononuclear cells (PBMC) were cultured with a combination of IL‐2, IL‐12 and IL‐15 for several days, a minor population of CD56bright NK cells expanded up to 15%, and also showed potent cytotoxicities against various cancer cells. Sorting experiments revealed that unconventional CD16− CD56+ NK cells (CD16− CD56dim NK cells and CD16− CD56bright NK cells, both of which are less than 1% in PBMC) much more vigorously proliferated after cytokine stimulation, whereas predominant CD16+ CD56dim NK cells proliferated poorly. In addition, many of the resting CD16− CD56bright NK cells developed into CD16+ CD56bright NK cells, and CD16− CD56dim NK cells developed into CD16− CD56bright NK cells and also further into CD16+ CD56bright NK cells by the cytokines. CSFE label experiments further substantiated the proliferation capacity of each subset and the developmental process of CD16+ CD56bright NK cells. Both CD16− CD56dim NK cells and CD16− CD56bright NK cells produced large amounts of IFN‐γ and Fas‐ligands. The CD16+ CD56bright NK cells showed strong cytotoxicities against not only MHC class I (−) but also MHC class I (+) tumours regardless of their expression of CD94/NKG2A presumably because they expressed NKG2D as well as natural cytotoxicity receptors. The proliferation of CD16+ CD56bright NK cells was also induced when PBMC were stimulated with penicillin‐treated Streptococcus pyogenes, thus suggesting their role in tumour immunity and bacterial infections.

Age-Associated Augmentation of the Synthetic Ligand- Mediated Function of Mouse NK1.1 Ag+ T Cells: Their Cytokine Production and Hepatotoxicity In Vivo and In Vitro

We recently reported that the direct antitumor effectors in the liver induced by α-galactosylceramide (α-GalCer) are NK cells that are activated by the IFN-γ produced from NK1.1 Ag+ T cells (NKT cells) specifically stimulated with α-GalCer, whereas NKT cells cause hepatocyte injury through the Fas-Fas ligand pathway. In the present study, we investigated how mouse age affects the α-GalCer-induced effect using young (6-wk-old), middle-aged (30-wk-old), and old (75-wk-old) mice. The serum IFN-γ and IL-4 concentrations as well as alanine aminotransferase levels after the α-GalCer injection increased in an age-dependent manner. An α-GalCer injection also induced an age-dependent increase in the Fas ligand expression on liver NKT cells. Under the stimulus of α-GalCer in vitro, the liver mononuclear cells from old and middle-aged mice showed vigorous proliferation, remarkable antitumor cytotoxicity, and enhanced production of both IFN-γ and IL-4 in comparison to those of young mice, all of which were mediated mainly by NK1.1+ cells. Furthermore, liver mononuclear cells from old mice stimulated with α-GalCer showed a more potent Fas-Fas ligand-mediated cytotoxicity against primary cultured hepatocytes than did those from young mice. Most α-GalCer-injected old mice, but no young mice, died, while anti-IFN-γ Ab pretreatment completely inhibited mouse mortality. However, α-GalCer-induced hepatic injury did not improve at all by anti-IFN-γ Ab treatment, and the Fas-ligand expression of liver NKT cells did not change. Taken together, the synthetic ligand-mediated function of NKT cells is age-dependently up-regulated, and the produced IFN-γ is responsible for α-GalCer-induced antitumor immunity and the mouse mortality, while hepatic injury was unexpectedly found to be independent of IFN-γ.

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.