Hisami Watanabe

Details of an isolation method for hepatic lymphocytes in mice

The liver comprises a unique lymphocyte population, i.e., extrathymic alpha beta T cells with TcR of intermediate intensity. In the present study, we attempted to determine what pretreatments were appropriate to isolate hepatic mononuclear cells (MNC) containing such intermediate alpha beta TcR cells in mice. Hepatic MNC were isolated from untreated mice and mice subjected to either bleeding or liver perfusion, and the intermediate alpha beta TcR cells in each preparation were identified. For reasons of simplicity, cell purity and cell yields, hepatic lymphocytes should be obtained from mice subjected to total bleeding. Additional information on extrathymic alpha beta T cells obtained by using the recommended method is also presented.

TNF‐α, nitric oxide and IFN‐γ are all critical for development of necrosis in the small intestine and early mortality in genetically susceptible mice infected perorally with Toxoplasma gondii

We previously reported that genetic susceptibility of mice to peroral infection with T. gondii is associated with CD4+ T cell‐dependent, interferon (IFN)‐γ‐mediated necrosis of their small intestine. We examined the role of tumour necrosis factor (TNF)‐α and nitric oxide (NO), in addition to IFN‐γ. At 7 days after infection, a marked increase in CD4+ T cells was observed in lamina propria mononuclear cells (LPC) of the small intestine as compared with normal mice, and significantly greater amounts of mRNA for IFN‐γ, TNF‐α, and inducible NO synthase (iNOS) were detected in LPC of the small intestine of infected than uninfected animals. Treatment of infected mice with anti‐TNF‐α monoclonal antibody (mAb) or the iNOS inhibitor, aminoguanidine, prevented necrosis and prolonged time to death. Infected iNOS‐targeted mutant mice did not develop the disease whereas infected, control mice did. Treatment with anti‐TNF‐α mAb did not affect the expression of IFN‐γ in the LPC but inhibited expression of iNOS in the infected mice, indicating the role of TNF‐α in the induction of iNOS. These results suggest that NO induced by a combination of IFN‐γ and TNF‐α through activation of iNOS is a critical mediator of intestinal pathology and contributes to early mortality in genetically susceptible mice.

Physiological responses of extrathymic T cells in the liver

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Functional alteration of granulocytes, NK cells, and natural killer T cells in centenarians

The immune system in centenarians was characterized as elevated levels in the proportion and number of granulocytes, NK cells, and extrathymic T cells (including NKT cells) in the peripheral blood. Conventional T cells, abundant in youth, were decreased in proportion and number. In addition to this numerical change in centenarians, the function was significantly altered in comparison with that in middle-aged subjects. The phagocytic function and cytokine production of granulocytes in centenarians increased whereas the production of superoxides from granulocytes decreased. This tendency was almost the same in both healthy and unhealthy centenarians. IFN gamma production by NK and extrathymic T cells in centenarians seemed to be augmented and resulted in an elevated level of serum IFN gamma. Possibly due to the effect of this endogenous IFN gamma, the proportion of CD64(+) (Fc gamma RI) cells among granulocytes was elevated. The expansion of CD64 antigens on granulocytes is known to be regulated by IFN gamma and to be associated with their induction of phagocytosis. These results suggest that the immune system of centenarians is not merely impaired, but altered in terms of the number and functions of granulocytes, NK cells, NKT cells.

Neutrophil depletion exacerbates experimental Chagas' disease in BALB/c, but protects C57BL/6 mice through modulating the Th1/Th2 dichotomy in different directions.

To elucidate the roles of neutrophils in experimental Chagas disease, we depleted the peripheral neutrophils in BALBu2009/u2009c and C57BLu2009/u20096 mice with a monoclonal antibody 1 day before Trypanosoma cruzi infection. Neutrophil depletion in BALBu2009/u2009c mice resulted in exacerbation of the disease and decreased expression of mRNA for Th1 cytokines, including IL‐2 and IFN‐γ, IL‐12p40 and TNF‐α in their spleens after the infection, while a Th2 cytokine, IL‐10, increased especially 1 day after infection. Neutrophils from infected BALBu2009/u2009c mice expressed mRNA for IL‐12p40, IFN‐γ, TNF‐α and Th1 chemoattractive chemokines, monokine induced by IFN‐γ (MIG) and macrophage inflammatory protein‐1α (MIP‐1α). In contrast, in C57BLu2009/u20096 mice neutrophil depletion induced resistance to the disease and enhanced the expression of the above Th1 cytokines, although IL‐10 mRNA in neutrophil‐depleted C57BLu2009/u20096 mice was also higher than in control mice. Neutrophils from C57BLu2009/u20096 mice did not express IL‐12p40, IFN‐γ and MIG but expressed TNF‐α, MIP‐1α and IL‐10. Therefore, neutrophils may play opposite roles in these two strains of mice with respect to protection versus exacerbation of T.u2009cruzi infection, possibly through modulating the Th1u2009/u2009Th2 dichotomy in different directions.

Numerical and functional characteristics of lymphocyte subsets in centenarians

The immune system in the aged is a very interesting subject for study. In this study, analysis was extended to extrathymic T cells as well as NK cells and “conventional” T cells (i.e., thymus-derived T cells) in terms of their constitution and function in both healthy and unhealthy centenarians. Middle-aged persons were used as controls. Healthy and unhealthy centenarians showed lower levels in the proportion and absolute number of lymphocytes. The major change in the constitution of lymphocyte subsets was increased levels in the proportion of NK cells (CD56+/CD57+) and extrathymic T cells (CD3+CD57+). Inversely, conventional T cells decreased in proportion and function (i.e., proliferative response to mitogen). Although NK cells increased in centenarians, NK activity by whole lymphocytes and the purified NK fraction decreased. The difference between healthy and unhealthy centenarians was small in all parameters, the only difference being a lower level of expression of CD56 antigens on CD57+ T cells in unhealthy centenarians. These results indicate that there is a major shift in lymphcyte population from conventional T cells to NK cells and extrathymic T cells with aging. Concerning the age-associated increases in CD56+ T and CD57+ T cells, these cells correspond to NK1+ T cells in mice.

Role of macrophage scavenger receptor in endotoxin shock.

Lipopolysaccharide (LPS) is known to bind to several receptors on macrophages, including CD14 and macrophage scavenger receptor class A types I and II (MSR‐A), and stimulates macrophages to release various inflammatory mediators. MSR‐A recognizes a broad range of polyanionic ligands such as chemically modified lipoproteins, LPS of Gram‐negative bacteria, and lipoteichoic acid of Gram‐positive bacteria, suggesting a role in host defence. In this study, mice lacking MSR‐A were used to elucidate the role of MSR‐A in endotoxin shock. Peritoneal macrophages from MSR‐A‐deficient (MSR‐A−/−) mice bound less remarkably to LPS than those from wild‐type (MSR‐A+/+) mice and the binding activity of MSR‐A+/+ macrophages to LPS was reduced by the addition of an anti‐MSR‐A antibody. Clearance of LPS in serum was retarded in MSR‐A−/− mice after intraperitoneal administration of LPS. LPS‐induced expression of cytokines in the liver was similar in MSR‐A+/+ and MSR‐A−/− mice, but levels of interleukin (IL)‐1β expression and serum IL‐1β were lower in MSR‐A−/− mice. Administration of large doses of LPS resulted in a higher mortality of MSR‐A+/+ mice and pretreatment with an IL‐1 receptor antagonist reduced the mortality. Thus, MSR‐A‐mediated macrophage activation plays a negative role in protecting mice from endotoxin shock by enhancing IL‐1β production by macrophages. Copyright

Tissue-specific expansion of NKT and CD5+B cells at the onset of autoimmune disease in (NZB×NZW)F1 mice

Natural killer T (NKT) cells and CD5+B cells were searched for in various immune organs of autoimmune prone (NZB×NZW)F1 (NZB/W F1) mice. The number of lymphocytes increased in the liver, spleen, and peritoneal cavity after the onset of disease (at the age of 30u2004weeks) while the number of thymocytes decreased at that time. Prominent changes of lymphocyte subsets were seen in the liver and peritoneal cavity, namely, expansion of IL‐2Rβ+TCRαu2009βint cells in the liver and of CD5+B220+ cells in the peritoneal cavity. The majority of TCRαu2009βint cells in the liver were NK1.1+, and CD5+B cells in the peritoneal cavity were CD1d+. Proteinuria became prominent in NZB/W F1 mice with the progression of disease. In parallel with this progression, the proportion of NKT cells decreased slightly in the liver, but their absolute number remained at a high level in this organ. These NKT cells were CD4+ and used an invariant chain of Vα14Jα281 for TCRα. Reflecting the elevation of CD5+B cells, autoantibodies against hepatocyte cytoplasmand denatured DNA were detected in sera. Although NKT cells are known to be immunoregulatory cells in some autoimmune mice, the present results raise the possibility that NKT cells as well as CD5+B cells might be associated with the onset of autoimmune diseases in NZB/W F1 mice. Indeed, NKT cells in F1 mice had a high potential to induce autoimmune‐like inflammationwhen α–galactosylceramide was administered or when active NKT cells were transferred into young F1 mice.

Essential Role of Extrathymic T Cells in Protection Against Malaria

Athymic nude mice carry neither conventional T cells nor NKT cells of thymic origin. However, NK1.1−TCRint cells are present in the liver and other immune organs of athymic mice, because these lymphocyte subsets are truly of extrathymic origin. In this study, we examined whether extrathymic T cells had the capability to protect mice from malarial infection. Although B6-nu/nu mice were more sensitive to malaria than control B6 mice, these athymic mice were able to survive malaria when a reduced number of parasitized erythrocytes (5 × 103 per mouse) were injected. At the fulminant stage, lymphocytosis occurred in the liver and the major expanding lymphocytes were NK1.1−TCRint cells (IL-2Rβ+TCRαβ+). Unconventional CD8+ NKT cells (Vα14−) also appeared. Similar to the case of B6 mice, autoantibodies (IgM type) against denatured DNA appeared during malarial infection. Immune lymphocytes isolated from the liver of athymic mice which had recovered from malaria were capable of protecting irradiated euthymic and athymic mice from malaria when cell transfer experiments were conducted. In conjunction with the previous results in euthymic mice, the present results in athymic mice suggest that the major lymphocyte subsets associated with protection against malaria might be extrathymic T cells.

Enforced Expression of Bcl-2 Restores the Number of NK Cells, But Does Not Rescue the Impaired Development of NKT Cells or Intraepithelial Lymphocytes, in IL-2/IL-15 Receptor β-Chain-Deficient Mice

IL-2/IL-15Rβ-deficient mice display impaired development of NK cells, NKT cells, and intraepithelial lymphocytes of the intestine and skin. To determine the role of survival signals mediated by IL-2/IL-15R in the development of these innate lymphocytes, we introduced a bcl-2 transgene into IL-2/IL-15Rβ-deficient mice. Enforced expression of Bcl-2 restored the number of NK cells in IL-2/IL-15Rβ-deficient mice, but the rescued NK cells showed no cytotoxic activity. The numbers of NKT cells and intestinal intraepithelial lymphocytes did not increase significantly, and skin intraepithelial lymphocytes remained undetectable in the bcl-2 transgenic IL-2/IL-15Rβ-deficient mice. These results indicate an essential role of IL-2/IL-15R-mediated survival signals in the development of NK cells, but they also show that additional nonsurvival signals from IL-2/IL-15R are necessary for innate lymphocyte development.