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Identifying the specific functional regulator of integrin family molecules in cancer cells is critical because they are directly involved in tumor invasion and metastasis. Here we report high expression of PLOD2 in oropharyngeal squamous cell carcinomas (SCCs) and its critical role as a stabilizer of integrin β1, enabling integrin β1 to initiate tumor invasion/metastasis. Integrin β1 stabilized by PLOD2-mediated hydroxylation was recruited to the plasma membrane, its functional site, and accelerated tumor cell motility, leading to tumor metastasis in vivo, whereas loss of PLOD2 expression abrogated it. In accordance with molecular analysis, examination of oropharyngeal SCC tissues from patients corroborated PLOD2 expression associated with integrin β1 at the invasive front of tumor nests. PLOD2 is thus implicated as the key regulator of integrin β1 that prominently regulates tumor invasion and metastasis, and it provides important clues engendering novel therapeutics for these intractable cancers.

DOI： 10.1016/j.isci.2020.100850

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Organized tissue structure in the secondary lymphoid organs (SLOs) tightly depends on the development of fibroblastic stromal cells (FSCs) of mesenchymal origin; however, the mechanisms of this relationship are poorly understood. In this study, we specifically inactivated the canonical NF-κB pathway in FSCs in vivo by conditionally inducing IκBα mutant in a Ccl19-IκBSR mouse system in which NF-κB activity is likely to be suppressed in fetal FSC progenitors. Given that NF-κB activation in fetal FSCs is essential for SLO development, the animals were expected to lack SLOs. However, all SLOs were preserved in Ccl19-IκBSR mice. Instead, the T cell area was severely disturbed by the lack of CCL21-expressing FSCs, whereas the follicles and associated FSC networks were formed. Fate mapping revealed that IκBSR-expressing cells constituted only a small fraction of stromal compartment outside the follicles. Taken together, our findings indicate an essential role of the canonical NF-κB pathway activity in the development of three FSC subsets common to SLOs and suggest transient or stochastic CCL19 expression in FSC progenitors and a compensatory differentiation program of follicular FSCs.

DOI： 10.4049/jimmunol.1800539

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Allogeneic organ transplants are rejected by the recipient immune system within several days or weeks. However, the rejection process of allogeneic T (allo-T) cells is poorly understood. In this study, using fluorescence-based monitoring and two-photon live imaging in mouse adoptive transfer system, we visualized the fate of allo-T cells in the in vivo environment and showed rapid elimination in secondary lymphoid organs (SLOs). Although i.v. transferred allo-T cells efficiently entered host SLOs, including lymph nodes and the spleen, ∼70% of the cells had disappeared within 24 h. At early time points, allo-T cells robustly migrated in the T cell area, whereas after 8 h, the numbers of arrested cells and cell fragments were dramatically elevated. Apoptotic breakdown of allo-T cells released a large amount of cell debris, which was efficiently phagocytosed and cleared by CD8+ dendritic cells. Rapid elimination of allo-T cells was also observed in nu/nu recipients. Depletion of NK cells abrogated allo-T cell reduction only in a specific combination of donor and recipient genetic backgrounds. In addition, F1 hybrid transfer experiments showed that allo-T cell killing was independent of the missing-self signature typically recognized by NK cells. These suggest the presence of a unique and previously uncharacterized modality of allorecognition by the host immune system. Taken together, our findings reveal an extremely efficient and dynamic process of allogeneic lymphocyte elimination in SLOs, which could not be recapitulated in vitro and is distinct from the rejection of solid organ and bone marrow transplants.

DOI： 10.4049/jimmunol.1700219

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The spatiotemporal regulation of immune responses in the lymph node (LN) depends on its sophisticated tissue architecture, consisting of several subcompartments supported by distinct fibroblastic stromal cells (FSCs). However, the intricate details of stromal structures and associated FSC subsets are not fully understood. Using several gene reporter mice, we sought to discover unrecognized stromal structures and FSCs in the LN. The four previously identified FSC subsets in the cortex are clearly distinguished by the expression pattern of reporters including PDGFRβ, CCL21-ser, and CXCL12. Herein, we identified a unique FSC subset expressing both CCL21-ser and CXCL12 in the deep cortex periphery (DCP) that is characterized by preferential B cell localization. This subset was clearly different from CXCL12highLepRhigh FSCs in the medullary cord, which harbors plasma cells. B cell localization in the DCP was controlled chiefly by CCL21-ser and, to a lesser extent, CXCL12. Moreover, the optimal development of the DCP as well as medulla requires B cells. Together, our findings suggest the presence of a unique microenvironment in the cortex-medulla boundary and offer an advanced view of the multi-layered stromal framework constructed by distinct FSC subsets in the LN.

DOI： 10.3389/fimmu.2018.02196

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press  

Lymphadenopathy is a frequently observed symptom in systemic lupus erythematosus, although the immunological role of lymph nodes (LNs) in systemic autoimmunity remains largely unknown. Here, we performed comprehensive and systematic analyses of LNs in lupus-prone NZB × NZW F1 (BWF1) mice, demonstrating extensive tissue re-organization of the systemic LNs with follicular expansion, hyper germinal center (GC) formation, atrophy of the paracortical T-cell area and expansion of the medulla in aged BWF1 mice bearing glomerulonephritis. The proportion of B cells was significantly increased in these reactive LNs but not in the spleen, and lymphocyte subsets involved in antibody production, i.e. GC B cells, follicular helper T cells and plasma cells, were elevated. Draining LNs of the affected organs, such as the renal and cervical nodes, showed enhanced tissue re-organization and accumulation of effector lymphocytes, suggesting the presence of a positive feedback loop of regional responses. LN cells isolated from disease-bearing animals produced anti-DNA antibody, indicating activation of autoreactive lymphocytes in situ. The substantial development of disease and LN alterations in mice that received a splenectomy at a young age points to the importance of other secondary lymphoid organs, most likely LNs, for the progression of autoimmune responses independent of the spleen. Taken together, our findings highlight the value of taking LN alterations and activities into consideration for understanding the pathogenesis of systemic autoimmunity.

DOI： 10.1093/intimm/dxx066

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Ras GTPase-activating proteins negatively regulate the Ras/Erk signaling pathway, thereby playing crucial roles in the proliferation, function, and development of various types of cells. In this study, we identified a novel Ras GTPase-activating proteins protein, RASAL3, which is predominantly expressed in cells of hematopoietic lineages, including NKT, B, and Tcells. We established systemic RASAL3-deficient mice, and the mice exhibited a severe decrease in NKT cells in the liver at 8 weeks of age. The treatment of RASAL3-deficient mice with -GalCer, a specific agonist for NKT cells, induced liver damage, but the level was less severe than that in RASAL3-competent mice, and the attenuated liver damage was accompanied by a reduced production of interleukin-4 and interferon- from NKT cells. RASAL3-deficient NKT cells treated with -GalCer in vitro presented augmented Erk phosphorylation, suggesting that there is dysregulated Ras signaling in the NKT cells of RASAL3-deficient mice. Taken together, these results suggest that RASAL3 plays an important role in the expansion and functions of NKT cells in the liver by negatively regulating Ras/Erk signaling, and might be a therapeutic target for NKT-associated diseases.

DOI： 10.1002/eji.201444977

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CHURCHILL LIVINGSTONE  

Capillary vessel flow in the base of the fingernail can be observed by microscopy. This flow is switched off under some conditions, such as coldness, surprise, and anger and is switched on again under other conditions, such as warming, relaxation, and mild exercise. In other words, capillary vessels perform two functions: switching flow on and off. It is speculated that the switch-off function is necessary to direct energy production to the glycolysis pathway, while the switch-on function is necessary for the mitochondrial pathway. This is because glycolysis takes place under anaerobic conditions, while oxidative phosphorylation in the mitochondria proceeds under aerobic conditions in the body. To switch off circulation, the negative electric charges on the surface of erythrocytes and the capillary wall may be decreased by stimulation of the sympathetic nerves and secretion of steroid hormones. Negative charge usually acts as repulsive force between erythrocytes and between erythrocytes and the capillary wall. By decreasing the negative charge, erythrocytes can aggregate and also adhere to the capillary wall. These behaviors may be related to the capillary flow switch-off function. Here, it is emphasized that the capillary vessels possess not only a switch-on function but also a switch-off function for circulation. (C) 2014 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.mehy.2014.03.035

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

In order to further examine the reactivity of autoantibodies, mice were infected with a non-lethal strain of Plasmodium yoelii. Parasitemia appeared between days 10 and 21. During this period, hyperglycemia and hypothermia were serially obeserved and this phenomenon resembled stress-associated responses. In parallel with parasitemia, autoantibodies appeared against nucleus and double-stranded DNA in the sera. To examine further the reactivity of autoantibodies against tissues, immunohistochemical staining using sera from mice with or without malaria was conducted. Autoantibodies contained reactivity to erythrocytes in the spleen, bone marrow and peripheral blood, especially against tissues obtained from mice with malaria. In the liver and intestine, autoantibodies reacted with hepatocytes and intestinal epithelial cells, respectively. These results suggested that the reactivity of autoantibodies against erythrocytes and hepatocytes might be associated with the modulation of the disease course in malaria. (C) 2014 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.cellimm.2014.04.008

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Macrophages are the major source of the chemokines macrophage inflammatory protein-2 (MIP-2) and keratinocyte-derived chemokine (KC), which play a major role in neutrophil migration to sites of inflammation. Although extracellular ATP from inflammatory tissues induces several immune responses in macrophages, it is unclear whether ATP-stimulated macrophages affect neutrophil migration. Therefore, the aim of the present study was to investigate the role of ATP-induced MIP-2 production by macrophages. When ATP was injected intraperitoneally into mice, the number of neutrophils within the peritoneal cavity markedly increased, along with the levels of MIP-2 and KC in the peritoneal lavage fluid. Consistent with this, ATP induced MIP-2 production, but not that of KC, by peritoneal exudate macrophages (PEMs) in vitro. This occurred via interactions with the P2X7 receptor and P2Y2 receptor. Furthermore, treatment of PEMs with ATP led to the production of reactive oxygen species. The ATP-induced MIP-2 production was inhibited by treatment with the antioxidant N-acetyl-l-cysteine. Also, MIP-2 production was inhibited by pre-incubating PEMs with inhibitors of extracellular signal-regulated kinase 1/2 or p38 mitogen-activated protein kinase. The MIP-2 neutralization reduced the increase in neutrophil numbers observed in ATP-treated mice. Taken together, these results suggest that increased production of reactive oxygen species by ATP-stimulated macrophages activates the signalling pathways that promote MIP-2 production which, in turn, induces neutrophil migration.

DOI： 10.1111/j.1365-2567.2012.03601.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER HEIDELBERG  

Natural killer (NK) T cells are well known to play important roles in both tumor rejection and the defense against infectious. Therefore, the antitumor potential of NKT cell-activating antigens have been the focus for the development of NKT cell-based immunotherapies. Up to now, several studies have revealed that the administrations of glycolipids (e.g. alpha-galactosylceramide) can successfully treat certain metastatic tumors. However, liver injuries appeared upon the application of these antigens. We previously examined the potential of using beta-glucosylceramide (beta-GlcCer) to inhibit tumor metastasis to the liver. The aim of this study was to determine the antimetastatic effects of beta-GlcCer and its impact on the activation of NKT cells. Intraperitoneal administration of beta-GlcCer enhanced the production of interferon-gamma from hepatic lymphocytes containing NKT cells, and increased the cytotoxicity of hepatic lymphocytes against tumor cells. Moreover, beta-GlcCer administration suppressed the hepatic metastasis of tumors in wild type (WT) mice, but not in CD1d (-/-) or J alpha 18 (-/-) mice. The drawback associated with the other glycolipids in liver injury was not noted in WT mice treated with the continuous daily administration of beta-GlcCer for 2 weeks. The present study demonstrated that beta-GlcCer treatment activates invariant NKT cells, thus resulting in the inhibition of tumor metastasis.

DOI： 10.1007/s11745-012-3666-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Wild-type (WT) and CD1d-/- [without natural killer (NK) T cells] mice were treated with zymosan A to induce granuloma formation in the liver. Increased granuloma formation was seen in NKT-less mice on days 7 and 14 after administration. WT mice showed limited granuloma formation, and zymosan A eventually induced NKT cell accumulation as identified by their surface marker (e.g. CD1d-tetramer). Zymosan A augmented the expression of Toll-like receptor 2 on the cell surface of both macrophages and NKT cells. One possible reason for accelerated granuloma formation in NKT-less mice was increased production of interferon- ? (IFN-?); a theory that was confirmed using IFN-?-/- mice. Also, zymosan A increased interleukin-10 production in WT mice, which suppresses IFN-? production. Taken together, these results suggest that NKT cells in the liver have the potential to suppress zymosan A-mediated granuloma formation.

DOI： 10.1111/j.1365-2567.2012.03562.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

P>Natural killer T (NKT) cells are known to be specifically activated by alpha-galactosylceramide (alpha-GalCer) via their interaction with CD1d. At that time, NKT cells mediate autoreactivity and eventually induce hepatic injury. As these immune responses resemble acute autoimmune hepatitis, it was examined whether autoantibody production and the activation of autoantibody-producing B-1 cells were accompanied by this phenomenon. Autoantibodies against Hep-2 cells and double-stranded DNA were detected in sera as early as day 3 (showing a peak at day 14) when mice were treated with alpha-GalCer. On day 3, B220low cells appeared in the liver. These B220low cells were CD5- (i.e. B-1b cells) and CD69+ (an activation marker). Primarily, such B220low cells were present in the peritoneal cavity, but the proportion of B220low cells increased with the administration of alpha-GalCer even at this site. In parallel with the appearance of B220low cells in the liver, hepatic lymphocytes acquired the potential to produce autoantibodies in in vitro cell culture in the presence of lipopolysaccharide. These results suggested that hepatic injury induced by alpha-GalCer administration resembled acute autoimmune hepatitis and that the major effector lymphocytes were NKT cells with autoreactivity and autoantibody-producing B-1 cells.

DOI： 10.1111/j.1365-2567.2011.03405.x

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Mice with malaria showed unique immunological responses, including the expansion of NK1.1(-)TCR(int) cells (extrathymic T cells). Since TCR(int) cells with autoreactivity and autoantibody-producing B cells (B-1 cells) are often simultaneously activated under autoimmune conditions, it was examined whether B-1 cells were activated in the course of malarial infection. From days 14 after infection, B220(low) B-1 cells appeared in the liver and spleen. The number of B220(low) B cells was highest at day 14, but the ratio was highest at days 28-35. In parallel with the appearance of B220(low) cells, autoantibodies against HEp-2 cells and double-stranded DNA were detected in sera. These B220(low) cells had phenotypes of CD44(high), CD23(-) and CD62L(-). In sharp contrast, conventional B220(high) B cells (B-2 cells) were CD44(low), CD23(+) and CD62L(+). These results suggested that malaria immune responses were not mediated by conventional T and B cells but resembled the responses during autoimmune diseases. (C) 2010 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.cellimm.2010.02.015

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Severe hepatic injury is induced by Concanavalin A (Con A) administration in mice, the major effector cells being CD4(+) T cells, NKT cells and macrophages. Since autologous lymphocyte subsets are associated with tissue damage, Con A-induced hepatic injury is considered to be autoimmune hepatitis. However, it has remained to be investigated how autoantibodies and B-1 cells are responsible for this phenomenon. In this study, it was demonstrated that autoantibodies which were detected using Hep-2 cells in immunofluorescence tests and using double-strand (ds) DNA in the ELISA method, appeared after Con A administration (a peak at day 14). Moreover, autoantibody-producing B220(low) cells (i.e., B-1 cells) also appeared at this time. Purified B220(low) cells were found to have a potential to produce autoantibodies. These results suggest that Con A-induced hepatic injury indeed includes the mechanism of autoimmune hepatitis. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.cellimm.2009.09.009

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DOI： 10.1111/j.1365-2249.2008.03709.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

It is still controversial whether malaria protection is mediated by conventional immunity associated with T and B cells or by innate immunity associated with extrathymic T cells and autoantibody-producing B cells. Given this situation, it is important to examine the mechanism of malaria protection in beta(2)-microglobulin-deficient (beta(2)m(-/-)) mice. These mice lack major histocompatibility complex class I and CD1d antigens, which results in the absence of CD8(+) T cells and natural killer T (NKT) cells. When C57BL/6 and beta(2)m(-/-) mice were injected with parasitized (Plasmodium yoelii 17XNL) erythrocytes, both survived from the infection and showed a similar level of parasitaemia. The major expanding T cells were NK1.1(-) alpha beta T-cell receptor(int) cells in both mice. The difference was a compensatory expansion of NK and gamma delta T cells in beta(2)m(-/-) mice, and an elimination experiment showed that these lymphocytes were critical for protection in these mice. These results suggest that malaria protection might be events of the innate immunity associated with multiple subsets with autoreactivity. CD8(+) T and NKT cells may be partially related to this protection.

DOI： 10.1111/j.1365-2567.2007.02661.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMEDICAL RESEARCH PRESS LTD  

Since high levels of hyperthermia induce immunosuppression to a certain extent (i.e., granulocytosis and lymphocytopenia) in patients, we applied mild hyperthermia in volunteers using equipment enabling well-controlled hyperthermia. Restricted control of rectal temperature at 39.4 (+/- 0.2)degrees C for 30 min was conducted and various parameters of the body were examined. The most prominent change observed during exposure to hyperthermia was elevated levels of pH and PO2 in the blood, even in the venous blood. A transient elevation of ACTH, cortisol and growth hormone in the blood was also seen during this time. In parallel with this phenomenon, the number of total lymphocytes and those of its subsets (especially CD57(+) or CD56(+) NK cells and NKT cells) increased. More interestingly, the proportion of HLA-DR (MHC class II antigens) increased in NK and NKT cells, and their intensity on the Surface of CD20(+) B cells increased. These results suggest that mild hyperthermia is important for modulation of the functions of the circulatory, endocrine and immune systems.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：HUMANA PRESS INC  

It is well-known that physiological phenomena and certain diseases, including neonatal granulocytosis, age-associated granulocytosis, periodontitis, pancreatitis, Crohn's disease, ulcerative colitis, hemorrhoids, endometriosis, and NSADs-enteritis, are accompanied by tissue destruction and granulocytosis. We investigated what is a key factor connecting tissue destruction and granulocytosis, attention being, focused on adrenergic receptors on granulocytes and stress-induced sympathetic nerve stimulation. If we introduce the concept that "granulocytosis and subsequent tissue destruction are induced by sympathetic nerve stimulation," the mechanisms underlying many physiological phenomena and the etiology of several uncurable diseases in humans can be clearly understood.

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Language：Japanese   Publisher：(NPO)日本免疫学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Among digestive organs, the liver and the large intestine are abundant in T cells expressing NK1.1. NK1.1(+) T cells in the liver are mostly CD1d-dependent whereas those in the large intestine are CD1d-independent. In this study, we investigated the effects of Lactobacilli on NK1.1(+) T cells in the digestive organs of mice. C57BL/6 mice were orally given a dietary supplement prepared from mixed cultures of eight strains of Lactobacilli. Oral administration of Lactobacilli to mice resulted in the selective expansion of NK1.1(+) T cells in the large intestine. These colon NK1.1(+) T cells activated by Lactobacilli were found to express IFN-gamma mRNA. The level of IFN-gamma in the serum was also elevated by the administration of Lactobacilli. Our results suggest that Lactobacilli selectively activate CD1d-independent NK1.1(+) T cells in the large intestine to produce IFN-gamma and therefore modulate Th1 immune responses. (c) 2005 Published by Elsevier B.V.

DOI： 10.1016/j.imlet.2005.07.003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

Peyer's patches (PP) are important inductive sites for the mucosal immune response. It is well known that lymphocytes that migrate into PP are mainly of T-cell lineage from thymus-derived cells (i.e. alpha beta TCRhigh cells). In this study, we further characterized the properties of PP lymphocytes in mice using a mouse model of colitis induced by dextran sulphate sodium (DSS). Although the major site of the inflammation induced by DSS is known to be the large intestine, the small intestine was also damaged. When mice developed DSS-induced colitis, CD3(+)CD8(+)B220(+)gamma delta T cells increased in PP in the small intestine. These gamma delta T cells, which are not seen in the PP of normal mice, resembled intraepithelial lymphocytes (IEL) in the small intestine in terms of their expression of CD5, CD103 and Thy1.2. In addition, the V gamma/delta repertoire of these gamma delta T cells was similar to that of gamma delta IEL. When DSS-treated mice were injected with IEL isolated from normal mice, IEL including gamma delta T cells preferentially migrated to PP, raising the possibility that B220(+) T cells seen in PP of diseased mice may derive from IEL in the small intestine. Our present study suggests that PP might be able to accept T-cell lineages from intestinal IEL as well as from thymus-derived T cells.

DOI： 10.1111/j.1440-1711.2005.01361.x

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