Yasuhiro Kanda

Extracellular ATP-stimulated macrophages produce macrophage inflammatory protein-2 which is important for neutrophil migration.

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.

Identification and characterization of autoantibody-producing B220low B (B-1) cells appearing in malarial infection

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.

Appearance of B220low autoantibody-producing B-1 cells at neonatal and older stages in mice

In this study, normal adult mice carried B220high conventional B cells in the spleen and liver, but carried both B220high and B220low in the bone marrow. However, at the neonatal stage, only B220low unconventional B cells were found in all these organs. This pattern continued up to 2 weeks after birth, and at this stage autoantibodies were detected in the sera. This phenomenon was seen in all tested young mice (1–2 weeks), irrespective of their gender. Furthermore, at older stages (more than 20 weeks), B220low cells reappeared in the spleen and liver, and these B220low cells became dominant in the bone marrow. Autoantibodies also reappeared in the sera of these older mice. Cell‐sorting experiments revealed that B220low cells were able to produce autoantibodies upon lipopolysaccharide stimuli in vitro. These results suggest that B220low cells appear at both neonatal and older stages as physiological responses and eventually produce autoantibodies.

Malaria protection in β2-microglobulin-deficient mice lacking major histocompatibility complex class I antigens: essential role of innate immunity, including γδT cells

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 β2‐microglobulin‐deficient (β2m(–/–)) 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 β2m(–/–) 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– αβΤ‐cell receptorint cells in both mice. The difference was a compensatory expansion of NK and γδT cells in β2m(–/–) 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.

Coincidence of autoantibody production with the activation of natural killer T cells in α-galactosylceramide-mediated hepatic injury

Natural killer T (NKT) cells are known to be specifically activated by α‐galactosylceramide (α‐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 α‐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 α‐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 α‐GalCer administration resembled acute autoimmune hepatitis and that the major effector lymphocytes were NKT cells with autoreactivity and autoantibody‐producing B‐1 cells.

Relationship between diseases accompanied by tissue destruction and granulocytes with surface adrenergic receptors

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 stressinduced 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.

Co-appearance of autoantibody-producing B220low B cells with NKT cells in the course of hepatic injury

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.

RASAL3, a novel hematopoietic RasGAP protein, regulates the number and functions of NKT cells.

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 T cells. 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 α‐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‐γ from NKT cells. RASAL3‐deficient NKT cells treated with α‐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.

Natural killer T cells suppress zymosan A-mediated granuloma formation in the liver by modulating interferon-γ and interleukin-10

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‐ γ (IFN‐γ); a theory that was confirmed using IFN‐γ−/− mice. Also, zymosan A increased interleukin‐10 production in WT mice, which suppresses IFN‐γ production. Taken together, these results suggest that NKT cells in the liver have the potential to suppress zymosan A‐mediated granuloma formation.

On/off switching of capillary vessel flow controls mitochondrial and glycolysis pathways for energy production

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.