Reactivity of autoantibodies against not only erythrocytes but also hepatocytes in sera of mice with malaria.

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.

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 P2X(7) receptor and P2Y(2) 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.

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.

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, B220(low) cells appeared in the liver. These B220(low) cells were CD5(-) (i.e. B-1b cells) and CD69(+) (an activation marker). Primarily, such B220(low) cells were present in the peritoneal cavity, but the proportion of B220(low) cells increased with the administration of α-GalCer even at this site. In parallel with the appearance of B220(low) 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.

NKG2A inhibits invariant NKT cell activation in hepatic injury.

Activation of invariant NKT (iNKT) cells in the liver is generally regarded as the critical step for Con A-induced hepatitis, and the role of NK cell receptors for iNKT cell activation is still controversial. In this study we show that blockade of the NKG2A-mediated inhibitory signal with antagonistic anti-NKG2A/C/E mAb (20d5) aggravated Con A-induced hepatitis in wild-type, Fas ligand (FasL)-mutant gld, and IL-4-deficient mice even with NK cell and CD8 T cell depletion, but not in perforin-, IFN-gamma-, or IFN-gamma- and perforin-deficient mice. Consistently, 20d5 pretreatment augmented serum IFN-gamma levels and perforin-dependent cytotoxicity of liver mononuclear cells following Con A injection, but not their FasL/Fas-dependent cytotoxicity. However, blockade of NKG2A-mediated signals during the cytotoxicity effector phase did not augment cytotoxic activity. Activated iNKT cells promptly disappeared after Con A injection, whereas NK1(-) iNKT cells, which preferentially expressed CD94/NKG2A, predominantly remained in the liver. Pretreatment with 20d5 appeared to facilitate disappearance of iNKT cells, particularly NK1(-) iNKT cells. Moreover, Con A-induced and alpha-galactosylceramide-induced hepatic injury was very severe in CD94/NKG2A-deficient DBA/2J mice compared with CD94/NKG2A-intact DBA/2JJcl mice. Overall, these results indicated that a NKG2A-mediated signal negatively regulates iNKT cell activation and hepatic injury.

Identification and characterization of autoantibody-producing B220(low) 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.

Co-appearance of autoantibody-producing B220(low) 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.

Resistance and augmentation of innate immunity in mice exposed to starvation.

Mice were exposed to starvation for 3 days. Body temperature and various parameters were examined. By starvation, body temperature, blood glucose and ACTH decreased, especially on days 2 and 3. The level of corticosterone increased at this time. On the other hand, the number of lymphocytes yielded by the liver, spleen and thymus decreased from day 1 to 3. The change of the distribution of lymphocyte subsets was unique because NK, NKT and extrathymic T cells were stress-resistant in the liver. Conventional T and B cells were stress-sensitive. Reflecting the increased proportion of NK and NKT cells, NK and NKT activities were augmented. The increased proportion of NKT cells produced both IFNgamma and IL-4 (Th0-type profile). The proportion and some functions of granulocytes and macrophages increased on Day 1 after starvation. These results suggest that starvation has a potential to increase the functions of unconventional lymphocytes and myeloid cells.

Role of alpha-adrenergic stimulus in stress-induced modulation of body temperature, blood glucose and innate immunity.

Mice were exposed to restraint stress for 3h. During this period, low body temperature (hypothermia, 39 degrees C-->less than 37 degrees C) and high blood glucose levels (hyperglycemia, 150 mg/dl-->up to 220 mg/dl) were simultaneously induced. Reflecting a stress-induced phenomenon, blood levels of catecholamines increased at that time. Administration of adrenaline (alpha-stimulus), but neither noradrenaline (alpha but less than adrenaline) nor isoproterenol (beta), induced a similar stress-induced pattern of body temperature and blood glucose variations. This alpha-adrenergic effect was confirmed using alpha- and beta-blockers in adrenaline-induced hypothermia and hyperglycemia. By applying this alpha-stimulus, the effect on immunoparameters was then investigated. Stress-resistant lymphocyte populations were found to be NK cells, extrathymic T cells and NKT cells, especially in the liver. Functional assays showed that both NK-cell cytotoxicity and NKT-cell cytotoxicity were augmented by alpha-stimulus. These results suggest that alpha-stimulus is one of the important factors in the stress-induced phenomenon and that it eventually produces hypothermia, hyperglycemia and innate-immunity activation seen during stress.

Malaria protection in beta 2-microglobulin-deficient mice lacking major histocompatibility complex class I antigens: essential role of innate immunity, including gammadelta T 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 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(-) alphabeta T-cell receptor(int) cells in both mice. The difference was a compensatory expansion of NK and gammadelta 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.

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 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.

Modulation of the endocrine and immune systems by well-controlled hyperthermia equipment.

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 PO(2) 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.

Potentiation of intestinal immunity by micellary mushroom extracts.

Mushroom (shiitake) extracts were dispersed with lecithin micelles to prepare superfine particles (0.05 to 0.2 microm in diameter) of beta-1,3-glucan (micellary mushroom extracts). When mice were fed with these micelles of beta-glucan (0.75 mg/day/mouse, smaller amounts of beta-glucan), the number of lymphocytes yielded by the small intestine increased by up to 40%. More interestingly, the ratio of CD8alphabeta(+)TCRalphabeta(+) cells/CD8alphaalpha(+)TCRalphabeta(+) cells increased prominently. In parallel with this deviation in the distribution of lymphocyte subsets, tumor cytotoxicity against P815 cells and cytokine productions were also augmented. In other words, phylogenetically developed lymphocytes (CD8alphabeta(+), TCRalphabeta(+)) were much more effectively activated by the oral administration of micellary beta-glucan. These results suggest that smaller amounts of micellary beta-glucan might be useful for the potentiation of intestinal immunity.

Activation of CD1d-independent NK1.1+ T cells in the large intestine by Lactobacilli.

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.

Protection against malaria due to innate immunity enhanced by low-protein diet.

Mice were fed ad libitum with a normal diet (25% protein) or low-protein diets (0-12.5% protein) for a wk and then infected with a nonlethal or lethal strain of Plasmodium yoelii, that is, blood stage infection. The same diet was continued until recovery. Mice fed with a normal diet showed severe parasitemia during nonlethal infection, but survived the infection. They died within 2 wk in the case of lethal infection. However, all mice fed with low-protein diets survived without apparent parasitemia (there were small peaks of parasitemia) in cases of both nonlethal and lethal strains. These surviving mice were found to have acquired potent innate immunity, showing the expansion of NK1.1 -TCRint cells and the production of autoantibodies during malarial infection. Severe combined immunodeficiency (scid) mice, which lack TCRint cells as well as TCRhigh cells, did not survive after malarial infection of lethal strain of P. yoelii, even when low-protein diets were given. These results suggest that low-protein diets enhanced innate immunity and inversely decreased conventional immunity, and that these immunological deviations rendered mice resistant against malaria. The present outcome also reminds us of our experience in the field study of malaria, in which some inhabitants eventually avoided contracting malaria even after apparent malarial infection.

Immunologic states of autoimmune diseases.

The etiology and immunologic states of autoimmune diseases have mainly been discussed without consideration of extrathymic T cells, which exist in the liver, intestine, and excretion glands. Because extrathymic T cells are autoreactive and are often simultaneously activated along with autoantibody-producing B-1 cells, these extrathymic T cells and B-1 cells should be introduced when considering the immunologic states of autoimmune diseases. The immunologic states of autoimmune diseases resemble those of aging, chronic GVH disease, and malarial infection. Namely, under all these conditions, conventional T and B cells are rather suppressed concomitant with thymic atrophy or involution. In contrast, extrathymic T cells and B-1 cells are inversely activated at this time. These facts suggest that the immunologic states of autoimmune diseases should be reevaluated by introducing the concept of extrathymic T cells and autoantibody-producing B-1 cells, which might be primordial lymphocytes in phylogeny.

The effect of repetitive mild hyperthermia on body temperature, the autonomic nervous system, and innate and adaptive immunity.

The effect of repetitive mild hyperthermia on body temperature, the autonomic nervous system, and innate and adaptive immunity was investigated using a new hyperthermia treatment system, nanomist sauna (NMS). Six healthy volunteers participated and the concentration of catecholamines and cortisol, and the frequency and function of leukocytes in the peripheral blood were investigated before and after successive 7 days of hyperthermia treatment (20 min/day, 40°C, 100% relative humidity). After treatment, the blood level of adrenaline and cortisol on the 7th day was decreased compared with the 1st day, indicating the suppression of the sympathetic nervous system activity. Moreover, the frequency of CD56(+)NK, CD56(+)NKT and B cells on the 7th day tended to be increased compared with the 1st day. The frequency of HLA-DR-positive NK and NKT cells and expression of HLA-DR on B and T cells increased. The cytotoxicity of NK cells and proliferative response of B cells were also elevated. The results indicate that repetitive mild hyperthermia treatment might suppress excessive sympathetic dominance and modify immunity. Additionally, because it can provide the same effects as conventional hyperthermia treatments with minimal burden to the body, NMS may be a novel patient- and elderly-friendly hyperthermia treatment for health promotion.

Association of CD8+ natural killer T cells in the liver with neonatal tolerance phenomenon.

BACKGROUND: Neonatal tolerance is a very interesting phenomenon, because even allogeneic skin grafts are not rejected in these mice at the adult stage. However, the underlying mechanism remains unclear. METHODS: In this study we prepared such tolerant C57BL/6 (B6) mice (H-2b) by the injection of allogeneic lymphocytes of BALB/c origin (H-2d) at the neonatal stage. RESULTS: The total number of liver lymphocytes in these tolerant mice was found to increase when it was examined at the adult stage. Nevertheless, the retention of allogeneic lymphocytes that were injected at the neonatal stage was highest in the spleen. It is speculated that these allogeneic lymphocytes stimulate the hepatic immune system via the portal vein and that such stimulation maintains the tolerance phenomenon. Indeed, these tolerant mice showed elevated levels of IL-2R beta+ CD3int cells (i.e., extrathymic T cells) and NK1.1+ CD3int cells (i.e., NKT cells) in the liver. Even more interestingly, the number and proportion of CD8+ NKT cells, which are usually a minor population in normal mice, increased among NKT cells in the liver of tolerant mice. This became much more prominent when tolerant mice were grafted with allogeneic (H-2d) skin. CONCLUSION: In conjunction with additional data from a cell-transfer experiment and a splenectomy experiment, our results suggest that CD8+ NKT cells in the liver of tolerant mice might be intimately associated with the neonatal tolerance phenomenon.

Unique immunomodulation by electro-acupuncture in humans possibly via stimulation of the autonomic nervous system.

Cumulative evidence suggests that immunologic responses are under the regulation of the autonomic nervous system. Since acupuncture has recently been reported to modulate the autonomic nervous system, we investigated the possibility that acupuncture eventually modulates the immune system. In the present study, electro-acupuncture was applied in young volunteer subjects. As for the proportions of granulocytes and lymphocytes in the blood, there were three groups: (1) granulocytosis and lymphocytopenia; (2) granulocytopenia and lymphocytosis; and (3) normal pattern. Interestingly, with the administration of acupuncture, the status of subjects with relatively low levels of granulocytes and high levels of lymphocytes shifted to Group 1, whereas that of subjects with high levels of granulocytes and low levels of lymphocytes shifted to Group 2. In other words, acupuncture tended to normalize the pattern of leukocytes. We confirmed that acupuncture induced parasympathetic nerve stimulation, resulting in a decrease in the heart rate. These results suggest possible mechanisms underlying how acupuncture ameliorates the condition of patients with many chronic diseases.

Mechanisms underlying the activation of cytotoxic function mediated by hepatic lymphocytes following the administration of glycyrrhizin.

Stronger neo-minophagen C (SNMC), a glycyrrhizin (GL) preparation, has been used for the treatment of chronic viral hepatitis. It has been reported that a single administration of SNMC induced the activation of hepatic lymphocytes in number and function in animal studies. However, it is still unknown how SNMC augments the cytotoxic function and why such augmentation of cytotoxicity occurs in the liver and other organs. In this study, SNMC was daily injected into mice (2 mg GL/day/mouse) for 2 weeks. A significant augmentation of cytotoxicity mediated by NK cells, NKT cells and TNFalpha was demonstrated mainly in the liver. The presence of TNFalpha-mediated cytotoxicity in the liver was demonstrated for the first time. In contrast to CD8+ cytotoxic T cells (CD8+ CTL), all these cytotoxicities were preexistent in lymphocytes without the immunization of a specific antigen or alloantigens. NK cytotoxicity was mediated by a perforin system, while NKT cytotoxicity was mediated by a Fas ligand system. The present results suggest that the entire cytotoxic function mediated by hepatic lymphocytes was simultaneously augmented by SNMC.