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Importin α1/KPNA1 is a member of the Importin α family widely present in the mammalian brain and has been characterized as a regulator of neuronal differentiation, synaptic functionality, and anxiety-like behavior. In humans, a de novo mutation of the KPNA1 (human Importin α5) gene has been linked with schizophrenia; however, the precise roles of KPNA1 in disorder-related behaviors are still unknown. Moreover, as recent studies have highlighted the importance of gene-environment interactions in the development of psychiatric disorders, we investigated the effects of Kpna1 deletion and social isolation stress, a paradigm that models social stress factors found in human patients, on psychiatric disorder-related behaviors in mice. Through assessment in a behavioral battery, we found that Kpna1 knockout resulted in the following behavioral phenotype: (1) decreased anxiety-like behavior in an elevated plus maze test, (2) short term memory deficits in novel object recognition test (3) impaired sensorimotor gating in a prepulse inhibition test. Importantly, exposure to social isolation stress resulted in additional behavioral abnormalities where isolated Kpna1 knockout mice exhibited: (1) impaired aversive learning and/or memory in the inhibitory avoidance test, as well as (2) increased depression-like behavior in the forced swim test. Furthermore, we investigated whether mice showed alterations in plasma levels of stress-associated signal molecules (corticosterone, cytokines, hormones, receptors), and found that Kpna1 knockout significantly altered levels of corticosterone and LIX (CXCL5). Moreover, significant decreases in the level of prolactin were found in all groups except for group-housed wild type mice. Our findings demonstrate that Kpna1 deletion can trigger widespread behavioral abnormalities associated with psychiatric disorders, some of which were further exacerbated by exposure to adolescent social isolation. The use of Kpna1 knockout mice as a model for psychiatric disorders may show promise for further investigation of gene-environment interactions involved in the pathogenesis of psychiatric disorders.

Because AA (L-ascorbic acid) scavenges various types of free radicals to form MDAA (monodehydroascorbic acid) and DAA (dehydroascorbic acid), its regeneration from the oxidized metabolites is critically important for humans and other animals that lack the ability to synthesize this antioxidant. To study the dynamic aspects of AA metabolism in the circulation, a long acting AOase (ascorbate oxidase) derivative was synthesized by covalently linking PEG [poly(ethylene glycol)] to the enzyme. Fairly low concentrations of the modified enzyme (PEG–AOase) rapidly decreased AA levels in isolated fresh plasma and blood samples with a concomitant increase in their levels of MDAA and DAA. In contrast, relatively high doses of PEG–AOase were required to decrease the circulating plasma AA levels of both normal rats and ODS (osteogenic disorder Shionogi) rats that lack the ability to synthesize AA. Administration of 50 units of PEG–AOase/kg of body weight rapidly decreased AA levels in plasma and the kidney without affecting the levels in other tissues, such as the liver, brain, lung, adrenal grand and skeletal muscles. PEG–AOase slightly, but significantly, decreased glutathione (GSH) levels in the liver without affecting those in other tissues. Suppression of hepatic synthesis of GSH by administration of BSO [L-buthionin-(S,R)-sulfoximine] enhanced the PEG–AOase-induced decrease in plasma AA levels. These and other results suggest that the circulating AA is reductively regenerated from MDAA extremely rapidly and that hepatic GSH plays important roles in the regeneration of this antioxidant.

Microglia are the main resident immunocompetent and phagocytic cells in the central nervous system (CNS). Activated microglia could play phagocytic roles as well as mediate inflammatory processes in the CNS. Involvement of activated microglia in the pathogenesis has been demonstrated in several neurological diseases including Alzheimer's disease (AD). Juzen-taiho-to (JTT), a traditional herbal medicine, has been reported to have effects on activating immune responses and phagocytosis. So far, little is known about the effects of this Kampo formulation JTT on microglia and in AD. In this report, we studied the effects of JTT on the activation and phagocytic functions of mouse microglia and bone marrow-derived macrophages (BMM). JTT could activate microglia, which was confirmed by the prominent morphological change and increased surface expression of an activation marker CD11b. In addition, JTT was revealed to induce microglial proliferation, and enhance microglial phagocytosis of, without eliciting an excessive production of nitric oxide. Furthermore, when mice were administrated with JTT in vivo, their BMM showed more effective phagocytosis of fibrillar Abeta(1-42). These findings implicate the therapeutic potential of JTT in AD and other neurological diseases accompanied by microglial activation.

Role of IL-18 on proliferation and survival of CD8+ T cells, activated by immobilized anti-CD3 antibody (anti-CD3), was examined. Proliferation and survival of activated T cells, especially that of CD8+ T cells, were impaired by IL-18 deficiency [IL-18 knockout (KO)]. After 3 days of culture with anti-CD3, the number of living CD8+ T cells from IL-18KO mice was ∼25% of that from wild-type (WT) mice but was increased to the same level as WT cells by the addition of IL-18. The expression of IL-18 receptors (IL-18Rs), particularly IL-18Rβ chain, in naïve CD8+ T cells was very low but elevated after stimulation with anti-CD3. Blockade of IL-18R by anti-IL-18R antibody on activated WT CD8+ T cells resulted in reduction of living cells, suggesting that IL-18 promotes survival of proliferating CD8+ T cells. Levels of Bcl-2 in activated IL-18KO CD8+ T cells were lower than those in WT cells but were raised by exogenous IL-18. Blockade of IL-18R on WT CD8+ T cells decreased the expression of surface markers CD122 and CD94, which are related to cell viability, and the expression of these markers was increased by exogenous IL-18 in IL-18KO cells. These results suggest that IL-18 acts directly on activated CD8+ T cells through IL-18Rs and promotes their survival to expand the population.

The role of interleukin (IL)-18 in the protection from interstitial pneumonia and pulmonary fibrosis induced by bleomycin (BLM) was investigated by comparing the severity of BLM-induced lung injuries between wild-type and C57BL/6 mice with a targeted knockout mutation of the IL-18 gene (IL-18−/− mice). IL-18−/− mice showed much worse lung injuries than wild-type mice, as assessed by the survival rate, histological images, and leukocyte infiltration in the bronchoalveolar lavage fluid and myeloperoxidase activity. In wild-type mice, administration of IL-18 before BLM instillation resulted in suppression of lung injuries, increases in the hydroxyproline content, and decreases in the granulocyte-macrophage colony-stimulating factor content in the lung. Preadministration of IL-18 also resulted in prevention of the reduction of the lung IL-10 content caused by BLM-induced damage of alveolar epithelial. BLM instillation suppressed superoxide dismutase (SOD) activity in IL-18−/− mice to a greater extent than in wild-type mice. Pretreatment of IL-18 augmented Mn-containing superoxide dismutase (Mn-SOD) messenger RNA expression and SOD activity in the lung and prevented the reduction of SOD activity caused by BLM in both wild-type and IL-18−/− mice. These results suggest that IL-18 plays a protective role against BLM-induced lung injuries by upregulating a defensive molecule, Mn-SOD.

SummaryPsychological/physical stresses are known to cause relapses of autoimmune and inflammatory diseases. To reveal a mechanism by which noninflammatory stresses affect host defenses, responses to immobilization stress in mice were investigated, focusing on the role of a multifunctional cytokine, interleukin-18 (IL-18). In the adrenal cortex, the stress induced IL-18 precursor proteins (pro-IL-18) via adrenocorticotropic hormone (ACTH) and a superoxide-mediated caspase-1 activation pathway, resulting in conversion of pro-IL-18 to the mature form, which was released into plasma. Inhibitors of caspase-1, reactive oxygen species, and P38 mitogen-activated protein kinase (MAPK) suppressed stress-induced accumulation of plasma IL-18. These inhibitors also blocked stress-induced IL-6 expression. This, together with the observation that IL-6 was not induced in IL-18-deficient mice, showed that IL-6 induction by stress is dependent on IL-18. In stressed organisms, IL-18 may influence pathological and physiological processes. Controlling the caspase-1 activating pathway to suppress IL-18 levels may provide preventative means against stress-related disruption of host defenses.

Psychological/physical stresses have been reported to exacerbate auto-immune and inflammatory diseases. To clarify a mechanism by which non-inflammatory stresses disrupt host defenses, responses to immobilization stress in mice were investigated, focusing on the role of a multifunctional cytokine, interleukin-18 (IL-18). In the adrenal cortex, the stress induced IL-18 precursor proteins (pro-IL-18) via ACTH and a superoxide-mediated caspase-1 activation pathway, resulting in conversion of pro-IL-18 to the mature form which was released into plasma. Inhibitors of caspase-1, reactive oxygen species and P38 MAPK prevented stress-induced accumulation of plasma IL-18. These inhibitors also blocked stress-induced IL-6 expression. This, together with the observation that IL-6 was not induced in stressed-IL-18 deficient mice, showed that IL-6 induction by stress is dependent on IL-18. In stressed organisms, IL-18 may influence pathological and physiological processes. Controlling the caspase-1 activating pathway to suppress IL-18 levels may provide preventative means against stress-related disruption of host defenses.

Objective: Although psychological and/or physiological stress has been well documented to influence immune responses, the precise mechanism for immunomodulation remains to be elucidated. The present work describes the role of the hypothalamic-pituitary-adrenal (HPA) axis in the mechanism of stress-mediated enhanced-resistance to lethality after lipopolysaccharide (LPS) injection. Methods/Results: Preconditioning with restraint stress (RS) resulted in enhanced activation of the HPA axis in response to LPS injection and suppressed LPS-induced release of proinflammatory cytokines and nitric oxide metabolites. Melanocortin 2 receptor-deficient mice (MC2R-/-) failed to increase plasma levels of glucocorticoids in response to LPS injection, and exhibited high sensitivity to LPS-induced lethality with enhanced release of proinflammatory cytokines as compared with MC2R+/- mice. Real-time PCR analysis revealed that RS induced upregulation of uncoupling protein-2 (UCP2) in macrophages in the lung and the liver of MC2R+/-, but not of MC2R-/-, mice. In addition, RS increased UCP2-dependent uncoupling activity of isolated mitochondria from the liver of MC2R+/-, but not of MC2R-/-, mice. In vitro study revealed that corticosterone and dexamethasone directly increased UCP2 expression in mouse RAW 264.7 macrophages and suppressed the generation of LPS-induced mitochondrial reactive oxygen species (ROS) and TNF-α production. Knockdown of UCP2 by small interfering RNA blunted the dexamethasone action for suppressing LPS-induced mitochondrial ROS and TNF-α production. Conclusion: The present work suggests that RS enhances activation of the HPA axis to release glucocorticoids and upregulation of UCP2 in macrophages, thereby increasing the resistance to endotoxin-induced systemic inflammation and death.

To estimate the potent immunomodulating effects of different types of traditional Japanese millet, we analyzed the effect of bran extracts of foxtail millet (Awa in Japanese), barnyard millet (Hie) and proso millet (Mochi-kibi) on nitric oxide (NO) and inflammatory cytokine production induced by lipopolysaccharide (LPS) in a mouse macrophage cell line (RAW264.7 cells). All methanol extracts of these millet brans showed suppressive activities against the production of NO and inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-6 in LPS-stimulated macrophages, which were not responsible for their cytotoxic activities. These immunosuppressive activities were roughly proportional to the contents of the phenolic compounds in their extracts. Especially, the extract of proso millet exhibited the strongest immunosuppressing effect, which was associated with the highest content of phenolic compound. However, the extracts did not exhibit significant suppressive effects on the production of an anti-inflammatory cytokine, IL-10, in the same macrophage culture system. These results suggest that traditional Japanese millets have potent immunomodulating activities against the production of NO and cytokine production in activated macrophages.

To elucidate the possible involvement of nitric oxide (NO) derived from inducible NO synthase (iNOS) in the pathogenesis of patients with allergic rhinitis, we used an animal model of atopic dermatitis (AD) induced by epicutaneous sensitization and analysed the differences in ear thickness, the frequency of scratching and plasma levels of ovalbumin-specific immunoglobulin E (OVA-IgE), transforming growth factor (TGF)-β, tumor necrosis factor (TNF)-α, adrenocorticotropic hormone (ACTH) and α-melanocyte-stimulating hormone (α-MSH) between control and iNOS(-/-) mice. Eight-week-old control and iNOS(-/-) male C57BL/6j mice were sensitized three times with OVA antigen. Before and after the last skin sensitization, the number of scratching incidents and the thickness of the ear were examined, and the plasma levels of OVA-IgE, α-MSH, ACTH, TGF-β and TNF-α were analysed by ELISA. Sensitization of mice with OVA resulted in increased plasma levels of OVA-IgE, α-MSH, ACTH, TGF-β and TNF-α in control, but not in iNOS(-/-) mice. The administration of l-nitro-arginine-methyl ester (l-NAME) abolished all the above changes that occurred in the control mice. In addition, iNOS(-/-) mice given α-MSH exhibited a change similar to that seen in the control, whereas iNOS(-/-) mice given ACTH, TGF-β or TNF-α did not demonstrate any changes. These results indicate that symptoms of AD such as scratching can be exacerbated by α-MSH, which is induced by iNOS-derived NO.

Psychological stress is an environmental factor considered to be a precipitating factor of inflammatory bowel disease. Interleukin (IL)-18 plays a role in stress-induced aggravation in some diseases. The aim of this study was to establish a model of murine colitis exacerbated by psychological stress and to clarify the role of IL-18 in this model. Male C57Bl/6 mice and IL-18−/−mice were used for this study. The mice received dextran sulfate sodium (DSS) for induction of colitis. Some mice were exposed to psychological stress using a communication box. Body weight, colonic length, and histological inflammation were measured for assessment of colitis. Tumor necrosis factor (TNF)-α and IL-18 expression in the colon and IL-18 expression in the adrenal gland were analyzed using real-time PCR. The effect of anti-IL-18 antibody was also investigated. Effects of TNF-α and IL-18 on cytokine expressions were studied using the colonic epithelial cell line LS174T. Induction of psychological stress in DSS-treated wild-type mice significantly exacerbated colitis with enhanced expression of proinflammatory cytokines and IL-18. However, induction of psychological stress in DSS-treated IL-18−/−mice did not aggravate colitis compared with that in the IL-18−/−group given only DSS treatment. Stress-induced aggravation of colitis was ameliorated significantly by anti-IL-18 antibody treatment. IL-18 did not enhance TNF-α-induced expression of intercellular adhesion molecule-1 or IL-8 in LS174T. We established a model of colitis exacerbated by psychological stress. Psychological stress enhanced IL-18 expression and plays a proinflammatory role in stress-induced aggravation of colitis.

We investigated the role of mitochondrial reactive oxygen species (ROS) in the response of macrophages to lipopolysaccharide (LPS) using RAW 264.7 cells and their ρ(o) cells lacking mitochondria. Mitochondrial density, respiratory activity and related proteins in ρ(o) cells were significantly lower than those in RAW cells. LPS rapidly stimulated mitochondrial ROS prior to cytokine secretion, such as TNF-α and IL-6, from RAW 264.7 cells by activating the MAPK pathway, while the response was attenuated in ρ(o) cells. Exposure of ρ(o) cells to H(2)O(2) partially restored the secretion of cytokines induced by LPS. These results suggest that mitochondrial density and/or the respiratory state contribute to intracellular oxidative stress, which is responsible for the stimulation of LPS-induced MAPK signaling to enhance cytokine release from macrophages.

The gastrointestinal epithelium functions as physical and innate immune barriers against commensal or pathogenic microbes. NADPH oxidase 1 (Nox1) and dual oxidase 2 (Duox2), highly expressed in the colon, are suggested to play a potential role in host defense. Guinea-pig gastric pit cells and human colonic epithelial cells (T84 cells) express Nox1. With regard to activation of Nox1, the gastric epithelial cells are primed with Helicobacter pylori lipopolysaccharide, whereas T84 cells preferentially use the Toll-like receptor (TLR) 5, rather than TLR4, against Salmonella enteritidis infection. Thus, gastric and colonic epithelial cells may use different TLR members to discern pathogenicities among bacteria, depending on their environments and to activate Nox1 appropriately for host defense. Nox1-derived reactive oxygen species (ROS) have been implicated in the pathogenesis of inflammation-associated tumor development. The human stomach does not express Nox1. Helicobacter pylori infection alone does not induce it, whereas Nox1 is specifically expressed in gastric adenocarcinomas. In the human colon, Nox1 is differentiation-dependently expressed, and its expression is upregulated in adenomas and well-differentiated adenocarcinomas. Although Nox1 expression may not be directly linked to mitogenic activity, Nox1-derived ROS may exert a cancer-promoting effect by increasing resistance to programmed cell death of tumor cells.

Treatment of Nylon wool-passed cells (NWC) prepared from the spleen of C57BL/6 mice with IL-18 and IL-12, but not with IL-18 alone, resulted in induction of IFN-gamma, a Th1 cytokine, and GM-CSF at 24 h, and IL-13, a Th2 cytokine at 72 h. The induction of IL-13 was suppressed by anti-GM-CSF antibody, indicating involvement of GM-CSF in IL-13 production. When NWC incubated with IL-18 and IL-12 for 72 h ("primary treatment") were treated again with the same cytokines ("secondary treatment"), IL-13 was induced much more quickly than observed in the primary treatment. Flow cytometric analysis of NWC after the primary treatment showed marked increases in the CD4(-)CD8(-) non-T cell population bearing CD25(+), CD45RB(super high) and CD122(+). These cells were positive for CD49b but negative for NK1.1, indicating that they were not typical but NK-like cells. The NK-like cells produced IL-13 in response to the treatment with IL-18 alone, indicating that the generation of these cells in the primary treatment likely accounts for the quick production of IL-13 in the secondary treatment. These results show that IL-18 and IL-12 generates the NK-like cells in NWC by a process mediated by GM-CSF that are ready for producing IL-13.