Hisayasu Wada

Lack of Interleukin-1β Decreases the Severity of Atherosclerosis in ApoE-Deficient Mice

Objective—Atherosclerosis is considered to be a chronic inflammatory disease and many cytokines participate in the development of atherosclerosis. We focused on the role of interleukin-1&bgr; (IL-1&bgr;), one of the proinflammatory cytokines secreted by monocytes/macrophages, in the progression of atherosclerosis. Methods and Results—We generated mice lacking both apoE and IL-1&bgr;. The sizes of atherosclerotic lesions at the aortic sinus in apoE−/−/IL-1&bgr;−/−mice at 12 and 24 weeks of age showed a significant decrease of approximately 30% compared with apoE−/−/IL-1&bgr;+/+ mice, and the percentage of the atherosclerotic area to total area of apoE−/−/IL-1&bgr;−/− at 24 weeks of age also showed a significant decrease of about 30% compared with apoE−/−/IL-1&bgr;+/+. The mRNA levels of vascular cell adhesion molecule (VCAM)-1 and monocyte chemotactic protein-1 in the apoE−/−/IL-1&bgr;−/− aorta were significantly reduced compared with the apoE−/−/IL-1&bgr;+/+. Furthermore, VCAM-1 was also reduced at the protein level in apoE−/−/IL-1&bgr;−/− aorta compared with apoE−/−/IL-1&bgr;+/+. Conclusions—The lack of IL-1&bgr; decreases the severity of atherosclerosis in apoE deficient mice, possibly through increased expressions of VCAM-1 and monocyte chemotactic protein-1 in the aorta.

Lipopolysaccharide induction of indoleamine 2,3‐dioxygenase is mediated dominantly by an IFN‐γ‐independent mechanism

Indoleamine 2,3‐dioxygenase (IDO) is a rate‐limiting enzyme in the L‐tryptophan‐kynurenine pathway, which converts an essential amino acid, L‐tryptophan, to N‐formylkynurenine. It has been speculated that IFN‐γ is a dominant IDO inducer in vivo. The present study used IFN‐γ or TNF‐α gene‐disrupted mice and IFN‐γ antibody‐treated mice to demonstrate that lipopolysaccharide (LPS)‐induced systemic IDO is largely dependent on TNF‐α rather than IFN‐γ. IFN‐γ‐independent IDO induction was also demonstrated in vitro with LPS‐stimulated monocytic THP‐1 cells. These findings clearly indicate that there is an IFN‐γ‐independent mechanism of IDO induction in addition to the IFN‐γ‐dependent mechanism.

Improved myocardial ischemia/reperfusion injury in mice lacking Tumor necrosis factor-α

OBJECTIVES This study sought to assess the role of tumor necrosis factor-alpha (TNF-alpha) in myocardial ischemia/reperfusion (I/R) injury using TNF-alpha knockout (KO) mice. BACKGROUND Tumor necrosis factor-alpha is thought to be involved in the pathogenesis of myocardial I/R injury by promoting leukocyte infiltration of the myocardium. However, the precise role of TNF-alpha in I/R injury is still unknown. METHODS The hearts in TNF-alpha KO and wild-type (WT) mice were exposed by left lateral thoracotomy, and the left coronary artery was occluded for 30 min then reperfused for 120 min. RESULTS The infarct size in TNF-alpha KO mice was significantly reduced compared with WT mice. The frequency of arrhythmia was decreased, and cardiac function during reperfusion was significantly improved in TNF-alpha KO mice compared with WT mice. The activation of nuclear factor-kappaB (NF-kappaB), the expression of chemokines and adhesion molecules and the infiltration of leukocytes were also significantly reduced in TNF-alpha KO mice, compared with WT mice. These findings provide evidence that TNF-alpha aggravates I/R injury. CONCLUSIONS Tumor necrosis factor-alpha exacerbates myocardial I/R injury at an early stage of reperfusion by activating NF-kappaB, thereby inducing chemokines and adhesion molecules and facilitating leukocyte infiltration.

L-tryptophan-L-kynurenine pathway metabolism accelerated by Toxoplasma gondii infection is abolished in gamma interferon-gene-deficient mice: cross-regulation between inducible nitric oxide synthase and indoleamine-2,3-dioxygenase.

ABSTRACT l-Tryptophan degradation by indoleamine 2,3-dioxygenase (IDO) might have an important role in gamma interferon (IFN-γ)-induced antimicrobial effects. In the present study, the effects of Toxoplasmagondii infection on IDO were investigated by using wild-type and IFN-γ-gene-deficient (knockout) (IFN-γ KO) mice. In wild-type C57BL/6J mice, enzyme activities and mRNA levels for IDO in both lungs and brain were markedly increased and lung l-tryptophan concentrations were dramatically decreased following T. gondii infection. In contrast, these metabolic changes did not occur in T. gondii-infected IFN-γ KO mice or in uninfected IFN-γ KO mice. The levels of inducible nitric oxide synthase (iNOS) induction in infected IFN-γ KO mice were high in lungs and low in brain compared to those in infected wild-type mice. The extent of increased mRNA expression of T. gondii surface antigen gene 2 (SAG2) induced in lungs and brain by T. gondii infection was significantly enhanced in IFN-γ KO mice compared to wild-type mice on day 7 postinfection. Treatment with N-nitro-l-arginine methyl ester, an iNOS inhibitor, increased the levels of SAG2 mRNA in brain but not in lungs and of plasma l-kynurenine after T. gondii infection. This in vivo study provides evidence that l-tryptophan depletion caused by T. gondii is directly mediated by IFN-γ in the lungs, where iNOS is not induced by IFN-γ. This study suggests that there is an antitoxoplasma mechanism of cross-regulation between iNOS and IDO and that the expression of the main antiparasite effector mechanisms for iNOS and/or IDO may vary among tissues.

Tumor Necrosis Factor-α (TNF-α) Plays a Protective Role in Acute Viral Myocarditis in Mice A Study Using Mice Lacking TNF-α

BACKGROUND It has been reported that tumor necrosis factor-alpha (TNF-alpha) is expressed in the heart with viral myocarditis and that its expression aggravates the condition. The pathophysiological effects of TNF-alpha on viral myocarditis, however, have not been fully elucidated. METHODS AND RESULTS To investigate the role of TNF-alpha in the progression of viral myocarditis, we used TNF-alpha gene-deficient mice (TNF-alpha(-/-)) and induced acute myocarditis by infection with encephalomyocarditis virus (EMCV). The survival rate of TNF-alpha(-/-) mice after EMCV infection was significantly lower than that of TNF-alpha(+/+) mice (0% versus 67% on day 14). Injection of recombinant human TNF-alpha (0.2 to 4.0 microg/mouse IV) improved the survival of TNF-alpha(-/-) mice in a dose-dependent manner, indicating that TNF-alpha is essential for protection against viral myocarditis. The levels of viral titer and viral genomic RNA of EMCV in the myocardium were significantly higher in TNF-alpha(-/-) than in TNF-alpha(+/+) mice. Histopathological examination showed that the inflammatory changes of the myocardium were less marked in TNF-alpha(-/-) than in TNF-alpha(+/+) mice. Immunohistochemical analysis revealed that the levels of immunoreactivity of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in the myocardium were decreased in TNF-alpha(-/-) mice compared with TNF-alpha(+/+) mice. CONCLUSIONS These observations suggested that TNF-alpha is necessary for adhesion molecule expression and to recruit leukocytes to inflammatory sites, and thus, the lack of this cytokine resulted in failure of elimination of infectious agents. We concluded that TNF-alpha plays a protective role in the acute stage of viral myocarditis.Background—It has been reported that tumor necrosis factor-α (TNF-α) is expressed in the heart with viral myocarditis and that its expression aggravates the condition. The pathophysiological effects of TNF-α on viral myocarditis, however, have not been fully elucidated. Methods and Results—To investigate the role of TNF-α in the progression of viral myocarditis, we used TNF-α gene–deficient mice (TNF-α−/−) and induced acute myocarditis by infection with encephalomyocarditis virus (EMCV). The survival rate of TNF-α−/− mice after EMCV infection was significantly lower than that of TNF-α+/+ mice (0% versus 67% on day 14). Injection of recombinant human TNF-α (0.2 to 4.0 μg/mouse IV) improved the survival of TNF-α−/− mice in a dose-dependent manner, indicating that TNF-α is essential for protection against viral myocarditis. The levels of viral titer and viral genomic RNA of EMCV in the myocardium were significantly higher in TNF-α−/− than in TNF-α+/+ mice. Histopathological examination showed that the inflam...

3-Hydroxyanthranilic acid, an L-tryptophan metabolite, induces apoptosis in monocyte-derived cells stimulated by interferon-γ

3-Hydroxyanthranilic acid (3-HAA), a metabolite of L-tryptophan, accumulates in monocyte-derived cells (THP-1),but not in other celllines tested(MRC9, H4, U373MG, Wil-NS), following immune stimulation that induces indoleamine 2,3-dioxygenase (IDO), a rate-limiting enzyme in the L-tryptophan-kynurenine pathway. We examined whether metabolites of the L-tryptophan-kynurenine pathway act to induce apoptosis in monocytes/macrophages. Of the L-tryptophan metabolites tested, only 3-HAA at a concentration of 200µmol/L was found to induce apoptosis in THP-1 and U937 cells. The addition of ferrous or manganese ions further enhanced apoptosis and free radical formation by 3-HAA in these two types of cells. The apoptotic response induced by 3-HAA was significantly attenuated by the addition of antioxidant, α-tocopherol or Trolox (a water-soluble analogue of vitamin E), and the xanthine oxidase inhibitor, allopurinol. In addition, the 3-HAA-induced apoptotic response was slightly attenuated by catalase, but not by superoxide dismutase (SOD), indicating that generation of hydrogen peroxide is involved in this response. Interferon-γ (IFN-γ), an inducer of IDO, potently induced apoptosis in THP-1 cells, but not in U937 cells, in the presence of ferrous or manganese ions. This different susceptibility to apoptosis inducer between THP-1 and U937 cells may depend on the capacity of the cells for 3-HAA synthesis following IDO induction by IFN-γ. Furthermore, apoptosis was suppressed by cycloheximide in THP-1 cells, suggesting that newly synthesized proteins may be essential for apoptotic events. These results suggest that 3-HAA induces apoptosis in monocytes/macrophages under inflammatory or other pathophysiological conditions.

Increases in tumor necrosis factor-α following transient global cerebral ischemia do not contribute to neuron death in mouse hippocampus

The actions of tumor necrosis factor‐α (TNF‐α) produced by resident brain cells and bone marrow‐derived cells in brain following a transient global ischemia were evaluated. In wild‐type mice (C57Bl/6J) following 20 min ischemia with bilateral common carotid artery occlusion (BCCAo), TNF‐α mRNA expression levels in the hippocampus were significantly increased at 3 h and 36 h and exhibited a biphasic expression pattern. There were no hippocampal TNF‐α mRNA expression levels at early time points in either wild‐type mice bone marrow transplanted (BMT)‐chimeric‐TNF‐α gene‐deficient (T/W) or TNF‐α gene‐deficient mice BMT‐TNF‐α gene‐deficient mice (T/T), although TNF‐α mRNA levels were detectable in T/W BMT mice at 36 h. Histopathological findings showed no intergroup differences between wild‐type and TNF‐α gene‐deficient mice at 4 and 7 days after transient ischemia. In addition, nuclear factor‐κB (NF‐κB) was activated within 12 h after global cerebral ischemia, but electrophoretic mobility shift assays (EMSA) showed no intergroup differences between wild type and TNF‐α gene‐deficient mice. In summary, early hippocampal TNF‐α mRNA expression may not be related to bone marrow‐derived cells, and secondary TNF‐α expression as early as 36 h after ischemia probably resulted mainly from endogenous brain cells and possibly a few bone marrow‐derived cells. Although we cannot exclude the possibility of the TNF‐α contribution to the physiologic changes of hippocampus after transient global ischemia, these results indicate that TNF‐α does not influence the morphological changes of the hippocampal neurons under our study condition.

Suppression of neurocognitive damage in LP-BM5-infected mice with a targeted deletion of the TNF-α gene

Brain levels of TNF‐α increase in many inflammatory conditions, including HIV‐1 infection, and may contribute to neurodegenerative processes. The paucity of agents that can selectively and potently block TNF‐α processing or its receptors has led us to investigate the role of TNF‐α in chronic neurodegeneration associated with retroviral infection using mice with targeted deletions of the TNF‐α gene. Infection of wild‐type C57BL/6 mice with the LP‐BM5 murine leukemia retrovirus mixture leads to the development of a severe immunodeficiency as well as cognitive deficits and neuronal damage. TNF‐α‐(‐/‐) mice infected with LP‐BM5 developed a systemic immunopathology indistinguishable in severity from that observed in contemporaneously infected wild‐type mice. In contrast, the performance of infected TNF‐α‐(‐/‐) mice in the γ‐maze and Morris water maze was not different from that of uninfected TNF‐α‐(‐/‐) mice. The extent of glial activation in the striatum, as indicated by the increase in density of peripheral benzodiazepine receptors, was equivalent in both groups of LP‐BM5‐infected mice. However, the decrease in striatal MAP‐2 expression, a marker of neurodegeneration observed in infected wild‐type mice, was not found in infected TNF‐α‐(‐/‐) mice. While the loss of TNF‐α appeared to have no effect on the course or severity of the central or peripheral immunopathology resulting from LP‐BM5 infection, the behavioral and biochemical manifestations were substantially curtailed in the TNF‐α‐(‐/‐) mice. These findings directly support a role for TNF‐α in the neurodegenerative processes associated with viral infections such as HIV‐1.—Iida, R., Saito, K., Yamada, K., Basile, A. S., Sekikawa, K., Takemura, M., Fujii, Wada, H. H., Seishima, M., Nabeshima, T. Suppression of neurocognitive damage in LP‐BM5‐infected mice with a targeted deletion of the TNF‐α gene. FASEBJ. 14, 1023–1031 (2000)

L-Tryptophan-Kynurenine Pathway Metabolite 3-Hydroxyanthranilic Acid Induces Apoptosis in Macrophage-Derived Cells Under Pathophysiological Conditions

Accumulation of L-kynurenine and 3-hydroxyanthranilic acid (3HAA) occurs in the monocyte-derived cells following immune stimulation, and may derive from L-tryptophan following induction of indoleamine-2,3-dioxygenase. In the present study, we evaluate the possibility that 3HAA acts as an endogenous inducer of monocyte/macrophage apoptosis. Supplementation with 200 microM of 3HAA, but not other L-tryptophan metabolites tested, significantly increased the number of apoptotic cells in both THP-1 and U937 cells. Catalase, superoxide dismutase and manganese ions markedly enhanced apoptosis in the presence of 3HAA in these cells. The present results suggest that 3HAA induces the macrophage/monocyte apoptosis under certain conditions, which may be relevant to pathophysiology of inflammatory conditions.

Autonomic regulation after exercise evidenced by spectral analysis of heart rate variability in asthmatic children

BACKGROUND Bronchial asthma is associated with abnormal autonomic nervous function in childhood. Exercise is one of the most common precipitating factors of acute asthmatic crises although the exact mechanism of autonomic regulation in asthmatic children after exercise is unclear. OBJECTIVE The aim of this study was to investigate the features of autonomic regulation after exercise in asthmatic and control children. METHODS Pulmonary function tests and heart rate variability spectral analysis were performed in 15 asthmatic children and 7 control children (age 6 to 15 years) during and after an exercise challenge. RESULTS The maximum % fall of forced expiratory volume in 1 second (FEV1) was significantly greater (P < .01) in asthmatic subjects (9.1 +/- 5.1%) than in normal control subjects (1.0 +/- 2.5%). The high frequency band (HF) amplitude, an index of cardiac vagal tone, 5 minutes after exercise was significantly higher (P < .05) in the asthmatic subjects (14.4 +/- 7.9 msec) than in control subjects (5.9 +/- 2.6 msec). Furthermore, the difference in the HF amplitude between the control group and the exercise-induced asthma group was significant both 5 minutes (P < .01) and 10 minutes (P < .05) after challenge. There was a significant correlation (P = .565, P = .0165) between HF amplitude 5 minutes after exercise and the magnitude of the decrease in FEV1. On the other hand, no significant difference was observed in the low frequency band amplitude between the controls and the asthmatic subjects. The ratio of low frequency to high frequency power, which is suggested to correlate with cardiac sympathetic activity, did not differ between the two groups. CONCLUSION These findings suggest that autonomic nervous activities, particularly vagal response after exercise, in asthmatic children is different from that in control children.