Yoshinori Seko

A critical link between Toll-like receptor 3 and type II interferon signaling pathways in antiviral innate immunity

A conundrum of innate antiviral immunity is how nucleic acid-sensing Toll-like receptors (TLRs) and RIG-I/MDA5 receptors cooperate during virus infection. The conventional wisdom has been that the activation of these receptor pathways evokes type I IFN (IFN) responses. Here, we provide evidence for a critical role of a Toll-like receptor 3 (TLR3)-dependent type II IFN signaling pathway in antiviral innate immune response against Coxsackievirus group B serotype 3 (CVB3), a member of the positive-stranded RNA virus family picornaviridae and most prevalent virus associated with chronic dilated cardiomyopathy. TLR3-deficient mice show a vulnerability to CVB3, accompanied by acute myocarditis, whereas transgenic expression of TLR3 endows even type I IFN signal-deficient mice resistance to CVB3 and other types of viruses, provided that type II IFN signaling remains intact. Taken together, our results indicate a critical cooperation of the RIG-I/MDA5-type I IFN and the TLR3-type II IFN signaling axes for efficient innate antiviral immune responses.

Hypoxia and Hypoxia/Reoxygenation Activate Raf-1, Mitogen-Activated Protein Kinase Kinase, Mitogen-Activated Protein Kinases, and S6 Kinase in Cultured Rat Cardiac Myocytes

In response to hypoxia and reoxygenation, mammalian cells are known to express a variety of genes to adapt to these external stresses or lead to further cell damage. We investigated the intracellular signaling cascades in cultured rat cardiac myocytes subjected to hypoxia followed by reoxygenation (hypoxia/reoxygenation). Here, we show that both hypoxia and hypoxia/reoxygenation caused rapid activation of the mitogen-activated protein kinase kinase kinase (MAPKKK), activity of Raf-1. This was followed by the sequential activation of mitogen-activated protein kinase kinase (MAPKK), mitogen-activated protein (MAP) kinases, and S6 kinase (p90rsk). Furthermore, hypoxia caused hyperphosphorylation of Raf-1. The maximal hyperphosphorylation of Raf-1 appeared to be accompanied by a significant decrease in MAPKKK activity. These results strongly suggest the following: (1) Intracellular signals initiated by both hypoxia and hypoxia/reoxygenation converge on Raf-1 and activate its MAPKKK activity. Then, Raf1 activates downstream serine/threonine kinases including MAPKK, MAP kinases and p90rsk. (2) Raf-1 is not only located upstream from MAPKK and MAP kinases but also may be phosphorylated by MAP kinases directly or indirectly, and at least Raf-1 kinase activity may be downregulated by this feedback mechanism.

Restricted Usage of T-Cell Receptor Vα-Vβ Genes in Infiltrating Cells in Aortic Tissue of Patients With Takayasu’s Arteritis

Background Infiltration by perforin-secreting killer lymphocytes, such as T cells and natural killer cells, has been shown to be involved in the pathogenesis of vascular cell damage in Takayasu’s arteritis. Methods and Results To investigate the immunological mechanisms involved, especially the nature of T-cell infiltration in Takayasu’s arteritis as well as atherosclerosis, we analyzed the expression of T-cell receptor (TCR) Vα and Vβ genes in infiltrating cells in the aortic tissue of patients with Takayasu’s arteritis and atherosclerotic aortic aneurysm by polymerase chain reaction (PCR). We also analyzed the expression of cytokine genes by PCR. We found that the repertoires of TCR Vα as well as Vβ gene transcripts in Takayasu’s arteritis were restricted. The infiltrating cells expressing Vα2, Vα16, Vα17, Vβ7, and Vβ13.1 were found in 3 of 4 patients. In contrast, TCR Vα-Vβ repertoires in atherosclerotic aortic aneurysm were polyclonal. There was no significant difference in the pattern of cytokine gene expression between the two diseases. Conclusions The restricted usage of TCR Vα as well as Vβ genes by infiltrating T cells in Takayasu’s arteritis may indicate that a specific antigen in the aortic tissue was targeted. Our findings provide the evidence that distinct immunological mechanisms are involved in the pathogenesis of Takayasu’s arteritis and atherosclerotic aortic aneurysm.

Vascular Endothelial Growth Factor Induces Activation and Subcellular Translocation of Focal Adhesion Kinase (p125FAK) in Cultured Rat Cardiac Myocytes

Vascular endothelial growth factor (VEGF) has been proposed to be among the candidate factors with the most potential to play a role in ischemia-induced collateral vessel formation. Recently, we found that VEGF activated the mitogen-activated protein kinase cascade in cultured rat cardiac myocytes. To elucidate how VEGF affects adhesive interaction of cardiac myocytes with the extracellular matrix (ECM), one of the important cell functions, we investigated the molecular mechanism of activation of focal adhesion-related proteins, especially focal adhesion kinase (p125(FAK)), in cultured rat cardiac myocytes. We found that the 2 VEGF receptors, KDR/Flk-1 and Flt-1, were expressed in cardiac myocytes and that KDR/Flk-1 was significantly tyrosine phosphorylated on VEGF stimulation. VEGF induced tyrosine phosphorylation and activation of p125(FAK) as well as tyrosine phosphorylation of paxillin; this was accompanied by subcellular translocation of p125(FAK) from perinuclear sites to the focal adhesions. This VEGF-induced activation of p125(FAK) was inhibited partially by the tyrosine kinase inhibitors genistein and tyrphostin. Activation of p125(FAK) was accompanied by its increased association with adapter proteins GRB2, Shc, and nonreceptor type tyrosine kinase p60(c-src). Furthermore, we confirmed that VEGF induced a significant increase in adhesive interaction between cardiac myocytes and ECM using an electric cell-substrate impedance sensor. These results strongly suggest that p125(FAK) is one of the most important components in VEGF-induced signaling in cardiac myocytes, playing a critical role in adhesive interaction between cardiac myocytes and ECM.

Expression of cytokine mRNAs in murine hearts with acute myocarditis caused by coxsackievirus B3

In murine acute viral myocarditis, natural killer (NK) cells infiltrate the heart first, followed by activated T‐cells, which play an important role in the pathogenesis of the myocardial damage. Because of their multipotential effects, cytokines are thought to play a role in the induction and development of these immune processes. To clarify in more detail the precise mechanism of the cytokine networks involved, the expression of various cytokine mRNAs has been investigated in myocardial cells infected with Coxsackievirus B3 (CVB3) in vivo and in vitro by a semiquantitative polymerase chain reaction (PCR) method. Interleukin (IL)‐1α, IL‐1β, IL‐6, tumour necrosis factor (TNF)‐α, and TNF‐β were expressed almost throughout the early phase of virus infection with some variations. IL‐2, IL‐3, IL‐4, IL‐10, interferon (IFN)‐γ, granulocyte/macrophage colony stimulating factor (GM‐CSF), and IL‐2 receptor (IL‐2R) were mainly expressed by the infiltrating cells. TNF‐α, TNF‐β, and IL‐1β were also expressed partly by the infiltrating cells. T‐helper (Th)1‐related cytokines (IL‐2, IFN‐γ, and TNF‐β) were more strongly expressed than Th2‐related cytokines (IL‐4 and IL‐10) in vivo, indicating that the Th cells which infiltrated the heart and mediated the immune responses in the early phase of acute myocarditis were mainly of Th1‐type.

Expression of perforin in infiltrating cells in murine hearts with acute myocarditis caused by coxsackievirus B3.

BackgroundCell-mediated autoimmunity has been strongly implicated in the pathogenesis of viral myocarditis. Methods and ResultsUsing a murine model of acute myocarditis caused by coxsackievirus B3, we analyzed the phenotypes and morphology of the infiltrating cells in the hearts by immunofluorescence and electron microscopy. We also examined the expression of a cytolytic factor, perforin, in the infiltrating cells by immunoperoxidase and in situ hybridization. We found that the dominant population of the infiltrating cells were asialo GM1 positive, were negative for T-cell markers, and had electron-dense cytoplasmic granules, which is consistent with a morphology of large granular lymphocytes. Perforin was found in the cytoplasmic granules of the infiltrating cells expressing perforin messenger RNA. These findings provide for the first time the direct evidence that the first wave of cell infiltration in hearts mainly consists of killer cells and strongly suggests that perforin plays, in part, an important role in myocardial cell damage involved in acute viral myocarditis. T-helper cells and cytotoxic T lymphocytes made up the second wave of cell infiltration. ConclusionsAs we previously reported, the expression of major histocompatibility complex class I antigen on cardiac myocytes induced by the infiltrating cells, such as killer cells, may facilitate the interaction between cardiac myocytes and cytotoxic T lymphocytes, and may lead to further myocardial cell damage in a later phase.

Expression of tumour necrosis factor (TNF) ligand superfamily co-stimulatory molecules CD30L, CD27L, OX40L, and 4-1BBL in murine hearts with acute myocarditis caused by Coxsackievirus B3.

Antigen‐specific T‐cells infiltrate the heart and play an important role in the myocardial damage involved in acute myocarditis and dilated cardiomyopathy. To investigate the roles of the co‐stimulatory molecules CD30/CD30L, CD27/CD27L, OX40/OX40L, and 4‐1BB/4‐1BBL, which belong to the tumor necrosis factor (TNF) receptor/ligand superfamily, in the development of acute viral myocarditis, the expression of these molecules was first analysed in the hearts of mice with acute myocarditis induced by Coxsackievirus B3 (CVB3) in vivo. Secondly, the induction of these molecules was evaluated on cultured cardiac myocytes treated with interferon (IFN)‐γ and the interleukin (IL)‐6 production by cultured cardiac myocytes was analysed by stimulation with monoclonal antibodies (MAbs) against these molecules in vitro. Thirdly, the effects of in vivo administration of anti‐CD30L, anti‐CD27L, anti‐OX40L, or anti‐4‐1BBL MAb on the development of acute viral myocarditis were examined. CVB3‐induced myocarditis resulted in the induction of CD30L and 4‐1BBL on the surface of cardiac myocytes, confirmed by treatment with IFN‐γ in vitro. CD27L and OX40L were constitutively expressed on cardiac myocytes in vivo and in vitro. Anti‐CD30L and anti‐4‐1BBL MAbs stimulated IL‐6 production by cardiac myocytes in vitro. Furthermore, in vivo anti‐4‐1BBL MAb treatment significantly decreased the myocardial inflammation, whereas the other MAbs did not. These findings suggest that TNF ligand superfamily co‐stimulatory molecules, especially 4‐1BBL, play an important role in the development of acute viral myocarditis and raise the possibility that immunotherapy with anti‐4‐1BBL MAb may be of benefit in acute viral myocarditis. Copyright

Treatment of Focal Cerebral Ischemia With Synthetic Oligopeptide Corresponding to Lectin Domain of Selectin

BACKGROUND AND PURPOSE Synthetic oligopeptides with amino acid sequences of the lectin domain of selectin block selectin-mediated cell adhesion in vitro, which may be applied to a therapeutic intervention to attenuate acute inflammatory reactions. To evaluate the efficacy of such treatment against ischemic brain injury, the effects of administering a selectin oligopeptide that selectively blocks selectin-mediated cell adhesion on histological outcome and on cerebral blood flow (CBF) were studied in models of rodent focal cerebral ischemia. METHODS Spontaneously hypertensive rats were anesthetized with halothane. Permanent focal cerebral ischemia was induced by tandem left middle cerebral artery (MCA) and common carotid artery (CCA) occlusion. Focal cerebral ischemia with partial reperfusion was introduced by reperfusing the CCA after 2 hours of tandem MCA/CCA occlusion. A synthetic oligopeptide (amino acid residues 23-30 from N terminal) of E-selectin was dissolved in physiological saline and was injected intravenously at a dosage of 2 mg/kg or 10 mg/kg before artery occlusion. Control animals received equivalent volumes of physiological saline or 10 mg/kg of synthetic oligopeptide with a scrambled amino acid sequence. Twenty-four hours after the occlusion, seven coronal brain slices were stained with 2,3,5-triphenyltetrazolium chloride, and the volume of ischemic injury was calculated. In a separate set of animals, regional CBF was monitored with laser-Doppler flowmetry at the dorsolateral cerebral cortex during 2-hour ischemia and 30 minutes after partial reperfusion. RESULTS The volume of ischemic injury did not differ among groups in permanent ischemia. In ischemia with partial reperfusion, 10 mg/kg selectin oligopeptide, but not the same dosage of scrambled oligopeptide, significantly reduced the volume of ischemic injury (95 +/- 13, 73 +/- 11, 55 +/- 6, and 111 +/- 14 mm3 for saline [n = 11]; 2 mg/kg [n = 10] and 10 mg/kg [n = 16] selectin oligopeptide and 10 mg/kg scrambled oligopeptide [n = 6], respectively; P < .01 by one-way ANOVA followed by the Tukey test). Laser-Doppler flowmetry demonstrated a larger increase in CBF after reperfusion of the CCA in the 10-mg/kg selectin oligopeptide group. CONCLUSIONS Our data demonstrate that administration of a synthetic oligopeptide corresponding to the lectin domain of selectin decreases the size of ischemic injury after transient, but not after permanent, focal cerebral ischemia as evaluated at 24 hours after onset of ischemia. These effects were associated with an improved CBF at the dorsolateral cerebral cortex after partial reperfusion.

Expression of major histocompatibility complex class I antigen in murine ventricular myocytes infected with Coxsackievirus B3.

Evidence has accumulated that T cell-mediated autoimmunity plays an important role in the pathogenesis of viral myocarditis. T lymphocytes are known to recognize antigen-presenting cells, such as virus-infected cells, being restricted by syngeneic major histocompatibility complex (MHC) antigens. To clarify in more detail the immunological mechanisms involved, we induced acute viral myocarditis in C3H/He mouse ventricles with coxsackievirus B3 (CVB3) and examined, by immunofluorescence, the expression of MHC class I and II antigens, previously reported not to be expressed by normal cardiac myocytes. Furthermore, to confirm the expression of MHC class I (H-2Kk) antigens at the cellular level, we treated cultured cardiac myocytes with interferon gamma and examined the antigen expression by immunofluorescence and Northern blot hybridization, using an antisense RNA probe for MHC messenger RNA. Our observations demonstrated 1) CVB3-induced myocarditis resulted in the enhanced expression of MHC class I (H-2Kk) gene product on the surface of cardiac myocytes but low or undetectable levels of MHC class II or H-2Dk gene products, and moderate focal transient (days 5-7) expression of both MHC class I (Kk + Dk) region gene products and MHC class II antigens were induced on capillary endothelial cells; 2) murine fetal cardiac myocytes cultured in vitro in the presence of interferon gamma similarly were shown to express marked levels of MHC class I (H-2Kk) but low to undetectable levels of the H-2Dk gene product; however, weak to moderate MHC Class II antigens were expressed by these cultured myocytes; and 3) the expression of MHC antigens in cardiac myocytes was modulated at the transcriptional level.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of Costimulatory Molecules (4-1BBL and Fas) and Major Histocompatibility Class I Chain-Related A (MICA) in Aortic Tissue with Takayasu’s Arteritis

To further investigate the immunological mechanisms involved, we analyzed the expression of costimulatory molecules in aortic tissue and their counterpart molecules on infiltrating cells of patients with Takayasu’s arteritis. We also examined the expression of major histocompatibility complex (MHC) class I chain-related (MIC) A in aortic tissue, which is known to be induced by external stress, and its counterpart NKG2D receptors on infiltrating cells. Among these costimulatory molecules, strong expression of 4-1BBL and Fas was induced in the aortic tissue, and most of the infiltrating cells expressed 4-1BB and FasL, suggesting these pathways play critical roles in T-cell-mediated vascular injury. We also found that MICA was strongly induced in the aortic tissue and that at least part of the infiltrating cells expressed NKG2D receptors. Some infiltrating cells – but not vascular smooth muscle cells – seemed to have undergone apoptosis. Our findings strongly suggest that 4-1BB/4-1BBL and Fas/FasL pathways play important roles in vascular injury in Takayasu’s arteritis. We assume that γδ T cells infiltrated aortic tissue recognizing MICA, resulting in the induction of MHC antigens and costimulatory molecules, and then αβ T-cells infiltrated recognizing some auto-antigens presented by MHC antigens, leading to chronic inflammation.