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Tsukasa Seya

Publications and source records attributed to Tsukasa Seya.

At least 37 records · Page 2Linked to original sources
PubMed2005

Surface-expressed TLR6 participates in the recognition of diacylated lipopeptide and peptidoglycan in human cells.

Recognition of microbial components by TLR2 requires cooperation with other TLRs. TLR6 has been shown to be required for the recognition of diacylated lipoproteins and lipopeptides derived from mycoplasma and to activate the NF-kappaB signaling cascade in conjunction with TLR2. Human TLR2 is expressed on the cell surface in a variety of cells, including monocytes, neutrophils, and monocyte-derived, immature dendritic cells (iDCs), whereas the expression profile of TLR6 in human cells remains obscure. In this study we produced a function-blocking mAb against human TLR6 and analyzed TLR6 expression in human blood cells and cell lines and its participation in ligand recognition. TLR6 was expressed, although at a lower level than TLR2, on the cell surface in monocytes, monocyte-derived iDCs, and neutrophils, but not on B, T, or NK cells. Confocal microscopic analysis revealed that TLR6 was colocalized with TLR2 at the plasma membrane of monocytes. Importantly, TLR2/6 signaling did not require endosomal maturation, and anti-TLR6 mAb inhibited cytokine production in monocytes and iDCs stimulated with synthetic macrophage-activating lipopeptide-2 or peptidoglycan, indicating that TLR6 recognized diacylated lipopeptide and peptidoglycan at the cell surface. In addition, TLR2 mutants C30S and C36S (Cys(30) and Cys(36) in TLR2 were substituted with Ser), which were expressed intracellularly in HEK293 cells, failed to induce NF-kappaB activation upon macrophage-activating lipopeptide-2 stimulation even in the presence of TLR6. Thus, coexpression of TLR2 and TLR6 at the cell surface is crucial for recognition of diacylated lipopeptide and peptidoglycan and subsequent cellular activation in human cells.

Acylation↗
PubMed2005

Cutting Edge: NF-kappaB-activating kinase-associated protein 1 participates in TLR3/Toll-IL-1 homology domain-containing adapter molecule-1-mediated IFN regulatory factor 3 activation.

TLRs signal the presence of microbial patterns and activate transcription factors. In TLR3 and TLR4, the adapter Toll-IL-1R homology domain-containing adapter molecule (TICAM-1) (also called Toll/IL-1R domain-containing adapter inducing IFN-beta (TRIF)) mediates IFN regulatory factor 3 (IRF3) phosphorylation followed by IFN-beta production. The regulatory subunit TNFR-associated factor family member-associated NF-kappaB activator (TANK) couples with the kinase complex IkappaB kinase-related kinase epsilon/NF-kappaB-activating kinase (NAK) (TANK-binding kinase 1 (TBK1)) that involveTICAM-1-dependent IFN-beta induction. There are several TANK-homologous proteins. We tested whether TICAM-1 binds and coprecipitates with TANK or its family proteins. The results are: 1) the TANK family protein NAK-associated protein 1 (NAP1), but not TANK, coprecipitates withTICAM-1; 2) NAP1 overexpression markedly enhances TBK1-mediated IFN-beta promoter activation; 3) a dominant-negative form, NAP (158-270), suppresses IRF3 activation in response to poly(I:C) or LPS; 4) RNA interference targeting of the NAP1 message results in a failure of poly(I:C)-mediated IRF3 polymerization and IFN-beta production. Thus, NAP1 is the kinase subunit responsible for TLR3/4-mediated IFN-beta induction in the TICAM-1 pathway.

Adaptor Proteins, Signal Transducing↗
PubMed2005

TICAM-1 and TICAM-2: toll-like receptor adapters that participate in induction of type 1 interferons.

Toll-like receptor (TLR) 3 and 4 mediate the expression of many genes, including NF-kappaB- and interferon-regulatory factor (IRF)-3/interferon (IFN)-inducible genes, in macrophages and dendritic cells (DCs) in response to their ligand stimuli, polyI:C and lipopolysaccharide (LPS). Toll-IL-1 receptor homology domain (TIR)-containing adapter molecule 1 (TICAM-1) facilitates expression of IFN-inducible genes via TLR3. Although MyD88 and Mal/TIRAP adapters function downstream of TLR4, they barely induce IFN-beta. In addition, DC maturation as well as IFN-beta induction are largely independent of MyD88 and Mal/TIRAP. TICAM-1 is the functional adapter for both TLR3 and TLR4 that induces type 1 IFN and MyD88-independent DC maturation. In LPS-mediated TLR4 activation, a complex of TICAM-1 and an additional TLR4-binding adapter serves as the adapter. We named this TLR4-TICAM-1-bridging adapter TICAM-2. Our results reveal the details of MyD88-independent pathways which separately recruit the distinct adapters downstream of TLR3 and TLR4 and variations of the TLR output are in part regulated by the two additional adapters in DCs.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Interferon-beta induction through toll-like receptor 3 depends on double-stranded RNA structure.

Type I interferons (IFN-alpha/beta) play an essential role in both innate and adaptive antiviral immune responses. IFN- beta is produced by fibroblasts and myeloid dendritic cells (DCs) upon viral infection or in response to doublestranded RNA (dsRNA). Several intracellular molecules having a dsRNA-binding motif such as dsRNA-dependent protein kinase recognize dsRNA in a sequence-independent manner and induce antiviral innate responses. Toll-like receptor (TLR) 3, a member of TLR family proteins, recognizes extracellular dsRNA and activates NF- kappaB and the IFN-beta promoter leading to the induction of IFN-beta production. Here we analyzed the dsRNA structure capable of inducing TLR3-mediated IFN-beta production using various synthetic RNA duplexes. In contrast to the recognition of dsRNA by intracellular molecules, TLR3 preferentially recognizes polyriboinocinic:polyribocytidylic acid (poly(I:C)) rather than synthetic virus-derived dsRNAs. 2'-O-methyl or 2'-fluoro modification of cytidylic acid abolished the IFN-beta-inducing ability of the poly(I:C) duplex, and these modified dsRNAs inhibited poly(I:C)-induced TLR3-mediated IFN-beta production by fibroblasts and DCs. In addition, poly(dI:dC), a non-IFN inducer, also blocked poly(I:C)-induced IFN-beta induction. Since TLR3 is localized in the intracellular compartment of DCs where signaling occurs, modified dsRNAs may compete with poly(I:C) for binding to the cell-surface receptor that transfers dsRNA into TLR3-enriched vesicles. Thus, TLR3 recognizes a unique dsRNA structure that largely differs from those recognized by other dsRNA-binding proteins.

Cells, Cultured↗
PubMed2005

Antibodies against human Toll-like receptors (TLRs): TLR distribution and localization in human dendritic cells.

We have produced monoclonal antibodies (mAbs) against human Toll-like receptors (TLRs). These mAbs recognize the natural conformation of the extracellular domain leucine-rich repeats and, thereby, are suitable for immunoprecipitation, flow cytometric analysis and immunostaining. Using these mAbs, we determined the distribution of TLRs in a variety of human cell populations. Human TLRs that recognize bacterial components, particularly TLR-1, TLR-2, TLR-4 and TLR-6, reside on the cell surface and are expressed in myeloid and monocyte-derived dendritic cells (DCs) but not in plasmacytoid DCs (pDCs). Human TLR-3 resides in putative endosomes in myeloid DCs. Thus, the human myeloid DC subset harbors a unique and distinctive TLR repertoire. These mAbs will be useful to test the localization and distribution of human TLRs in a variety of cells and organs. Functional studies of human TLRs will also be feasible since certain of them are function-blocking mAbs.

Antibodies, Monoclonal↗
PubMed2005

Fish soluble Toll-like receptor 5 (TLR5S) is an acute-phase protein with integral flagellin-recognition activity.

From an EST fragment of the rainbow trout that was predicted to contain leucine-rich repeats (LRR), we cloned the whole cDNA and identified a soluble form of TLR5 ortholog (rtTLR5S), which does not exist in the mouse and human. rtTLR5S was about 38% homologous to the extracellular domains of human (hu) and mouse (mo)TLR5, while rtTLR5S showed <25% homologous to those of other human or mouse TLRs. A chimera constructed of rtTLR5S and the intra-cellular TIR of huTLR5 expressed on HeLa cells signaled the presence of flagellin A and C from V. anguillarum, resulting in NF-kappaB activation. The mRNA of rtTLR5S was predominantly detected in the liver. The hepatoma cell line of the rainbow trout RTH149 that responded to flagellin, allowed to up-regulate rtTLR5S in response to V. anguillarum or its purified flagellin within 8 h. rtTLR5S, when co-expressed with membrane huTLR5 in HeLa cells, augmented huTLR5-mediated NF-kappaB activation in response to flagellin. These results, together with the genome information of the pufferfish Fugu (Fugu rubripes), suggest that in fish the soluble TLR5 is an acute-phase protein sensing bacterial infection via recognition of a variety of bacterial flagellins to augment NF-kappaB activation, and may be important for fish to survive from bacterial infection in the water.

Acute-Phase Proteins↗
PubMed2005

Differential-expression and tyrosine-phosphorylation profiles of caveolin isoforms in human T cell leukemia cell lines.

Caveolin, the essential structural component of caveolae, serves as a scaffolding protein onto which signaling molecules are assembled, and functions as a negative regulator for signal transduction. Caveolin-1 and -2 are expressed in most cell types, but are not expressed in normal blood cells and cell lines. We previously demonstrated that caveolin-1 is expressed in a panel of human leukemia cell lines that show an activated T cell phenotype. In that study, we detected two caveolin bands by Western blotting using a polyclonal antibody (pAb) reacting with caveolin-1, -2, and -3. We identified caveolin-1alpha by its large molecular weight, but did not discriminatively detect other caveolin families. Since anti-caveolin-1 monoclonal antibody (mAb) was reported not to detect caveolin-1 in some cases, here we developed a sensitive method for the discriminative detection of caveolin-1, -2, and -3 by modified Western blotting. Caveolins were solubilized using a two-step procedure and detected by immunoprecipitation with a pAb to caveolins followed by Western blotting with mAbs specific to each caveolin. Using this method we detected caveolin-1beta, -2alpha and -2beta, but not caveolin-3 in the leukemia cell lines. Caveolin-1alpha, which was identified by pAb, was not detected by this method. We show here that caveolin-1alpha and -2alpha, but not caveolin-1beta and -2beta, are tyrosine phosphorylated. This modification is likely to cause the lack of reactivity of caveolin-1alpha to the mAb, and suggests a possible close relationship to cell activation.

Antibodies↗
PubMed2004

Gene-inducing program of human dendritic cells in response to BCG cell-wall skeleton (CWS), which reflects adjuvancy required for tumor immunotherapy.

Adjuvants induce the expression of a number of genes in dendritic cells (DCs), which facilitate effective antigen-presentation and cytokine/chemokine liberation. It has been accepted that the toll-like receptor (TLR) family governs the adjuvant activity in DCs. An adjuvant with a long history is mycobacteria in an oil-in-water emulsion, namely Freund's complete adjuvant. Since the active center for the adjuvancy in mycobacteria is the cell-wall skeleton (CWS), we used the bacillus Calmette-Guerin cell-wall skeleton (BCG-CWS) to test DC maturation by GeneChip analysis. We identified the genes supporting an efficient DC response and output. Approximately 2000 genes were up-regulated by BCG-CWS stimulation. BCG-CWS-, peptidoglycan (PGN)- and lipopolysaccharide (LPS)-stimulation generally up-regulated some gene clusters including genes for inflammatory cytokines (TNF, IL1alpha, IL1beta, IL6, IL12 p40, IL23 p19, etc.), chemokines (CCL20, IL8, etc.), cell adhesion molecules (ICAM-1, etc.), apoptosis-related proteins (GADD45B, BCL2A1, etc.), metabolic enzymes (PTGS2, SOD2, etc.) and miscellaneous proteins (EHD1, TNFAIP6, etc.). LPS-stimulation, but not BCG-CWS- or PGN-stimulation, up-regulated the interferon-inducible antiviral proteins, including IFIT1, IFIT2, IFIT4, CXCL10, ISG15, OASL, IFITM1 and MX1. We also found that the BCG-CWS- or PGN-stimulation up-regulated CXCL5, MMP1, etc. We discussed their properties in association with TLRs and recently discovered TLR adapters.

Adjuvants, Immunologic↗
PubMed2004

Identification of intelectin overexpression in malignant pleural mesothelioma by serial analysis of gene expression (SAGE).

Malignant pleural mesothelioma (MPM) is a fatal neoplasm with no acceptable curative approaches. We used serial analysis of gene expression (SAGE) to compare the gene expression pattern of a surgically resected MPM to the autologous normal mesothelium. Intelectin gene overexpression (>139-fold) was found in the tumor. Online SAGE datasets revealed intelectin to be consistently present in mesothelioma(s), ovarian cancer, and colon cancer. Intelectin mRNA expression was found by RT-PCR in 4 of 5 resected MPM tumors, and Intelectin protein expression was confirmed by immunohistochemistry in 28 of 53 MPM tumors, and in 4 of 4 mesothelioma cell lines studied by Western blot. A marked induction in intelectin gene expression was observed among human primary mesothelial cells as a consequence of crocidolite asbestos exposure and simian virus 40 infection. Intelectin overexpression in mesothelioma could have potential screening, and therapeutic implications.

Asbestos, Crocidolite↗
PubMed2004

Sensing bacterial flagellin by membrane and soluble orthologs of Toll-like receptor 5 in rainbow trout (Onchorhynchus mikiss).

Rainbow trout (Onchorhynchus mikiss) possess two genes encoding putative leucine-rich repeat (LRR)-containing proteins similar to human TLR5. Molecular cloning of these two LRR proteins suggested the presence of a TLR5-like membrane form (rtTLR5M) and a soluble form (rtTLR5S). Here we elucidated the primary structures and the unique combinational functions of these fish versions of TLR5. The LRR regions of rtTLR5S and rtTLR5M exhibited 81% homology and relatively high (35.6 and 33.7%) homology to the extracellular domains of human TLR5 (huTLR5). Thus, two distinct genes encode the TLR5 orthologs in fish, one of which has a consensus intracellular domain (TIR). In order to test their functions, we constructed fusion proteins with the LRR region of rtTLR5S (S-chimera) or that of rtTLR5M and the TIR of huTLR5 (M-chimera). The S- and M-chimeras expressed in HeLa or CHO cells signaled the presence of Vibrio anguillarum flagellin, resulting in NF-kappaB activation. rtTLR5M was ubiquitously expressed, whereas rtTLR5S was predominantly expressed in the liver. In the hepatoma cell lines of the rainbow trout RTH-149, stimulation of rtTLR5M with V. anguillarum or its flagellin allowed the up-regulation of rtTLR5S. Flagellin-mediated NF-kappaB activation was more significant in the presence of or simultaneous expression of rtTLR5S. Therefore, a two-step flagellin response occurred for host defense against bacterial infection in fish: (a) flagellin first induced basal activation of NF-kappaB via membrane TLR5, facilitating the production of soluble TLR5 and minimal acute phase proteins, and (b) the inducible soluble TLR5 amplifies membrane TLR5-mediated cellular responses in a positive feedback fashion.

Amino Acid Sequence↗
PubMed2004

A short consensus repeat-containing complement regulatory protein of lamprey that participates in cleavage of lamprey complement 3.

The prototype of the short consensus repeat (SCR)-containing C regulatory protein is of interest in view of its evolutionary significance with regard to the origin of the C regulatory system. Lamprey is an agnathan fish that belongs to the lowest class of vertebrates. Because it does not possess lymphocytes, it lacks Ig and consequently the classical C pathway. We identified an SCR-containing C regulatory protein from the lamprey. The primary structure predicted from the cDNA sequence showed that this is a secretary protein consisting of eight SCRs. This framework is similar to the alpha-chain of C4b-binding protein (C4bp). SCR2 and -3 of human C4bp are essential for C4b inactivation, and this region is fairly well conserved in the lamprey protein. However, the other SCRs of this protein are similar to those of other human C regulatory proteins. The lamprey protein binds to the previously reported lamprey C3b/C3bi deposited on yeast and cleaves lamprey C3b-like C3 together with a putative serum protease. The scheme resembles the C regulatory system of mammals, where factor I and its cofactor inactivate C3b. Unlike human cofactors, the lamprey protein requires divalent cations for C3b-like C3 cleavage. Its artificial membrane-anchored form protects host cells from lamprey C attack via the lectin pathway. Thus, the target of this protein appears to be C3b and/or its family. We named this protein Lacrep, the lamprey C regulatory protein. Lacrep is a member of SCR-containing C regulators, the first of its kind identified in the lowest vertebrates.

Amino Acid Sequence↗
PubMed2004

The cytoplasmic 'linker region' in Toll-like receptor 3 controls receptor localization and signaling.

Toll-like receptor 3 (TLR3) recognizes double-stranded RNA and transmits signals to activate NF-kappaB and the interferon (IFN)-beta promoter via the newly identified adaptor, TICAM-1. The extracellular LRR domain of TLR3 is engaged in the ligand recognition, while the intracellular TIR domain is crucial for the adaptor binding and signal transduction upon ligand stimulation. Here, we analyzed TLR3 localization in human monocyte-derived immature dendritic cells (iDCs) and stable transfectants expressing human TLR3 by immunofluorescence staining and confocal microscopy. TLR3 was predominantly localized in specific but as yet unidentified intracellular vesicles where TLR3 signaling was initiated. Expression analysis of TLR3-tail-truncated mutants revealed that the cytoplasmic 'linker' region (residues 730-755) determines the intracellular localization of TLR3. Site-directed mutagenesis of the linker region allowed us to identify the relevant determinants as Arg(740) and Val(741) residues for intracellular expression of TLR3. Furthermore, alanine scanning of the linker region demonstrated that the Phe(732), Leu(742) and Gly(743) in the TLR3 cytoplasmic linker region are essential for ligand-induced NF-kappaB and IFN-beta promoter activation. Thus, the cytoplasmic linker region of TLR3 regulates receptor retention inside the organelle and signaling, which may be closely linked to TLR3 function in DCs.

Adaptor Proteins, Vesicular Transport↗
PubMed2004

Adjuvant-mediated tumor regression and tumor-specific cytotoxic response are impaired in MyD88-deficient mice.

The Mycobacterium bovis bacillus Calmette-Guérin cell-wall skeleton (BCG-CWS) activates Toll-like receptor (TLR) 2 and TLR4, but unlike the typical TLR4 agonist bacterial lipopolysaccharide barely induces type 1 IFN. BCG-CWS has been used for adjuvant immunotherapy for patients with cancer. We investigated the adjuvant potential of BCG-CWS for induction of CTLs subsequent to TLR-mediated dendritic cell (DC) maturation, using a syngeneic mouse tumor model (B16 melanoma in C57BL/6). We evaluated the retardation of tumor growth and cytotoxic response in wild-type and MyD88-/- mice immunized with tumor debris and/or BCG-CWS. Delays in tumor growth and cytotoxic response were induced by immunization with a mixture of BCG-CWS emulsion and the tumor. BCG-CWS was capable of activating DCs ex vivo by the criteria of CD80/CD86 up-regulation and cytokine (interleukin-12, tumor necrosis factor-alpha) induction. Efficient tumor suppression and ex vivo cytokine induction did not occur in MyD88-deficient mice and cells, suggesting that the MyD88 adapter is crucial for induction of tumor cytotoxicity. Because TLR4 is involved in both MyD88-dependent and -independent pathways and the latter affects DC maturation, our findings indicate that both pathways cooperate to induce CTL-based tumor immunity.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Toll-like receptor 3: a link between toll-like receptor, interferon and viruses.

Production of type I interferon (IFN-alpha/beta) by virus-infected cells is the central event in their antiviral immune responses. In mammalian cells, IFN-alpha/beta gene transcription is induced through distinct signaling pathways by viral infection or by treatment with double-stranded (ds) RNA, which is an intermediate of virus replication. Toll-like receptor 3 (TLR3) was found to recognize dsRNA and transmit signals to activate NF-kappaB and the IFN-beta promoter. Recent identification of the TLR3-adaptor protein and its downstream signaling molecules, which are involved in IFN-alpha/beta production, revealed a novel IFN-inducing pathway for an anti-viral immune response. Here, we summarize the current knowledge of TLR3-mediated immune responses.

Immunity, Cellular↗
PubMed2004

Infection of different cell lines of neural origin with subacute sclerosing panencephalitis (SSPE) virus.

Measles virus is the causative agent of subacute sclerosing panencephalitis (SSPE). The viruses isolated from brain cells of patients with SSPE (called SSPE viruses) are defective in cell-free virus production in vitro. To investigate the cell tropism of three strains of SSPE virus (Osaka-1, Osaka-2, Osaka-3), SSPE virus-infected cell cultures were treated with cytochalasin D to prepare virus-like particles (CD-VLPs). All CD-VLPs formed syncytia after infection in CHO cells expressing CD150 but not in those expressing CD46. In addition, an antibody to CD46 did not block the infection of Vero cells by SSPE CDVLPs. The results were consistent with our previous suggestion that one or more unidentified receptors might be involved in the entry process. Infection with the CD-VLPs from three SSPE strains was further examined in different human cell lines, including those of neural origin, and was found to induce syncytia in epithelial cells (HeLa and 293T) as well as neuroblastoma cells (IMR-32 and SK-N-SH) with varying efficiency. SSPE CD-VLPs also infected glioblastoma cells (A172) and astrocytoma cells (U-251) but syncytial formation was rarely induced. These epithelial and neural cell lines were not permissive for the replication of wild-type MV. Together with our previous observations, these results suggest that the cell entry receptor is the major factor determining the cell tropism of SSPE viruses. Further studies are necessary to identify other viral and/or cellular factors that might be involved in the replication of SSPE virus in specific neural cells and in the brain.

Animals↗