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Biomedical subjects

Osamu Takeuchi

Publications and source records attributed to Osamu Takeuchi.

At least 55 records · Page 3Linked to original sources
PubMed2004

Inhibitory effect of Toll-like receptor 4 on fusion between phagosomes and endosomes/lysosomes in macrophages.

Toll-like receptor 4 (TLR4) of macrophages recognizes LPS of Gram-negative bacteria in cooperation with CD14, which is also involved in the recognition of apoptotic cells. In this study we asked whether TLR4 plays a role in the phagocytic clearance of apoptotic cells by macrophages. Macrophages were prepared from peritoneal fluid of thioglycolate-treated mice carrying either a wild-type or a disrupted TLR4-encoding gene and were examined for their ability to phagocytose apoptotic mouse thymocytes, apoptotic Jurkat T cells, Ig-opsonized mouse thymocytes, Ig-opsonized zymosan particles, and latex beads. Both populations of macrophages equally expressed CD14 on their surfaces and showed almost equal activities of binding to and engulfing all these targets. However, apoptotic thymocytes, apoptotic Jurkat cells, and opsonized thymocytes disappeared more rapidly in TLR4-deficient macrophages than in wild-type macrophages, and the fusion between endosomes/lysosomes and phagosomes containing any target cells or particles was accelerated in mutant macrophages. Activation of the transcription factor NF-kappaB appeared not to occur in wild-type macrophages after engulfment, and the rate of apoptotic cell degradation in wild-type macrophages remained the same regardless of the activation of NF-kappaB. Finally, immunohistochemical analyses showed that ectopically expressed TLR4 was associated with phagosomes in a macrophage-derived cell line. All these results collectively indicate that TLR4 negatively regulates the degradation of engulfed cells in macrophages via a pathway independent of NF-kappaB.

Ammonium Chloride↗
PubMed2004

Induction of macrophage migration inhibitory factor precedes the onset of acute tonsillitis.

We investigated the serum macrophage migration inhibitory factor (MIF) levels of palmoplantar pustulosis patients, before and after the tonsillar provocation test. Higher serum MIF levels of palmoplantar pustulosis patients were decreased after the tonsillar provocation test (n=29). To confirm these phenomena, two patients with acute tonsillitis had their changes in body temperature, C-reactive protein (CRP) and serum MIF levels examined during the course of their illness. Surprisingly, increased MIF preceded fever and CRP elevation, and MIF subsequently decreased at the onset of fever and CRP elevation. Since MIF is an initiator of other proinflammatory cytokines, we suggest that the induction of MIF may precede other inflammatory conditions.

Acute Disease↗
PubMed2004

Limited role of the Toll-like receptor-2 in resistance to Mycobacterium avium.

The role of the Toll-like receptor (TLR)-2 in the generation of protective immunity to Mycobacterium avium was evaluated using gene-disrupted mice. TLR-2-/- mice were more susceptible than wild-type C57Bl/6 mice to M. avium strains that were able to proliferate in vivo before the development of protective immunity and mycobacteriostasis. In contrast, the elimination of non-virulent strains was not affected by the mutation. The generation of interferon-gamma (IFN-gamma)-producing T cells and the expression of the interleukin-12 p40 gene were reduced in TLR-2-deficient mice as compared to C57Bl/6 mice early during infection with M. avium strain 2447. The generation of protective CD4+ T cells was also compromised in the mutated mice as compared with the controls. Our data show that TLR-2 is required for optimal immunity against certain virulent M. avium strains.

Adoptive Transfer↗
PubMed2004

Toll-like receptor 4 mediates the antitumor host response induced by a 55-kilodalton protein isolated from Aeginetia indica L., a parasitic plant.

A 55-kDa protein named AILb-A, isolated from the seed extract of Aeginetia indica L., a parasitic plant, induces a Th1-type T-cell response and elicits a marked antitumor effect in tumor-bearing mice. In the present study, we examined the role of Toll-like receptors (TLRs), which have been implicated in pathogen-induced cell signaling, in AILb-A-induced immune responses. In the luciferase assay using a nuclear factor (NF)-kappaB-dependent reporter plasmid, AILb-A induced NF-kappaB activation in the cells transfected with TLR4, but not with those transfected with the TLR2 gene, in a dose-dependent manner. TLR4-mediated NF-kappaB activation induced by AILb-A but not by lipopolysaccharide (LPS) was also observed under serum-free conditions. In in vitro experiments using human peripheral blood mononuclear cells, AILb-A-induced cytokine production was markedly inhibited by anti-TLR4 but not by anti-CD14 antibody, while LPS-induced, TLR4-mediated cytokine production was inhibited by anti-CD14 as well as anti-TLR4 antibodies. Cytokine production, killer cell activities, maturation of dendritic cells, phosphorylation of mitogen-activated protein kinases, and nuclear translocation of interferon-regulatory factor 3 induced by AILb-A were severely impaired in TLR4-deficient but not TLR2-deficient mice. Transfection of TLR4-deficient mouse-derived macrophages with the TLR4 expression plasmid led AILb-A to induce cytokines. Finally, the antitumor effect of AILb-A was also impaired in TLR4-deficient and TLR4-mutated mice. These findings suggest that TLR4 mediates antitumor immunity induced by the plant-derived protein AILb-A.

Animals↗
PubMed2004

Interferon response induced by Toll-like receptor signaling.

Toll-like receptors (TLRs) are essential for the recognition of distinct pathogen-associated molecular patterns (PAMPs). Activation of TLRs induces intracellular signaling pathways which lead to the production of pro-inflammatory cytokines, chemokines, and interferon (IFN)-inducible genes. TIR domain containing adaptor molecules in turn determine the signaling specificity of the response. Recent studies demonstrated that serine/threonine kinases IKK-i/TBK1 are critical for the regulation of IFN-beta as well as IFN-inducible genes. In response to lipopolysaccharide (LPS), transfection of poly(I:C) and viral infection, embryonic fibroblasts (MEFs) derived from TBK1-deficient (TBK1-/-) mice show impaired production of IFN-inducible genes, but not proinflammatory cytokines. Although IKK-i-/- mice show normal production of these genes, MEFs from IKK-i/TBK1-doubly deficient mice were completely defective in the induction of IFN-beta as well as IFN-inducible genes in response to poly(I:C) stimulation. Activation of IFN-regulatory factor (IRF) 3 in response to LPS and poly(I:C) was abolished in IKK-i/TBK1 doubly deficient cells. Interestingly, intracellular transduction of poly(I:C) initiates activation of IFN response in a TLR3-independent manner. These observations demonstrate that IKK-i/TBK1 signaling is essential for both TLR3-dependent and TLR3-independent viral and dsRNA-induced IFN responses.

Animals↗
PubMed2003

Toll-like receptor-2 modulates ventricular remodeling after myocardial infarction.

BACKGROUND: Toll-like receptors (TLRs) are members of the interleukin-1 receptor family and transduce similar signals as interleukin-1 receptor in response to exogenous pathogens. Recent studies have demonstrated that TLRs are activated by endogenous signals, such as heat shock proteins and oxidative stress, that may contribute to ventricular remodeling after myocardial infarction. In this study, we determined whether TLR-2 was involved in cardiac remodeling after myocardial infarction. METHODS AND RESULTS: Myocardial infarction was induced by surgical left anterior descending coronary artery ligation on wild-type (WT) mice and TLR-2-knockout (KO) mice. The survival rate was significantly higher in KO mice than in WT mice 4 weeks after myocardial infarction (65% versus 43%, P<0.03). Infarct size and degree of inflammatory cell infiltration in infarct area were similar between WT and KO mice. However, myocardial fibrosis in the noninfarct area of KO mice was much less than in WT mice (P<0.01) and was accompanied by reduced transforming growth factor-beta1 and collagen type 1 mRNA expressions (P<0.01 and P<0.05, respectively). Left ventricular dimensions at end diastole were smaller in KO mice than in WT mice at 1 week (P<0.05) and 4 weeks (P<0.01) after surgery. Furthermore, fractional shortening was higher (27.7+/-2.5% versus 21.2+/-2.6%, P<0.05, at 1 week, and 24.3+/-2.0% versus 16.6+/-2.5%, P<0.01, at 4 weeks) in KO mice compared with WT mice. CONCLUSIONS: These data suggest that TLR-2 plays an important role in ventricular remodeling after myocardial infarction.

Animals↗
PubMed2003

TRAM is specifically involved in the Toll-like receptor 4-mediated MyD88-independent signaling pathway.

Recognition of pathogens by Toll-like receptors (TLRs) triggers innate immune responses through signaling pathways mediated by Toll-interleukin 1 receptor (TIR) domain-containing adaptors such as MyD88, TIRAP and TRIF. MyD88 is a common adaptor that is essential for proinflammatory cytokine production, whereas TRIF mediates the MyD88-independent pathway from TLR3 and TLR4. Here we have identified a fourth TIR domain-containing adaptor, TRIF-related adaptor molecule (TRAM), and analyzed its physiological function by gene targeting. TRAM-deficient mice showed defects in cytokine production in response to the TLR4 ligand, but not to other TLR ligands. TLR4- but not TLR3-mediated MyD88-independent interferon-beta production and activation of signaling cascades were abolished in TRAM-deficient cells. Thus, TRAM provides specificity for the MyD88-independent component of TLR4 signaling.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway.

Stimulation of Toll-like receptors (TLRs) triggers activation of a common MyD88-dependent signaling pathway as well as a MyD88-independent pathway that is unique to TLR3 and TLR4 signaling pathways leading to interferon (IFN)-beta production. Here we disrupted the gene encoding a Toll/IL-1 receptor (TIR) domain-containing adaptor, TRIF. TRIF-deficient mice were defective in both TLR3- and TLR4-mediated expression of IFN-beta and activation of IRF-3. Furthermore, inflammatory cytokine production in response to the TLR4 ligand, but not to other TLR ligands, was severely impaired in TRIF-deficient macrophages. Mice deficient in both MyD88 and TRIF showed complete loss of nuclear factor kappa B activation in response to TLR4 stimulation. These findings demonstrate that TRIF is essential for TLR3- and TLR4-mediated signaling pathways facilitating mammalian antiviral host defense.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Candida albicans phospholipomannan is sensed through toll-like receptors.

Candida albicans is a common, harmless yeast in the human digestive tract that also causes severe systemic fungal infection in hospitalized patients. Its cell-wall surface displays a unique glycolipid called phospholipomannan (PLM). The ability of PLM to stimulate tumor necrosis factor (TNF)-alpha production by J774 mouse cells correlates with the activation of nuclear factor (NF)-kappaB. We examined the involvement of Toll-like receptors (TLRs) in PLM-dependent stimulation. Compared with wild-type cells, which produced large amounts of TNF-alpha after incubation with PLM, the deletion of the TLR4 and TLR6 genes led to a limited alteration of the PLM-induced response. Deletion of the TLR2 gene completely abolished the cell response. Surface expression of PLM is a phylogenic trait of C. albicans, and the recognition of PLM by TLRs, together with the unique pathogenic potential of C. albicans, suggests that this molecule may be a member of the pathogen-associated molecular pattern family.

Animals↗
PubMed2003

CD19 regulates innate immunity by the toll-like receptor RP105 signaling in B lymphocytes.

Lipopolysaccharide (LPS) is a major gram-negative bacterial component that stimulates innate immune response and also induces B-lymphocyte activation. Recent studies have revealed that common molecular patterns of microorganisms such as LPS are recognized by toll-like receptors (TLRs). B cells have 2 known TLRs that mediate LPS signaling, TLR4 and RP105 (CD180). While TLR4 is expressed on immune cells of various types, RP105 is preferentially expressed on mature B cells. Here we demonstrate that CD19 plays a major role in regulating signal transduction through RP105. Anti-RP105 ligation induced normal proliferation of B cells from mice deficient for MyD88, an adaptor protein that mediates most TLR pathways. By contrast, the loss of CD19 resulted in modest B-cell proliferation against anti-RP105 stimulation as well as LPS stimulation. LPS induced tyrosine phosphorylation of CD19, which was RP105-dependent but TLR4-independent. CD19 formed a complex with Lyn and Vav following RP105 ligation, and CD19 expression was required for optimal Lyn activation and Vav phosphorylation. Consistently, B cells deficient for CD19 exhibited specific defect in the activation of c-Jun N-terminal kinases following RP105 ligation and LPS stimulation. In contrast, CD19 and phosphatidylinositol 3-kinase independently regulated intracellular calcium mobilization induced by anti-RP105 stimulation. Thus, signaling through the B-cell-specific LPS receptor RP105 is uniquely regulated by the B-cell-specific signaling component, Lyn/CD19/Vav complex.

Animals↗
PubMed2003

Involvement of Toll-like receptor 4 signaling in interferon-gamma production and antitumor effect by streptococcal agent OK-432.

BACKGROUND: The streptococcal agent OK-432 has been used for immunotherapy of head and neck cancer, among other malignancies, but its mechanism of action is unknown. Because the Toll-like receptor 4 (TLR4)/MD-2 complex is important in enabling the mammalian immune system to recognize bacterial components, we investigated whether expression of the TLR4 and MD-2 genes is associated with OK-432-induced anticancer immunity. METHODS: Peripheral blood mononuclear cells (PBMCs) from 28 patients with head and neck cancer were analyzed for TLR4 and MD-2 mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR) analysis. PBMCs were treated in vitro with OK-432 or with OK-PSA (a lipoteichoic-acid-related molecule that is an active component of OK-432), and interferon-gamma (IFN-gamma) mRNA expression, an immune response measure, was analyzed by RT-PCR. Patient sera collected 24 hours after OK-432 administration were examined for IFN-gamma protein using an enzyme-linked immunosorbent assay. Lewis lung carcinoma-bearing wild-type C57BL/6 and TLR4-deficient mice (four mice per group) received intraperitoneal injections of OK-432, and tumor volumes and sera IFN-gamma levels were measured over time. All statistical tests were two-sided. RESULTS: Twenty patients expressed both TLR4 and MD-2. Expression of TLR4 and MD-2 genes was associated with the in vivo IFN-gamma induction in 19 patients administered OK-432 (Fisher's exact test P<.001). Although both OK-432 and OK-PSA induced IFN-gamma expression from PBMCs in vitro, expression of TLR4 and MD-2 was associated only with IFN-gamma expression induced by OK-PSA (P<.001). In vivo intraperitoneal administration of OK-432 resulted in an increase of IFN-gamma in sera from wild-type mice but not in sera from TLR4-deficient mice. Tumors in wild-type mice treated with OK-432 were statistically significantly smaller than those in mice treated with saline (P =.007). By contrast, in TLR4-deficient mice, there was no difference in tumor volume between the two treatment groups. CONCLUSIONS: TLR4 and MD-2 may mediate OK-432-induced anticancer immunity.

Aged↗
PubMed2003

Hypercapnic acidosis attenuates endotoxin-induced nuclear factor-[kappa]B activation.

Although permissive hypercapnia improves the prognosis of patients with acute respiratory distress syndrome, it has not been conclusively determined whether hypercapnic acidosis (HA) is harmful or beneficial to sustained inflammation of the lung. The present study was designed to explore the molecular mechanism of HA in modifying lipopolysaccharide (LPS)-associated signals in pulmonary endothelial cells. LPS elicited degradation of inhibitory protein kappaB (IkappaB)-alpha, but not IkappaB-beta, resulting in activation of nuclear factor (NF)-kappaB in human pulmonary artery endothelial cells. Exposure to HA significantly attenuated LPS-induced NF-kappaB activation through suppressing IkappaB-alpha degradation. Isocapnic acidosis and buffered hypercapnia showed qualitatively similar but quantitatively smaller effects. HA did not attenuate the LPS-enhanced activation of activator protein-1. Following the reduced NF-kappaB activation, HA suppressed the mRNA and protein levels of intercellular adhesion molecule-1 and interleukin-8, resulting in a decrease in both lactate dehydrogenase release into the medium and neutrophil adherence to LPS-activated human pulmonary artery endothelial cells. In contrast, HA did not inhibit LPS-enhanced neutrophil expression of integrin, Mac-1. Based on these findings, we concluded that hypercapnic acidosis would have anti-inflammatory effects essentially through a mechanism inhibiting NF-kappaB activation, leading to downregulation of intercellular adhesion molecule-1 and interleukin-8, which in turn inhibits neutrophil adherence to pulmonary endothelial cells.

Acidosis, Respiratory↗
PubMed2003

TLR2 as an essential molecule for protective immunity against Toxoplasma gondii infection.

To investigate the role of the Toll-like receptor (TLR) family in host defense against Toxoplasma gondii, we infected TLR2-, TLR4- and MyD88-deficient mice with the avirulent cyst-forming Fukaya strain of T. gondii. All TLR2- and MyD88-deficient mice died within 8 days, whereas all TLR4-deficient and wild-type mice survived after i.p. infection with a high dose of T. gondii. Peritoneal macrophages from T. gondii-infected TLR2- and MyD88-deficient mice did not produce any detectable levels of NO. T. gondii loads in the brain tissues of TLR2- and MyD88-deficient mice were higher than in those of TLR4-deficient and wild-type mice. Furthermore, high levels of IFN-gamma and IL-12 were produced in peritoneal exudate cells (PEC) of TLR4-deficient and wild-type mice after infection, but low levels of cytokines were produced in PEC of TLR2- and MyD88-deficient mice. On the other hand, high levels of IL-4 and IL-10 were produced in PEC of TLR2- and MyD88-deficient mice after infection, but low levels of cytokines were produced in PEC of TLR4-deficient and wild-type mice. The most remarkable histological changes with infiltration of inflammatory cells were observed in lungs of TLR2-deficient mice infected with T. gondii, where severe interstitial pneumonia occurred and abundant T. gondii were found.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Mycobacterial infection in TLR2 and TLR6 knockout mice.

To investigate the role of TLR in the development of murine tuberculosis in vivo, TLR2 and TLR6 knockout (KO) mice were infected with Mycobacterium tuberculosis by placing them in the exposure chamber of an airborne infection apparatus. Both TLR2 and TLR6 KO mice survived until sacrifice at 12 weeks after infection. Infected TLR2 KO mice developed granulomatous pulmonary lesions with neutrophil infiltration, which were slightly larger in size than those in wild-type mice. Pulmonary levels of the mRNAs for inducible nitric oxide synthase (iNOS), TNF-alpha, TGF-beta, IL-1beta, and IL-2 were significantly lower, but levels of the mRNAs for IL-4 and IL-6 were higher, than in wild-type (WT) mice. No significant difference was recognized in cytokine mRNA expression between TLR2 KO and WT mice at 12 weeks after infection. DNA binding by NF-kappaB was low in TLR2 KO mice. On the other hand, TLR6 KO mice were not different from WT mice in terms of pulmonary histopathology, mRNA expression and CFU assay. Therefore, TLR2 does not play an essential role in the pathogenesis of murine tuberculosis, although it is important for defense against mycobacterial infection.

Animals↗
PubMed2003

Simultaneous blocking of human Toll-like receptors 2 and 4 suppresses myeloid dendritic cell activation induced by Mycobacterium bovis bacillus Calmette-Guérin peptidoglycan.

The Mycobacterium bovis bacillus Calmette-Guérin (BCG) cell wall skeleton (CWS) consists of mycolic acids, arabinogalactan, and peptidoglycan (PGN) and activates Toll-like receptor 2 (TLR2) and TLR4. Here we investigated the ability of the essential portion of highly purified BCG CWS to support the TLR agonist function by using the following criteria: myeloid dendritic cell (DC) maturation, i.e., tumor necrosis factor alpha (TNF-alpha) production and CD83/CD86 up-regulation. The purified PGN region was sufficient to activate TLR2 and TLR4 in mouse DCs and macrophages; in TLR2 and TLR4 double-knockout cells the BCG PGN-mediated TNF-alpha production ability was completely impaired. Likewise, stimulation with BCG CWS of HEK293 cells expressing either human TLR2 or TLR4, MD-2, and CD14 resulted in NF-kappa B activation as determined by a reporter assay. Notably, specific blockers of extracellular human TLR2 (an original cocktail of monoclonal antibodies TLR2.45 and TH2.1) and TLR4 (E5531) inhibited BCG CWS-mediated NF-kappa B activation by 80%. Using this human TLR blocking system, we tested whether human myeloid DC maturation was TLR2 and TLR4 dependent. BCG PGN-mediated DC maturation was blocked by 70% by suppression of both TLR2 and TLR4 and by 30 to 40% by suppression of either of these TLRs. Similar but less profound suppression of BCG CWS-mediated DC maturation was observed. Hence, the presence of BCG PGN is a minimal requirement for activation of both TLR2 and TLR4 in human DCs, unlike the presence of PGNs of gram-positive bacteria, which activate only TLR2. Unexpectedly, however, BCG PGN, unlike BCG CWS, barely activated NF-kappa B in HEK293 cells coexpressing TLR2 plus TLR1, TLR2 plus TLR4, TLR2 plus TLR6, or TLR2 plus TLR10, suggesting that PGN receptors other than TLR2 and TLR4 present on human DCs but not on HEK293 cells are involved in TLR signaling for DC activation.

Animals↗
PubMed2003

Synergistic effects of lipopolysaccharide and interferon-gamma in inducing interleukin-8 production in human monocytic THP-1 cells is accompanied by up-regulation of CD14, Toll-like receptor 4, MD-2 and MyD88 expression.

Lipopolysaccharide (LPS) and interferon (IFN)-gamma synergistically induced interleukin-8 (IL-8) production in human monocytic THP-1 cells. IFN-gamma-primed THP-1 cells produced higher levels of IL-8 on stimulation with LPS than non-primed cells and the level correlated with duration of priming up to 24 h, although the level of IL-8 induced was most comparable to that induced by co-stimulation with LPS and IFN-gamma. Unstimulated THP-1 cells were shown by flow cytometry to be practically devoid of membrane CD14 (mCD14). LPS and IFN-gamma enhanced mCD14 and Toll-like receptor (TLR) 4 expression in THP-1 cells, respectively, and co-stimulation with LPS and IFN-gamma induced higher levels of mCD14 and TLR4 expression than stimulation with either agent alone. LPS and IFN-gamma alone each augmented MD-2 and MyD88 mRNA expression in THP-1 cells, and co-stimulation with LPS and IFN-gamma markedly enhanced MD-2 and MyD88 mRNA expression in the cells compared to those with either LPS or IFN-gamma alone. Anti-CD14 and anti-TLR4 monoclonal antibodies almost completely inhibited IL-8 production induced by LPS plus IFN-gamma in THP-1 cells. These findings suggest that combined stimulation of THP-1 cells with LPS and IFN-gamma up-regulate mCD14, TLR4, MD-2 and MyD88 expression by these cells, which might be involved in synergistic IL-8 production by the cells.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Cutting edge: a novel Toll/IL-1 receptor domain-containing adapter that preferentially activates the IFN-beta promoter in the Toll-like receptor signaling.

MyD88 is a Toll/IL-1 receptor (TIR) domain-containing adapter common to signaling pathways via Toll-like receptor (TLR) family. However, accumulating evidence demonstrates the existence of a MyD88-independent pathway, which may explain unique biological responses of individual TLRs, particularly TLR3 and TLR4. TIR domain-containing adapter protein (TIRAP)/MyD88 adapter-like, a second adapter harboring the TIR domain, is essential for MyD88-dependent TLR2 and TLR4 signaling pathways, but not for MyD88-independent pathways. Here, we identified a novel TIR domain-containing molecule, named TIR domain-containing adapter inducing IFN-beta (TRIF). As is the case in MyD88 and TIRAP, overexpression of TRIF activated the NF-kappaB-dependent promoter. A dominant-negative form of TRIF inhibited TLR2-, TLR4-, and TLR7-dependent NF-kappaB activation. Furthermore, TRIF, but neither MyD88 nor TIRAP, activated the IFN-beta promoter. Dominant-negative TRIF inhibited TLR3-dependent activation of both the NF-kappaB-dependent and IFN-beta promoters. TRIF associated with TLR3 and IFN regulatory factor 3. These findings suggest that TRIF is involved in the TLR signaling, particularly in the MyD88-independent pathway.

Adaptor Proteins, Vesicular Transport↗