Biomedical subjects

Tsukasa Seya

Publications and source records attributed to Tsukasa Seya.

At least 19 recordsLinked to original sources

PubMed2007

Lamprey TLRs with properties distinct from those of the variable lymphocyte receptors.

Fish express mammalian-type (M-type) TLRs consisting of leucine-rich repeats (LRRs) and Toll-IL-1R (TIR) homology domain for immunity, whereas invertebrates in deuterostomes appear to have no orthologs of M-type TLRs. Lampetra japonica (lamprey) belongs to the lowest class of vertebrates with little information about its TLRs. We have identified two cDNA sequences of putative TLRs in the lamprey (laTLRs) that contain LRRs and TIR domains. The two laTLRs were 56% homologous to each other, and their TIRs were similar to those of members of the human TLR2 subfamily, most likely orthologs of fish TLR14. We named them laTLR14a and laTLR14b. We raised a rabbit polyclonal Ab against laTLR14b and identified a 85-kDa protein in a human HEK293 transfectant by immunoblotting using the Ab. FACS, histochemical, and confocal analyses showed that laTLR14b is expressed intracellularly in lamprey gill cells and that the overexpressed protein resides in the endoplasmic reticulum of human and fish (medaka) cell lines. Because natural agonists of TLR14 remained unidentified, we made a chimera construct of extracellular CD4 and the cytoplasmic domain of laTLR14. The chimera molecule of laTLR14b, when expressed in HEK293 cells, elicited activation of NF-kappaB and, consequently, weak activation of the IFN-beta promoter. laTLR14b mRNA was observed in various organs and leukocytes. This lamprey species expressed a variable lymphocyte receptor structurally independent of laTLR14 in leukocytes. Thus, the jawless vertebrate lamprey possesses two LRR-based recognition systems, the variable lymphocyte receptor and TLR, and the M-type TLRs are conserved across humans, fish, and lampreys.

Amino Acid Sequence↗

PubMed2007

TLR3-mediated synthesis and release of eotaxin-1/CCL11 from human bronchial smooth muscle cells stimulated with double-stranded RNA.

Respiratory infections with RNA viruses, such as rhinovirus or respiratory syncytial virus, are a major cause of asthma exacerbation, accompanied by enhanced neutrophilic and/or eosinophilic inflammation of the airways. We studied the effects of dsRNA synthesized during RNA virus replication, and of its receptor, TLR3, on the synthesis of eosinophilic chemokines in bronchial smooth muscle cells (BSMC). Synthetic dsRNA, polyinosinic-cystidic acid (poly(I:C)), induced the synthesis of eosinophilic chemokines, eotaxin-1/CCL11 and RANTES/CCL5, from primary cultures of human BSMC, and IL-4 increased synergistically the synthesis of poly(I:C)-induced CCL11. A robust eosinophil chemotactic activity was released from BSMC stimulated with poly(I:C) and IL-4, which was mostly inhibited by preincubation with an anti-CCL11, but not with an anti-CCL5 Ab. Although the immunoreactivity of TLR3 was detectable on the cellular surface of BSMC by flow cytometric analysis, pretreatment with an anti-TLR3-neutralizing Ab failed to block the poly(I:C)-induced synthesis of CCL11. We have determined by confocal laser-scanning microscopy that the immunoreactivity of TLR3 was aggregated intracellularly in poly(I:C)-stimulated BSMC, colocalizing with fluorescein-labeled poly(I:C). The synthesis of CCL11 was prominently inhibited by the transfection of TLR3-specific small interfering RNA or by bafilomycin A1, an endosomal acidification inhibitor, further supporting the essential role played by intracellular TLR3 in the synthesis of poly(I:C)-induced CCL11 in BSMC. In conclusion, these observations suggest that, by activating intracellular TLR3 in BSMC, respiratory RNA virus infections stimulate the production of CCL11 and enhance eosinophilic inflammation of the airways in the Th2-dominant microenvironment.

Bronchi↗

PubMed2006

Antitumor NK activation induced by the Toll-like receptor 3-TICAM-1 (TRIF) pathway in myeloid dendritic cells.

Myeloid dendritic cells (mDCs) recognize and respond to polyI:C, an analog of dsRNA, by endosomal Toll-like receptor (TLR) 3 and cytoplasmic receptors. Natural killer (NK) cells are activated in vivo by the administration of polyI:C to mice and in vivo are reciprocally activated by mDCs, although the molecular mechanisms are as yet undetermined. Here, we show that the TLR adaptor TICAM-1 (TRIF) participates in mDC-derived antitumor NK activation. In a syngeneic mouse tumor implant model (C57BL/6 vs. B16 melanoma with low H-2 expresser), i.p. administration of polyI:C led to the retardation of tumor growth, an effect relied on by NK activation. This NK-dependent tumor regression did not occur in TICAM-1(-/-) or IFNAR(-/-) mice, whereas a normal NK antitumor response was induced in PKR(-/-), MyD88(-/-), IFN-beta(-/-), and wild-type mice. IFNAR was a prerequisite for the induction of IFN-alpha/beta and TLR3. The lack of TICAM-1 did not affect IFN production but resulted in unresponsiveness to IL-12 production, mDC maturation, and polyI:C-mediated NK-antitumor activity. This NK activation required NK-mDC contact but not IL-12 function in in vivo transwell analysis. Implanted tumor growth in IFNAR(-/-) mice was retarded by adoptively transferring polyI:C-treated TICACM-1-positive mDCs but not TICAM-1(-/-) mDCs. Thus, TICAM-1 in mDCs critically facilitated mDC-NK contact and activation of antitumor NK, resulting in the regression of low MHC-expressing tumors.

Adaptor Proteins, Vesicular Transport↗

PubMed2006

NAK-associated protein 1 participates in both the TLR3 and the cytoplasmic pathways in type I IFN induction.

TLR3 and the cytoplasmic helicase family proteins (retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5)) serve as dsRNA pattern-recognition receptors. In response to poly(I:C), a representative of dsRNA, and viral infection, they have been shown to activate the transcription factor IFN regulatory factor (IRF)-3, which in turn induces activation of the IFN-beta promoter. RIG-I/MDA5 recognizes dsRNA in the cytoplasm, whereas TLR3 resides in the cell surface membrane or endosomes to engage in extracytoplasmic recognition of dsRNA. Recent reports suggest that TLR3 induces cellular responses in epithelial cells in response to respiratory syncytial virus (RSV). The modus for TLR3 activation by RSV, however, remains unresolved. By small interference RNA gene-silencing technology and human cell transfectants, we have revealed that knockdown of NAK-associated protein 1 (NAP1) leads to the down-regulation of IFN-beta promoter activation >24 h after poly(I:C) or virus (RSV and vesicular stomatitis virus) treatment. NAP1 is located downstream of the adapter Toll-IL-1R homology domain-containing adapter molecule (TICAM)-1 (Toll/IL-1R domain-containing adapter-inducing IFN-beta) in the TLR3 pathway, but TICAM-1 and TLR3 did not participate in the IRF-3 and IFN-beta promoter activation by RSV infection. Virus-mediated activation of the IFN-beta promoter was largely abrogated by the gene silencing of IFN-beta promoter stimulator-1 (mitochondria antiviral signaling (MAVS), VISA, Cardif), the adapter of the RIG-I/MDA5 dsRNA-recognition proteins. In both the TLR and virus-mediated IFN-inducing pathways, IkappaB kinase-related kinase epsilon and TANK-binding kinase 1 participated in IFN-beta induction. Thus, RSV as well as other viruses induces replication-mediated activation of the IFN-beta promoter, which is intracellularly initiated by the RIG-I/MDA5 but not the TLR3 pathway. Both the cytoplasmic and TLR3-mediated dsRNA recognition pathways converge upon NAP1 for the activation of the IRF-3 and IFN-beta promoter.

Cytoplasm↗

PubMed2006

Protein kinase Cdelta binds TIRAP/Mal to participate in TLR signaling.

Toll-like receptor (TLR) family members recognize specific molecular patterns within pathogens. Signaling through TLRs results in a proximal event that involves direct binding of adaptor proteins to the receptors. We observed that TIRAP/Mal, an adaptor protein for TLR2 and TLR4, binds protein kinase Cdelta (PKCdelta). TIRAP/Mal GST-fusion protein and a TIRAP/Mal antibody were able to precipitate PKCdelta from rat peritoneal macrophage and THP1 cell lysates. Truncation mutants of TIRAP/Mal showed that the TIR domain of TIRAP/Mal is responsible for binding. TLR2- and TLR4-mediated phosphorylation of p38 MAPK, IKK, and IkappaB in RAW264.7 cells were abolished by depletion of PKCdelta. These results suggest that PKCdelta binding to TIRAP/Mal promotes TLR signaling events.

Animals↗

PubMed2006

Intelectin: a novel lipid raft-associated protein in the enterocyte brush border.

Intelectin is a mammalian Ca2+-dependent, D-galactosyl-specific lectin expressed in Paneth and goblet cells of the small intestine and proposed to serve a protective role in the innate immune response to parasite infection. In addition, it is structurally identical to the intestinal lactoferrin receptor known to reside in the enterocyte brush border. To clarify this apparent discrepancy with regard to localization, the aim of this work was to study the cellular and subcellular distribution of small intestinal intelectin by immunofluorescence and immunogold electron microscopy. Secretory granules of lysozyme-positive Paneth cells in the bottom of the crypts as well as goblet cells along the crypt-villus axis were intensively labeled with intelectin antibodies, but quantitatively, the major site of intelectin deposition was the enterocyte brush border. This membrane is organized in stable glycolipid-based lipid raft microdomains, and like the divalent lectin galectin-4, intelectin was enriched in microvillar "superrafts", i.e., membranes that resist solubilization with Triton X-100 at 37 degrees C. This strategic localization suggests that the trimeric intelectin, like galectin-4, serves as an organizer and stabilizer of the brush border membrane, preventing loss of digestive enzymes to the gut lumen and protecting the glycolipid microdomains from pathogens.

Animals↗

PubMed2006

The short consensus repeats 1 and 2, not the cytoplasmic domain, of human CD46 are crucial for infection of subgroup B adenovirus serotype 35.

Human CD46 (membrane cofactor protein) has recently been identified to be an attachment receptor for subgroup B adenoviruses (Ads); however, the precise interaction between human CD46 and subgroup B Ads are just beginning to be understood. In this study, to characterize the interaction between human CD46 and subgroup B Ads, varieties of mutant CD46 were tested for their ability to act as a receptor for Ad serotype 35 (Ad35), which belongs to subgroup B. In addition, we determined Ad35 vector-mediated transgene expression and cellular uptake of Ad35 vectors in the presence of a set of anti-CD46 antibodies. Our data demonstrated that the short consensus repeats (SCRs) 1 and 2 in human CD46 are important for interaction with Ad35, whereas the cytoplasmic domain of human CD46 was found not to be required for the function as an Ad35 receptor. Rather, a complete deletion of the cytoplasmic domain of human CD46 increased the transduction efficiencies of Ad35 vectors. This information should help in elucidation of the mechanism of subgroup B Ad infection, as well in the improvement of the subgroup B Ad vectors.

Adenoviruses, Human↗

PubMed2006

Role of Toll-like receptors in adjuvant-augmented immune therapies.

Effective therapeutic vaccines contain two primary constituents, antigen and adjuvant. Adjuvants consisting of microbial pattern molecules play a central role in vaccination. Successful vaccine requires efficient induction of antibody (Ab), type I interferons (IFN), cytokines/chemokines, cytotoxic T lymphocytes (CTL) and/or NK cells. Toll-like receptors (TLRs) in myeloid dendritic cells (mDC) essentially act as adjuvant receptors and sustain the molecular basis of adjuvant activity. Current consensus is that TLRs and their adapters introduce signals to preferentially induce IFN-alpha/beta, chemokines and proinflammatory cytokines, and mature mDC to augment antigen presentation. Although most of these data were obtained with mice, the results are presumed to be adaptable to humans. Whenever TLR pathway is activated in mDC, NK and/or CTL activation is promoted. For induction of antigen-specific CTL toward phagocytosed material, cross-priming must be induced in mDC, which is also sustained by TLR signaling in mDC. Since the TLR responses vary with different adjuvants, mDC functions are skewed depending on adjuvant-specific direction of mDC maturation. It appears that the directed maturation of mDC largely relies on selection of appropriate sets of TLRs and their adapter signaling pathways. Synthetic chimera molecules consisting of TLR agonists and target antigens are found to be effective in induction of CTL to eliminate target cells in vivo. Here, we review the role of human TLRs and adapters in a variety of host immune responses. We will also describe the relevance of adjuvants in the manipulation of receptors and adapters in vaccine therapy.

Journal Article↗

PubMed2006

The kinase complex responsible for IRF-3-mediated IFN-beta production in myeloid dendritic cells (mDC).

Type I interferons (IFN) IFN-alpha and -beta play a central role in the induction of antiviral immunity, which involves up-regulation or activation of a large number of IFN-inducible genes in host immune competent cells. Initial events in the antiviral response may occur in myeloid dendritic cells (mDCs), and the proteins expressed provoke early responses to cope with concomitant infection in the host. The participation of transcription factors IRF-3/7, AP1 and NF-kappaB in IFN-beta promoter activation in mDCs is well established. An initial trigger of this event is a viral dsRNA that is recognized by proteins with an RNA-binding motif. Toll-like receptor (TLR) 3 on membranes and RIG-Iin the cytoplasm are molecules with dsRNA-recognition ability. Our main aim in the present review is to describe how IRF-3 and/or NF-kappaB are activated through the initial recognition of dsRNA by these pattern-recognition receptors. By analogy to the trimolecular complex of IKKgamma, IKKalpha and IKKbeta, thus far, IRF-3-activated kinases have been reported to be kinase complexes with trimolecular assembly. Two kinases, TBK1 and IKKepsilon, are thought to be linked to regulatory subunit TANK or NAP1 with no kinase activity like IKKgamma. The TLR3 and RIG-I pathways converge upstream of IRF-3, possibly at NAP1, the regulatory subunit of IRF-3-activating kinase. Thus, a novel function of the regulatory subunit has emerged. These proteins are involved in the TLR3 and RIG-I pathways, and act as adapters bridging on the dsRNA-recognition unit and IRF-3-activating kinases in addition to their kinase-regulatory function. Here, we summarize the properties of regulatory subunits NAP1 and TANK, and the mode of activation of NF-kappaB and IRF-3 in conjunction with the unique properties of the TLR3 function.

Animals↗

PubMed2006

Analysis of the interaction between human TLR3 ectodomain and nucleic acids.

Toll-like receptor 3 (TLR3) recognizes dsRNA of viral origin and polyriboinosine-polyribocytidylic acid (poly (I:C)). TLR3 mediates the activation of IRF-3 and NF-kappaB and thereby the secretion of type I interferons and inflammatory cytokines. However, the mechanism of this activation is poorly understood. To study the molecular recognition events and biochemical interactions between TLR3 and dsRNA, human TLR3 ectodomain (ECD) fused with a signal peptide at the N-terminus and 6 x His-tag at the C-terminus (TLR3-ECD) was obtained by the baculovirus expression system. To examine the various nucleic acids binding to TLR3-ECD in vitro, a filter binding assay was carried out at pH 4.2-7.6. Interaction of TLR3-ECD with various nucleic acids (particularly dsRNA, in vitro transcripts of tRNA and HCV NS3 aptamer) required an acidic pH.

Binding Sites↗

PubMed2006

Isolation of RNA aptamers against human Toll-like receptor 3 ectodomain.

Toll-like receptor 3 (TLR3) detects double-stranded RNA (dsRNA) known as a universal viral molecular pattern and activates the antiviral immune response. While TLR3 preferentially recognizes polyriboinosine-polyribocytidylic acid (poly (I:C)), a sequence-specific dsRNA has not yet been shown to activate TLR3. To determine whether TLR3 preferentially recognizes some specific sequence that acts on the signaling pathway of TLR3, in vitro selection against human TLR3 ectodomain (TLR3 ECD) was performed. After seventh selection cycle, two major classes, Family-I and -II, were emerged from 64 clones with binding constants of about 3 nM. Although these aptamers bound to TLR3 ECD with high affinity in vitro, they did not have agonist and antagonist effects on TLR3 signaling in TLR3-transfected HEK293 cells. Further analyses of the structure/function relationship of these aptamers will be carried out by mutagenesis, RNase mapping and competition assay using poly (I:C).

Aptamers, Nucleotide↗

PubMed2005

A novel cyclohexene derivative, ethyl (6R)-6-[N-(2-Chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-242), selectively inhibits toll-like receptor 4-mediated cytokine production through suppression of intracellular signaling.

Proinflammatory mediators such as cytokines and NO play pivotal roles in various inflammatory diseases. To combat inflammatory diseases successfully, regulation of proinflammatory mediator production would be a critical process. In the present study, we investigated the in vitro effects of ethyl (6R)-6-[N-(2-chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-242), a novel small molecule cytokine production inhibitor, and its mechanism of action. In RAW264.7 cells and mouse peritoneal macrophages, TAK-242 suppressed lipopolysaccharide (LPS)-induced production of NO, tumor necrosis factor-alpha (TNF-alpha), and interleukin (IL)-6, with 50% inhibitory concentration (IC50) of 1.1 to 11 nM. TAK-242 also suppressed the production of these cytokines from LPS-stimulated human peripheral blood mononuclear cells (PBMCs) at IC50 values from 11 to 33 nM. In addition, the inhibitory effects on the LPS-induced IL-6 and IL-12 production were similar in human PBMCs, monocytes, and macrophages. TAK-242 inhibited mRNA expression of IL-6 and TNF-alpha induced by LPS and interferon-gamma in RAW264.7 cells. The phosphorylation of mitogen-activated protein kinases induced by LPS was also inhibited in a concentration-dependent manner. However, TAK-242 did not antagonize the binding of LPS to the cells. It is noteworthy that TAK-242 suppressed the cytokine production induced by Toll-like receptor (TLR) 4 ligands, but not by ligands for TLR2, -3, and -9. In addition, IL-1beta-induced IL-8 production from human PBMCs was not markedly affected by TAK-242. These data suggest that TAK-242 suppresses the production of multiple cytokines by selectively inhibiting TLR4 intracellular signaling. Finally, TAK-242 is a novel small molecule TLR4 signaling inhibitor and could be a promising therapeutic agent for inflammatory diseases, whose pathogenesis involves TLR4.

Animals↗

PubMed2005

Physical and functional interactions between STAT3 and Kaposi's sarcoma-associated herpesvirus-encoded LANA.

The Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded latency-associated nuclear antigen (LANA) is known to modulate viral and cellular gene expression. We show that LANA directly associates with an interleukin-6 signal transducer, signal transducer and activator of transcription 3 (STAT3) and that LANA enhances the transcriptional activity of STAT3. Coimmunoprecipitation studies documented a physical interaction between LANA and STAT3 in transiently transfected 293T cells as well as the KSHV-infected primary effusion lymphoma (PEL) cell line. Furthermore, small-interfering RNA-mediated reduction of LANA expression decreased the STAT3-dependent transcription in KSHV-positive PEL cells, whereas overexpression of LANA enhanced STAT3 activity in KSHV-negative B lymphoma cells. These data demonstrate that LANA is a transcriptional co-activator of STAT3, and may have implications for the pathogenesis of KSHV-associated diseases.

Antigens, Viral↗

PubMed2005

Fish soluble Toll-like receptor (TLR)5 amplifies human TLR5 response via physical binding to flagellin.

Fish has a soluble form of TLR5 ortholog (TLR5S), which does not exist in mammals. We identified TLR5S from rainbow trout and named rtTLR5S, which was about 38% homologous to the extracellular domains of human (hu) and mouse TLR5. Adjuvancy of rtTLR5S to flagellin response by human TLR5 (huTLR5) was tested in this study. A chimera constructed of rtTLR5S and the intracellular TIR of huTLR5 expressed on HeLa cells signaled the presence of various species of bacterial flagellin resulting in NK-kappaB activation. huTLR5S, when co-expressed with rtTLR5S in HeLa cells, augmented response to flagellin resulting in robust huTLR5-mediated NF-kappaB activation. Physical binding of flagellin to rtTLR5S was detected under the conditions where huTLR5 induced rtTLR5S-amplified NF-kappaB activation. Signal amplification by rtTLR5S was specific to huTLR5: no other huTLRs tested were responded to rtTLR5S. These results suggest that the soluble TLR5 serves as an adjuvant augmenting flagellin-TLR5-mediated NF-kappaB activation even in human.

Amino Acid Sequence↗

PubMed2005

Wild-type measles virus infection in human CD46/CD150-transgenic mice: CD11c-positive dendritic cells establish systemic viral infection.

We generated transgenic (TG) mice that constitutively express human CD46 (huCD46) and/or TLR-inducible CD150 (huCD150), which serve as receptors for measles virus (MV). These mice were used to study the spreading and pathogenicity of GFP-expressing or intact laboratory-adapted Edmonston and wild-type Ichinose (IC) strains of MV. Irrespective of the route of administration, neither type of MV was pathogenic to these TG mice. However, in ex vivo, limited replication of IC was observed in the spleen lymphocytes from huCD46/huCD150 TG and huCD150 TG, but not in huCD46 TG and non-TG mice. In huCD150-positive TG mouse cells, CD11c-positive bone marrow-derived myeloid dendritic cells (mDC) participated in MV-mediated type I IFN induction. The level and induction profile of IFN-beta was higher in mDC than the profile of IFN-alpha. Wild-type IC induced markedly high levels of IFN-beta compared with Edmonston in mDC, as opposed to human dendritic cells. We then generated huCD46/huCD150 TG mice with type I IFN receptor (IFNAR1)-/- mice. MV-bearing mDCs spreading to draining lymph nodes were clearly observed in these triple mutant mice in vivo by i.p. MV injection. Infectious lymph nodes were also detected in the double TG mice into which MV-infected CD11c-positive mDCs were i.v. transferred. This finding suggests that in the double TG mouse model mDCs once infected facilitate systemic MV spreading and infection, which depend on mDC MV permissiveness determined by the level of type I IFN generated via IFNAR1. Although these results may not simply reflect human MV infection, the huCD150/huCD46 TG mice may serve as a useful model for the analysis of MV-dependent modulation of mDC response.

Animals↗

PubMed2005

Regulator of complement activation (RCA) locus in chicken: identification of chicken RCA gene cluster and functional RCA proteins.

A 150-kb DNA fragment, which contains the gene of the chicken complement regulatory protein CREM (formerly named Cremp), was isolated from a microchromosome by screening bacterial artificial chromosome library. Within 100 kb of the cloned region, three complete genes encoding short consensus repeats (SCRs, motifs with tandemly arranged 60 aa) were identified by exon-trap method and 3'- or 5'-RACE. A chicken orthologue of the human gene 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2, which exists in close proximity to the regulator of complement activation genes in humans and mice, was located near this chicken SCR gene cluster. Moreover, additional genes encoding SCR proteins appeared to be present in this region. Three distinct transcripts were detected in RNA samples from a variety of chicken organs and cell lines. Two novel genes named complement regulatory secretory protein of chicken (CRES) and complement regulatory GPI-anchored protein of chicken (CREG) besides CREM were identified by cloning corresponding cDNA. Based on the predicted primary structures and properties of the expressed molecules, CRES is a secretory protein, whereas CREG is a GPI-anchored membrane protein. CREG and CREM were protected host cells from chicken complement-mediated cytolysis. Likewise, a membrane-bound form of CRES, which was artificially generated, also protected host cells from chicken complement. Taken together, the chicken possesses an regulator of complement activation locus similar to those of the mammals, and the gene products function as complement regulators.

Amino Acid Sequence↗

PubMed2005

Dendritic cell maturation induced by muramyl dipeptide (MDP) derivatives: monoacylated MDP confers TLR2/TLR4 activation.

6-O-acyl-muramyldipeptides (MDP) with various lengths of fatty acid chains were examined for their dendritic cell (DC) maturation activity expressed through TLRs. Judging from anti-TLR mAb/inhibitor-blocking analysis, MDP derivatives with a single octanoyl or stearoyl fatty acid chain were found to activate TLR2 and TLR4 on human DCs, although intact and diacylated MDP expressed no ability to activate TLRs. Human DC activation profiles by the monoacylated MDP were essentially similar to those by Calmette-Guerin (BCG)-cell wall skeleton (CWS) and BCG-peptidoglycan (PGN) based on their ability to up-regulate costimulators, HLA-DR, beta(2)-microglobulin, and allostimulatory MLR. Monoacylated MDP induced cytokines with similar profiles to BCG-CWS or -PGN, although their potency for induction of TNF-alpha, IL-12p40, and IL-6 was less than that of BCG-CWS or -PGN. The MDP derivatives initiated similar activation in normal mouse macrophages, but exhibited no effect on TLR2/4-deficient or MyD88-deficient mouse macrophages. Mutation of d-isoGln to l-isoGln in monoacylated MDP did not result in loss of the DC maturation activity, suggesting marginal participation of nucleotide-binding oligomerization domain 2, if any, in monoacyl MDP-dependent DC maturation. These results define the adjuvant activity of 6-O-acyl MDP compounds at the molecular level. They target TLR2/TLR4 and act through the MyD88-dependent pathway in DCs and macrophages. Hence, the unusual combined activation of TLR2 and TLR4 observed with Mycobacterium tuberculosis is in part reflected in the functional properties of monoacylated MDP compounds. These findings infer that the essential minimal requirement for TLR2/4-mediated adjuvancy of BCG lies within a modified MDP.

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMed2005

Pathways participating in activation of mouse uterine natural killer cells during pregnancy.

Activated natural killer (NK) cells proliferate in large numbers in murine mesometrial endometrium from Day 6 to Day 12 of gestation (term = 19 gestation days) to become the most abundant uterine lymphocytes. Early human decidua contains analogous CD56+/CD16- cells. Murine uterine (u)NK cells localize to decidua basalis and mesometrial lymphoid aggregate of pregnancy (MLAp). Decidua and MLAp are transient, pregnancy-associated tissues traversed by maternal arteries to the placentas. Uterine NK cells sensitize these arteries, facilitating their structural changes into high-volume conduits by Gestation Day 10 through release of interleukin (IL)-18, interferon (IFN)-gamma, vascular endothelial growth factor (VEGF), and other molecules. Little information exists concerning where, when, or how murine or human uNK cells become activated. In murine lymphoid tissue, three NK cell adaptor-mediated activation pathways are known: FcRgamma/CD3zeta, DNAX-activating protein (DAP) 10, and DAP12 (genes Fcgr3/Cd3z, Hcst, and Tyrobp, respectively). Expression of ligands for these receptors was demonstrated in implantation sites of normal C57BL/6J mice. Then, histological and morphometric analyses of implantation sites in mice with genetic inactivation of each pathway were undertaken. Implantation sites in DAP10-/- (Hcst deleted) mice appeared normal, spiral artery modification occurred, and concentrations of IFN-gamma in MLAp and decidua basalis were similar to those in time-matched C57BL/6J. Implantation sites of FcRgamma-/-/CD3zeta-/- (Fcgr3/Cd3z double knockout), DAP12 (Tyrobp)-loss-of-function-mutant, and FcRgamma-/-/DAP12-/- (Fcgr3/Tyrobp double knockout) mice differentiated abundant but functionally impaired uNK cells that could not modify spiral arteries. These data reveal key importance of FcRgamma-/-/CD3zeta-/- and thus maternal IgG during activation of mouse uNK cells and assign DAP12 but not DAP10 signaling contributions.

Adaptor Proteins, Signal Transducing↗