RESUMEN
Clusterin/Apolipoprotein J is a protein that is upregulated in a broad spectrum of diverse pathological processes. The predominant form is a secreted glycoprotein (sCLU) with cytoprotective and anti-inflammatory properties which shows enhanced expression in vascular smooth muscle cells (VSMC) following aortic injury and in atherosclerotic disease. Recent evidence indicates that during atherosclerosis, Toll-like receptors (TLRs) are activated in vascular cells by endogenous ligands. Here, we analyzed whether CLU expression in VSMC is controlled by TLRs, and stimulated by factors associated with or released by necrotic cells. Activation of TLR3 by the synthetic RNA analogue polyinosinic-polycytidylic acid (poly(I:C)) in CRL2018 VSMC and in mice led to induction of CLU mRNA and protein synthesis, respectively. In TLR3-deficient 10A yolk sac cells, induction of CLU by poly(I:C) challenge depended on the ectopic expression of human TLR3. In mice lacking the TLR3-signaling adaptor protein TRIF (TIR-domain-containing adaptor protein inducing IFN-ß) CLU induction by poly(I:C) was abrogated. In addition to poly(I:C) CLU gene expression in CRL2018 cells was induced by purified cellular RNA and RNA present in necrotic cell lysate. Our data indicate that cellular RNA following its release from necrotic cells in atherosclerotic lesions can act as an endogenous TLR3 ligand to induce CLU expression in VSMC and in vivo. Thus, they expand the view on TLR2 and TLR4 as known pro-atherosclerotic effectors toward TLR3. Conclusively, TLR3 activation induces expression of cytoprotective and anti-inflammatory CLU by VSMC and mice, to potentially counteract atherosclerotic pathology.
Asunto(s)
Extractos Celulares/farmacología , Clusterina/metabolismo , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/metabolismo , Necrosis/metabolismo , ARN/farmacología , Receptor Toll-Like 3/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Extractos Celulares/química , Línea Celular , Quimiocina CCL2/genética , Cloroquina/farmacología , Clusterina/sangre , Clusterina/genética , Clusterina/farmacología , Medios de Cultivo Condicionados/farmacología , Perros , Endocitosis/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Calor , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos del Músculo Liso/efectos de los fármacos , Poli I-C/farmacología , Desnaturalización Proteica , Proteínas/química , Proteínas/metabolismo , Ratas , Receptor Toll-Like 3/agonistas , Receptor Toll-Like 3/genética , Receptores Toll-Like/agonistas , Receptores Toll-Like/genética , TransfecciónRESUMEN
BACKGROUND: Common genetic variations in toll-like receptor 2 (TLR2), an innate pathogen recognition receptor, may influence the development of atopic diseases. So far, very little is known about the role of rare TLR2 mutations in these diseases. OBJECTIVE: We investigated the functional properties of six rare amino acid changes in TLR2 (and one amino acid change in a TLR2 pseudogene) and studied their effect on atopic sensitization and disease. METHODS: We identified rare TLR2 mutations leading to amino acid changes from databases. Functional effects of TLR2 variants were analyzed by NF-kappaB-dependent luciferase reporter assay and interleukin-8 enzyme linked immunosorbent assay in vitro. The frequency of these mutations was determined in a random sample of the general population (n = 368). Association with atopic diseases were studied in a cross sectional German study population (n = 3099). RESULTS: Three out of six mutations in the TLR2 gene altered receptor activity in vitro. Out of these, only the minor allele of R753Q occurred reasonably frequent in the German population (minor allele frequency 3%). The risk to develop atopy increased by 50% in carriers of the 753Q allele (P = 0.021) and total (P = 0.040) as well as allergen specific serum IgE levels (P = 0.011) were significantly elevated. CONCLUSION: The rare but functionally relevant mutation R753Q in TLR2 may significantly affect common conditions such as atopic sensitization in the general population.
Asunto(s)
Predisposición Genética a la Enfermedad , Hipersensibilidad Inmediata/genética , Receptor Toll-Like 2/metabolismo , Niño , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Genes Reporteros , Genotipo , Humanos , Immunoblotting , Mutación , Reacción en Cadena de la Polimerasa , Factores de Riesgo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Receptor Toll-Like 2/genéticaRESUMEN
Toll-like receptors (TLRs) exist on both myeloid and intrinsic renal cells contributing to the initiation of innate immunity during renal infection with uropathogenic Escherichia coli. Toll-interleukin 1 receptor (IL-1R) (TIR)8/SIGIRR is an orphan receptor of the TLR/IL-1R family, which suppresses TLR signaling of immune cells and is highly expressed in the kidney. Lack of TIR8/SIGIRR is associated with enhanced renal chemokine signaling upon exposure to lipopolysaccharide (LPS). This was because of TIR8/SIGIRR expression on resident intrarenal myeloid cells rather than tubular epithelial cells which express it on basolateral and luminal membranes. The lack of TIR8/SIGIRR does not enhance TLR/IL-1R signaling in tubular epithelial cells as was observed in monocytes. TIR8/SIGIRR is induced in monocytes treated with LPS or tumor necrosis factor and interferon-gamma in a dose-dependent manner but was downregulated in treated tubule epithelial cells. This cell type-specific regulation and function did not relate to mRNA splice variants but was associated with N- and O-glycosylation of the receptor in renal cells of myeloid and nonmyeloid origin. Our studies show that resident myeloid cells contribute to TLR-mediated antimicrobial immunity in the kidney and that this function is controlled by Tir8/sigirr. TIR8/SIGIRR does not suppress TLR signaling in tubular epithelial cells, which supports their role as sensors of microbial infection in the kidney.
Asunto(s)
Células Presentadoras de Antígenos/metabolismo , Células Epiteliales/metabolismo , Riñón/citología , Receptores de Interleucina-1/fisiología , Receptores Toll-Like/metabolismo , Animales , Células Presentadoras de Antígenos/inmunología , Células Cultivadas , Células Epiteliales/inmunología , Inmunidad Innata , Riñón/inmunología , Lipopolisacáridos/farmacología , Ratones , Ratones Noqueados , Monocitos , Células Mieloides , Receptores de Interleucina-1/inmunología , Transducción de Señal/inmunología , Receptores Toll-Like/inmunologíaRESUMEN
Mycoplasma arthritidis mitogen (MAM) is a superantigen (SAg) from M. arthritidis, an agent of murine toxic shock syndrome and arthritis. We previously demonstrated that C3H/HeJ and C3H/HeSnJ mice that differ in expression of TLR4 differed in immune reactivity to MAM. We show here that MAM directly interacts with TLR2 and TLR4 by using monoclonal antibodies to TLR2 and TLR4 which inhibit cytokine responses of THP-1 cells to MAM. Also, using macrophages from C3H substrains and TLR2-deficient mice, we confirmed that both TLR2 and TLR4 are used by MAM. Levels of IL-6 in supernatants of MAM-challenged macrophages were higher in mice which expressed only TLR2, lesser with both TLR2 and TLR4, and absent in mice lacking both TLR2 and TLR4. In addition, expression of TLR2 and TLR4 was moderately upregulated in wild-type cells but cells lacking TLR4 showed a fivefold increase in TLR2 expression. Further, blockade of TLR4 on macrophages of C3H/HeN mice with antibody greatly increased expression of TLR2 and release of IL-12p40 in response to MAM. These results indicate that the SAg, MAM, interacts with both TLR2 and TLR4 and that TLR4 signalling might downregulate the MAM/TLR2 inflammatory response.
Asunto(s)
Antígenos Bacterianos/inmunología , Mitógenos/inmunología , Receptores Inmunológicos/metabolismo , Superantígenos/inmunología , Animales , Antígenos , Línea Celular , Cricetinae , Citocinas/metabolismo , Femenino , Regulación de la Expresión Génica , Lipopolisacáridos/metabolismo , Lipoproteínas/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Mutantes , Mycoplasma/metabolismo , Proteínas , Receptores Inmunológicos/genética , Transducción de Señal , Receptor Toll-Like 2 , Receptor Toll-Like 4RESUMEN
Entamoeba histolytica is a human pathogen that may invade the intestinal mucosa, causing amoebic colitis or hepatic abscesses when the trophozoites travel through the portal circulation to the liver. Lipopeptidophosphoglycan (LPPG) is a molecular pattern of E. histolytica recognized by the human immune system. Here we report that LPPG is exposed on the cell surface of E. histolytica trophozoites, and is recognized by the host through toll-like receptor (TLR) 2 and TLR4. Correspondingly, human embryonic kidney (HEK)-293 cells were rendered LPPG responsive through overexpression of TLR2 or TLR4/MD2. Moreover, co-expression of CD14 enhanced LPPG signal transmission through TLR2 and TLR4. The interaction of LPPG with TLR2 and TLR4 resulted in activation of NF-kappaB and release of interleukin (IL)-10, IL-12p40, tumour necrosis factor (TNF)-alpha, and IL-8 from human monocytes. Consistent with these findings, responsiveness of mouse macrophages lacking TLR2 expression (TLR2-/-) or functional TLR4 (TLR4d/d) to E. histolytica LPPG challenge was impaired while double deficient macrophages were unresponsive. In contrast to wild-type control and TLR2-/- animals succumbing to lethal shock syndrome, TLR4d/d mice were resistant to systemic LPPG challenge-induced pathology.
Asunto(s)
Antígenos de Protozoos/inmunología , Entamoeba histolytica/inmunología , Inmunidad Innata , Glicoproteínas de Membrana/inmunología , Peptidoglicano/inmunología , Fosfolípidos/inmunología , Receptores de Superficie Celular/inmunología , Receptores Inmunológicos/inmunología , Animales , Línea Celular , Femenino , Humanos , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Subunidad p40 de la Interleucina-12 , Interleucina-8/metabolismo , Macrófagos/inmunología , Ratones , Ratones Endogámicos C3H , Monocitos/inmunología , FN-kappa B/metabolismo , Subunidades de Proteína/metabolismo , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptores Toll-Like , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Aspergillus fumigatius is a ubiquitous saprophytic fungus that has become the most prevalent airborne fungal pathogen for immunocompromised patients during the last two decades. In this report we have analysed how macrophages recognize this microorganism. Using transfected human HEK 293 cells we demonstrate that NF-kappaB-dependent promoter activation triggered by A. fumigatus is mediated by Toll-like receptors TLR2 and TLR4, whereas no activation was observed in cells overexpressing other distinct TLR proteins (TLR1, TLR3, TLR5-10). Using macrophages derived from mice lacking TLR2 expression, expressing defective TLR4 or both we found that A. fumigatus conidia and hyphae induce NF-kappaB translocation, release of pro-inflammatory molecules, like TNFalpha, and the chemoattractant MIP-2 in a TLR2- and TLR4-dependent manner. Recognition of A. niger and A. fumigatus, was similar in terms of the parameters analysed, suggesting that pathogenic and non-pathogenic aspergilli are sensed by macrophages in a similar fashion. Finally, we found that recruitment of neutrophils is severely impaired in mice lacking both functional TLR2 and TLR4, but is less impaired in single TLR2- or TLR4-deficient mice, providing evidence that both receptors are required for an optimal immune response to Aspergillus in vivo.
Asunto(s)
Aspergillus fumigatus/patogenicidad , Activación de Macrófagos/fisiología , Glicoproteínas de Membrana/fisiología , Receptores de Superficie Celular/fisiología , Transporte Activo de Núcleo Celular , Animales , Línea Celular , Quimiocina CXCL2 , Quimiocinas/biosíntesis , Citocinas/biosíntesis , Humanos , Técnicas In Vitro , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C3H , Ratones Noqueados , FN-kappa B/metabolismo , Neutrófilos/fisiología , Óxido Nítrico/biosíntesis , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/genética , Receptor Toll-Like 1 , Receptor Toll-Like 2 , Receptor Toll-Like 3 , Receptor Toll-Like 4 , Receptor Toll-Like 5 , Receptores Toll-Like , TransfecciónRESUMEN
Toll-like receptor (TLR) 2 is a member of the vertebrate protein family of TLRs that has been studied in substantial detail over the last years. The extracellular domain of the type I receptor molecule TLR2 contains 18 to 20 leucine rich repeat (LRR) and LRR like motives. The intracellular domain of TLR2 contains a Toll/IL-1 receptor/resistance protein typical TIR domain. After the first implication of TLR4 in immunity thereinafter followed by the discovery of the lipopolysaccharide signal transducer function of TLR4, TLR2 was the first of ten mammalian TLRs proven to be directly involved in recognition of pathogen associated molecular patterns (PAMPs). Among the TLR2 specific agonists are microbial products representing broad groups of species such as Gram-positive and Gram-negative bacteria, as well as mycobacteria, spirochetes, and mycoplasm. PAMP induced phagosomal localization of TLR2 and TLR2 dependent apoptosis have been shown. Complex formation with other molecules involved in pattern recognition such as CD14, MD2, TLR1, and TLR6 has been implicated for TLR2. Surprisingly even proteinaceous host material such as heat shock protein (HSP) 60 has been demonstrated to activate cells through TLR2. Thus, TLR2 may be a sensor and inductor of specific defense processes, including oxidative stress and cellular necrosis initially spurred by microbial compounds. Here we summarize the current knowledge on the structure and function of TLR2, which is far from being complete. Detailed understanding of the biology of TLR2 will probably contribute to the characterization of a number of infectious diseases and potentially help in the development of novel intervention strategies.
Asunto(s)
Proteínas de Drosophila , Glicoproteínas de Membrana/inmunología , Receptores de Superficie Celular/inmunología , Secuencia de Aminoácidos , Animales , Drosophila melanogaster , Regulación de la Expresión Génica , Humanos , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Receptores de Superficie Celular/genética , Transducción de Señal , Receptor Toll-Like 1 , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptor Toll-Like 5 , Receptores Toll-Like , VertebradosRESUMEN
Innate immunity initiates protection of the host organism against invasion and subsequent multiplication of microbes by specific recognition. Germ line-encoded receptors have been identified for microbial products such as mannan, lipopeptide, peptidoglycan (PGN), lipoteichoic acid (LTA), lipopolysaccharide (LPS), and CpG-DNA. The Drosophila Toll protein has been shown to be involved in innate immune response of the adult fruitfly. Members of the family of Toll-like receptors (TLRs) in vertebrates have been implicated as pattern recognition receptors (PRRs). Ten TLRs are known and six of these have been demonstrated to mediate cellular activation by distinct microbial products. TLR4 has been implicated as activator of adaptive immunity, and analysis of systemic LPS responses in mice led to the identification of LPS-resistant strains instrumental in its identification as a transmembrane LPS signal transducer. Structural similarities between TLRs and receptor molecules involved in immune responses such as CD14 and the IL-1 receptors (IL-1Rs), as well as functional analysis qualified TLR2 as candidate receptor for LPS and other microbial products. Targeted disruption of the TLR9 gene in mice led to identification of TLR9 as CpG-DNA signal transducer. Involvement of TLR5 in cell activation by bacterial flagellin has been demonstrated. Further understanding of recognition and cellular signaling activated through the ancient host defense system represented by Toll will eventually lead to means for its therapeutic modulation.
Asunto(s)
Proteínas de Drosophila , Inmunidad Innata , Glicoproteínas de Membrana/inmunología , Receptores de Superficie Celular/inmunología , Transducción de Señal/inmunología , Secuencia de Aminoácidos , Animales , Bacterias/patogenicidad , Línea Celular , Humanos , Lipopolisacáridos/inmunología , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptor Toll-Like 5 , Receptor Toll-Like 9 , Receptores Toll-LikeRESUMEN
The Toll-like receptor (TLR) family consists of phylogenetically conserved transmembrane proteins, which function as mediators of innate immunity for recognition of pathogen-derived ligands and subsequent cell activation via the Toll/IL-1R signal pathway. Here, we show that human TLR9 (hTLR9) expression in human immune cells correlates with responsiveness to bacterial deoxycytidylate-phosphate-deoxyguanylate (CpG)-DNA. Notably "gain of function" to immunostimulatory CpG-DNA is achieved by expressing TLR9 in human nonresponder cells. Transfection of either human or murine TLR9 conferred responsiveness in a CD14- and MD2-independent manner, yet required species-specific CpG-DNA motifs for initiation of the Toll/IL-1R signal pathway via MyD88. The optimal CpG motif for hTLR9 was GTCGTT, whereas the optimal murine sequence was GACGTT. Overall, these data suggest that hTLR9 conveys CpG-DNA responsiveness to human cells by directly engaging immunostimulating CpG-DNA.
Asunto(s)
Islas de CpG , ADN Bacteriano/inmunología , Proteínas de Unión al ADN/inmunología , Receptores de Superficie Celular/inmunología , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN , ADN Complementario , Proteínas de Unión al ADN/genética , Prueba de Complementación Genética , Humanos , Ratones , Datos de Secuencia Molecular , Receptores de Superficie Celular/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Especificidad de la Especie , Receptor Toll-Like 9RESUMEN
Heat shock proteins (HSPs) require no adjuvant to confer immunogenicity to bound peptides, as if they possessed an intrinsic "danger" signature. To understand the proinflammatory nature of HSP, we analyzed signaling induced by human and chlamydial HSP60. We show that both HSP60s activate the stress-activated protein kinases p38 and JNK1/2, the mitogen-activated protein kinases ERK1/2, and the I-kappaB kinase (IKK). Activation of JNK and IKK proceeds via the Toll/IL-1 receptor signaling pathway involving MyD88 and TRAF6. Human fibroblasts transfected with TLR2 or TLR4 plus MD-2 gain responsiveness to HSP60, while TLR2- or TLR4-defective cells display impaired responses. Initiation of signaling requires endocytosis of HSP60 that is effectively inhibited by serum component(s). The results revealed that adjuvanticity of HSP60 operates similar to that of classical pathogen-derived ligands.
Asunto(s)
Proteínas Bacterianas , Chaperoninas/metabolismo , Proteínas de Drosophila , Endocitosis , Glicoproteínas de Membrana/fisiología , Receptores de Superficie Celular/fisiología , Receptores Inmunológicos , Receptores de Interleucina-1/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Antígenos de Diferenciación/fisiología , Línea Celular , Chaperonina 60 , Humanos , Ratones , Factor 88 de Diferenciación Mieloide , Proteínas/fisiología , Factor 6 Asociado a Receptor de TNF , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptores Toll-Like , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Recently Toll-like receptors (TLRs) have been found to be involved in cellular activation by microbial products, including lipopolysaccharide, lipoproteins, and peptidoglycan. Although for these ligands the specific transmembrane signal transducers TLR-4, TLR-2, or TLR-2 and -6 have now been identified, the molecular basis of recognition of lipoteichoic acids (LTAs) and related glycolipids has not been completely understood. In order to determine the role of TLRs in immune cell activation by these stimuli, experiments involving TLR-2-negative cell lines, TLR-expression plasmids, macrophages from TLR-4-deficient C3H/HeJ-mice, and inhibitory TLR-4/MD-2 antibodies were performed. Glycolipids from Treponema maltophilum and Treponema brennaborense, as well as highly purified LTAs from Staphylococcus aureus and Bacillus subtilis exhibited TLR-2 dependence in nuclear factor kappaB activation and cytokine induction; however, T. brennaborense additionally appeared to signal via TLR-4. Fractionation of the T. brennaborense glycolipids by hydrophobic interaction chromatography and subsequent cell stimulation experiments revealed two peaks of activity, one exhibiting TLR-2-, and a second TLR-4-dependence. Furthermore, we show involvement of the signaling molecules MyD88 and NIK in cell stimulation by LTAs and glycolipids by dominant negative overexpression experiments. In summary, the results presented here indicate that TLR-2 is the main receptor for Treponema glycolipid and LTA-mediated inflammatory response.
Asunto(s)
Proteínas de Drosophila , Glucolípidos/metabolismo , Lipopolisacáridos/metabolismo , Glicoproteínas de Membrana/fisiología , FN-kappa B/metabolismo , Receptores de Superficie Celular/fisiología , Ácidos Teicoicos/metabolismo , Treponema/metabolismo , Animales , Línea Celular , Interleucina-6/biosíntesis , Ratones , Transporte de Proteínas , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptores Toll-LikeRESUMEN
Toll-like receptors (TLRs) have been found to be key elements in pathogen recognition by the host immune system. Dendritic cells (DCs) are crucial for both innate immune responses and initiation of acquired immunity. Here we focus on the potential involvement of TLR ligand interaction in DC maturation. TLR2 knockout mice and mice carrying a TLR4 mutation (C3H/HeJ) were investigated for DC maturation induced by peptidoglycan (PGN), lipopolysaccharide (LPS), or lipoteichoic acids (LTAs). All stimuli induced maturation of murine bone marrow-derived DCs in control mice. TLR2(-)/- mice lacked maturation upon stimulation with PGN, as assessed by expression of major histocompatibility complex class II, CD86, cytokine, and chemokine production, fluorescein isothiocyanate-dextran uptake, and mixed lymphocyte reactions, while being completely responsive to LPS. A similar lack of maturation was observed in C3H/HeJ mice upon stimulation with LPS. DC maturation induced by LTAs from two different types of bacteria was severely impaired in TLR2(-)/-, whereas C3H/HeJ mice responded to LTAs in a manner similar to wild-type mice. We demonstrate that DC maturation is induced by stimuli from Gram-positive microorganisms, such as PGN and LTA, with similar efficiency as by LPS. Finally, we provide evidence that TLR2 and TLR4 interaction with the appropriate ligand is essential for bacteria-induced maturation of DCs.
Asunto(s)
Células Dendríticas/citología , Células Dendríticas/fisiología , Proteínas de Drosophila , Glicoproteínas de Membrana/fisiología , Receptores de Superficie Celular/fisiología , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Ligandos , Lipopolisacáridos/farmacología , Ratones , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptores Toll-LikeRESUMEN
This study was done to elucidate the signal transduction pathway of interleukin-8 (IL-8) induction by gram-positive bacteria. Bacteria (micrococci) and peptidoglycan (PGN) induced transcription of IL-8 in HEK293 cells expressing Toll-like receptor 2 (TLR2) and CD14 but not in those expressing TLR1 or TLR4. A mutation within the NF-kappaB site in the IL-8 promoter abrogated transcriptional induction of IL-8 by the two stimulants. Dominant negative myeloid differentiation protein (MyD88), IL-1 receptor-associated kinase (IRAK), NFkappaB-inducing kinase (NIK), and IkappaB kinase (IKK) mutant forms completely inhibited micrococcus- and PGN-induced activation of NF-kappaB and expression of the gene for IL-8. Induction of NF-kappaB was partially inhibited by dominant negative tumor necrosis factor receptor-associated kinase 6 (TRAF6) but not TRAF2, whereas induction of IL-8 gene was partially inhibited by both TRAF6 and TRAF2. These data indicate that micrococci and PGN induce TLR2-dependent activation of the gene for IL-8 and that this activation requires MyD88, IRAK, NIK, IKK, and NF-kappaB and may also utilize TRAF6 and, to a lesser extent, TRAF2.
Asunto(s)
Antígenos de Diferenciación/fisiología , Proteínas de Drosophila , Interleucina-8/genética , Glicoproteínas de Membrana/fisiología , Micrococcus/fisiología , FN-kappa B/fisiología , Peptidoglicano/farmacología , Proteínas Quinasas/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas/fisiología , Receptores de Superficie Celular/fisiología , Receptores Inmunológicos , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales , Línea Celular , Humanos , Quinasa I-kappa B , Quinasas Asociadas a Receptores de Interleucina-1 , Factor 88 de Diferenciación Mieloide , Factor 6 Asociado a Receptor de TNF , Receptor Toll-Like 1 , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptores Toll-Like , Activación Transcripcional , Quinasa de Factor Nuclear kappa BRESUMEN
Leptospira interrogans are zoonotic pathogens that have been linked to a recent increased incidence of morbidity and mortality in highly populated tropical urban centers. They are unique among invasive spirochetes in that they contain outer membrane lipopolysaccharide (LPS) as well as lipoproteins. Here we show that both these leptospiral outer membrane constituents activate macrophages through CD14 and the Toll-like receptor 2 (TLR2). Conversely, it seems that TLR4, a central component for recognition of Gram-negative LPS, is not involved in cellular responses to L. interrogans. We also show that for intact L. interrogans, it is LPS, not lipoprotein, that constitutes the predominant signaling component for macrophages through a TLR2 pathway. These data provide a basis for understanding the innate immune response caused by leptospirosis and demonstrate a new ligand specificity for TLR2.
Asunto(s)
Proteínas de Drosophila , Leptospira interrogans/inmunología , Leptospira interrogans/patogenicidad , Lipopolisacáridos/toxicidad , Activación de Macrófagos/efectos de los fármacos , Glicoproteínas de Membrana/inmunología , Receptores de Superficie Celular/inmunología , Animales , Células CHO , Línea Celular , Cricetinae , Humanos , Leptospirosis/inmunología , Receptores de Lipopolisacáridos/metabolismo , Lipopolisacáridos/inmunología , Lipoproteínas/inmunología , Activación de Macrófagos/inmunología , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/genética , Transducción de Señal , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptores Toll-LikeRESUMEN
MD-2 is associated with Toll-like receptor 4 (TLR4) on the cell surface and enables TLR4 to respond to LPS. We tested whether MD-2 enhances or enables the responses of both TLR2 and TLR4 to Gram-negative and Gram-positive bacteria and their components. TLR2 without MD-2 did not efficiently respond to highly purified LPS and LPS partial structures. MD-2 enabled TLR2 to respond to nonactivating protein-free LPS, LPS mutants, or lipid A and enhanced TLR2-mediated responses to both Gram-negative and Gram-positive bacteria and their LPS, peptidoglycan, and lipoteichoic acid components. MD-2 enabled TLR4 to respond to a wide variety of LPS partial structures, Gram-negative bacteria, and Gram-positive lipoteichoic acid, but not to Gram-positive bacteria, peptidoglycan, and lipopeptide. MD-2 physically associated with TLR2, but this association was weaker than with TLR4. MD-2 enhanced expression of both TLR2 and TLR4, and TLR2 and TLR4 enhanced expression of MD-2. Thus, MD-2 enables both TLR4 and TLR2 to respond with high sensitivity to a broad range of LPS structures and to lipoteichoic acid, and, moreover, MD-2 enhances the responses of TLR2 to Gram-positive bacteria and peptidoglycan, to which the TLR4-MD-2 complex is unresponsive.
Asunto(s)
Adyuvantes Inmunológicos/fisiología , Antígenos de Superficie/fisiología , Pared Celular/inmunología , Proteínas de Drosophila , Bacterias Gramnegativas/inmunología , Bacterias Grampositivas/inmunología , Lipopolisacáridos/inmunología , Glicoproteínas de Membrana/fisiología , Receptores de Superficie Celular/fisiología , Adyuvantes Inmunológicos/metabolismo , Antígenos de Superficie/metabolismo , Linfocitos B/inmunología , Línea Celular , Humanos , Lipopolisacáridos/aislamiento & purificación , Lipopolisacáridos/metabolismo , Lipopolisacáridos/toxicidad , Lipoproteínas/inmunología , Activación de Linfocitos , Antígeno 96 de los Linfocitos , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Peptidoglicano/inmunología , Polisacáridos/inmunología , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Solubilidad , Ácidos Teicoicos/inmunología , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptores Toll-Like , TransfecciónRESUMEN
A missense mutation in the cytoplasmic domain of the Toll-like receptor-4 (TLR-4) has been identified as the defect responsible for lipopolysaccharide (LPS) hyporesponsiveness in C3H/HeJ mice. TLR-4 and TLR-2 have recently been implicated in LPS signaling in studies where these receptors were overexpressed in LPS non-responsive 293 human embryonic kidney cells. However, the signaling role of TLR-4 or TLR-2 in human cells with natural LPS response remains largely undefined. Here we show that human dermal microvessel endothelial cells (HMEC) and human umbilical vein endothelial cells express predominantly TLR-4 but very weak TLR-2 and respond vigorously to LPS but not to Mycobacterium tuberculosis 19-kDa lipoprotein. Transient transfection of non-signaling mutant forms of TLR-4 and anti-TLR-4 monoclonal antibody inhibited LPS-induced NF-kappaB activation in HMEC, while a monoclonal antibody against TLR-2 was ineffective. In contrast to LPS responsiveness, the ability of HMEC to respond to 19-kDa lipoprotein correlated with the expression of TLR-2. Transfection of TLR-2 into HMEC conferred responsiveness to 19-kDa lipoprotein. These data indicate that TLR-4 is the LPS signaling receptor in HMEC and that human endothelial cells (EC) express predominantly TLR-4 and weak TLR-2, which may explain why they do not respond to 19-kDa lipoprotein. The differential expression of TLRs on human EC may have important implications in the participation of vascular EC in innate immune defense mechanisms against various infectious pathogens, which may use different TLRs to signal.
Asunto(s)
Proteínas de Drosophila , Endotelio Vascular/efectos de los fármacos , Lipopolisacáridos/farmacología , Glicoproteínas de Membrana/fisiología , FN-kappa B/metabolismo , Receptores de Superficie Celular/fisiología , Animales , Células Cultivadas , Endotelio Vascular/metabolismo , Humanos , Receptores de Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Piel/citología , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptores Toll-LikeRESUMEN
The agent of Lyme disease, Borrelia burgdorferi, produces membrane lipoproteins possessing potent inflammatory properties linked to disease pathology. The recent association of toll-like receptors (TLR) 2 and 4 with LPS responses prompted the examination of TLR involvement in lipoprotein signaling. The ability of human cell lines to respond to lipoproteins was correlated with the expression of TLR2. Transfection of TLR2 into cell lines conferred responsiveness to lipoproteins, lipopeptides, and sonicated B. burgdorferi, as measured by nuclear translocation of NF-kappaB and cytokine production. The physiological importance of this interaction was demonstrated by the 10-fold greater sensitivity of TLR2-transfected cells to lipoproteins than LPS. Futhermore, TLR2-dependent signaling by lipoproteins was facilitated by CD14. These data indicate that TLR2 facilitates the inflammatory events associated with Lyme arthritis. In addition, the widespread expression of lipoproteins by other bacterial species suggests that this interaction may have broad implications in microbial inflammation and pathogenesis.
Asunto(s)
Grupo Borrelia Burgdorferi/inmunología , Proteínas de Drosophila , Lipoproteínas/fisiología , Glicoproteínas de Membrana/fisiología , Receptores de Superficie Celular/fisiología , Transducción de Señal/inmunología , Antígenos de Superficie/genética , Antígenos de Superficie/metabolismo , Antígenos de Superficie/fisiología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Membrana Bacteriana Externa/fisiología , Vacunas Bacterianas , Transporte Biológico/inmunología , Línea Celular , Relación Dosis-Respuesta Inmunológica , Humanos , Inflamación/inmunología , Interleucina-8/biosíntesis , Lipopolisacáridos/farmacología , Lipoproteínas/inmunología , Lipoproteínas/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , FN-kappa B/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacología , Receptor Toll-Like 2 , Receptores Toll-Like , Transfección , Células Tumorales CultivadasRESUMEN
Toll-like receptor (TLR) 2 and TLR4 have been implicated in the responses of cells to LPS (endotoxin). CD14-transfected Chinese hamster ovary (CHO)-K1 fibroblasts (CHO/CD14) are exquisitely sensitive to endotoxin. Sequence analysis of CHO-TLR2, compared with human and mouse TLR2, revealed a single base pair deletion. This frameshift mutation resulted in an alternative stop codon, encoding a protein devoid of transmembrane and intracellular domains. CHO-TLR2 cDNA failed to enable LPS signaling upon transient transfection into human epithelial kidney 293 cells. Site-directed mutagenesis of CHO-TLR2 enabled expression of a presumed full-length hamster TLR2 that conferred LPS responsiveness in human epithelial kidney 293 cells. Genomic TLR2 DNA from primary hamster macrophages also contained the frameshift mutation found in CHO fibroblasts. Nevertheless, hamster peritoneal macrophages were found to respond normally to LPS, as evidenced by the induction of cytokines. These results imply that expression of TLR2 is sufficient but not essential for mammalian responses to endotoxin.
Asunto(s)
Alelos , Células CHO/metabolismo , Proteínas de Drosophila , Lipopolisacáridos/farmacología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO/inmunología , Clonación Molecular , Cricetinae , Femenino , Humanos , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/deficiencia , Ratones , Datos de Secuencia Molecular , Receptores de Superficie Celular/química , Receptores de Superficie Celular/deficiencia , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Transducción de Señal/genética , Transducción de Señal/inmunología , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptores Toll-LikeRESUMEN
The life-threatening complications of sepsis in humans are elicited by infection with Gram-negative as well as Gram-positive bacteria. Recently, lipopolysaccharide (LPS), a major biologically active agent of Gram-negative bacteria, was shown to mediate cellular activation by a member of the human Toll-like receptor family, Toll-like receptor (TLR) 2. Here we investigate the mechanism of cellular activation by soluble peptidoglycan (sPGN) and lipoteichoic acid (LTA), main stimulatory components of Gram-positive bacteria. Like LPS, sPGN and LTA bind to the glycosylphosphatidylinositol-anchored membrane protein CD14 and induce activation of the transcription factor NF-kappaB in host cells like macrophages. We show that whole Gram-positive bacteria, sPGN and LTA induce the activation of NF-kappaB in HEK293 cells expressing TLR2 but not in cells expressing TLR1 or TLR4. The sPGN- and LTA-induced NF-kappaB activation was not inhibited by polymyxin B, an antibiotic that binds and neutralizes LPS. Coexpression together with membrane CD14 enhances sPGN signal transmission through TLR2. In contrast to LPS signaling, activation of TLR2 by sPGN and LTA does not require serum. These findings identify TLR2 as a signal transducer for sPGN and LTA in addition to LPS.