RESUMO
Lipopolysaccharide (LPS) is the main inducer of shock and death in Gram-negative sepsis. Recent evidence suggests that LPS-induced signal transduction begins with CD14-mediated activation of 1 or more Toll-like receptors (TLRs). The lipid A analogues lipid IVa and Rhodobacter sphaeroides lipid A (RSLA) exhibit an uncommon species-specific pharmacology. Both compounds inhibit the effects of LPS in human cells but display LPS-mimetic activity in hamster cells. We transfected human TLR4 or human TLR2 into hamster fibroblasts to determine if either of these LPS signal transducers is responsible for the species-specific pharmacology. RSLA and lipid IVa strongly induced NF-kappaB activity and IL-6 release in Chinese hamster ovary fibroblasts expressing CD14 (CHO/CD14), but these compounds antagonized LPS antagonists in CHO/CD14 fibroblasts that overexpressed human TLR4. No such antagonism occurred in cells overexpressing human TLR2. We cloned TLR4 from hamster macrophages and found that human THP-1 cells expressing the hamster TLR4 responded to lipid IVa as an LPS mimetic, as if they were hamster in origin. Hence, cells heterologously overexpressing TLR4 from different species acquired a pharmacological phenotype with respect to recognition of lipid A substructures that corresponded to the species from which the TLR4 transgene originated. These data suggest that TLR4 is the central lipid A-recognition protein in the LPS receptor complex.
Assuntos
Proteínas de Drosophila , Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Células CHO , Cricetinae , Glicolipídeos/metabolismo , Humanos , Ligantes , Lipídeo A/análogos & derivados , Lipídeo A/metabolismo , Receptores de Lipopolissacarídeos/genética , Lipopolissacarídeos/antagonistas & inibidores , Macrófagos/citologia , Macrófagos/metabolismo , Glicoproteínas de Membrana/genética , Mimetismo Molecular , Receptores de Superfície Celular/genética , Proteínas Recombinantes/metabolismo , Rhodobacter sphaeroides , Transdução de Sinais , Especificidade da Espécie , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Receptores Toll-LikeRESUMO
In 1997, a human homologue of the Drosophila Toll protein was described, a protein later to be designated Toll-like receptor 4 (TLR4). Since that time, additional human and murine TLR proteins have been identified. Mammalian TLR proteins appear to represent a conserved family of innate immune recognition receptors. These receptors are coupled to a signaling pathway that is conserved in mammals, insects, and plants, resulting in the activation of genes that mediate innate immune defenses. Numerous studies have now identified a wide variety of chemically-diverse bacterial products that serve as putative ligands for TLR proteins. More recent studies have identified the first endogenous protein ligands for TLR proteins. TLR signaling represents a key feature of innate immune response to pathogen invasion.
Assuntos
Proteínas de Drosophila , Lipopolissacarídeos/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Drosophila/genética , Drosophila/metabolismo , Humanos , Proteínas de Insetos/fisiologia , Ligantes , Lipopolissacarídeos/química , Mamíferos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiologia , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Receptores de Superfície Celular/genética , Homologia de Sequência de Aminoácidos , Receptor 4 Toll-Like , Receptor 5 Toll-Like , Receptor 6 Toll-Like , Receptores Toll-LikeRESUMO
Stimulation of macrophages by lipopolysaccharide (LPS) leads to the rapid activation of MAP kinases (MAPK) and the subsequent induction of cytokine gene expression. We sought to determine whether LPS-inducible cytokine genes were differentially regulated in macrophages derived from different tissues. Our studies revealed that PD98059, an inhibitor of the extracellular-regulated kinase (ERK) pathway, blocked LPS-induced activation of tumor necrosis factor alpha (TNF-alpha) gene expression in a murine cell line derived from alveolar macrophages but not in a nonpulmonary macrophage cell line. These findings were confirmed using primary murine alveolar and peritoneal macrophages. This suggests that the TNF-alpha promoter contains MAPK-dependent and -independent regulatory elements that are used in a cell type-specific manner. We also found that differences in MAPK-regulated signaling were not mediated by NF-KB, LITAF, Egr-1, CREB, or ATF2/ c-Jun. Together, these studies demonstrate that transcriptional activation of the TNF-alpha gene requires the ERK signaling cascade in selected macrophage populations.
Assuntos
Proteínas Imediatamente Precoces , Sistema de Sinalização das MAP Quinases , Macrófagos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Transcrição Gênica , Fator de Necrose Tumoral alfa/genética , Fator 2 Ativador da Transcrição , Animais , Linhagem Celular , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína 1 de Resposta de Crescimento Precoce , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Lipopolissacarídeos/farmacologia , MAP Quinase Quinase 1 , Macrófagos/efeitos dos fármacos , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , NF-kappa B/metabolismo , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Capuzes de RNA , RNA Mensageiro/biossíntese , Elementos de Resposta , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
We previously reported that gram-negative bacterial lipopolysaccharide (LPS) activates cells via Toll-like receptor (TLR) 4, whereas the mycobacterial cell wall glycolipid lipoarabinomannan (LAM) activates cells via TLR2. We also identified a secreted TLR2 agonist activity in short-term culture filtrates of Mycobacterium tuberculosis bacilli, termed soluble tuberculosis factor (STF). Here we show that STF contains mannosylated phosphatidylinositol (PIM) and that purified PIM possesses TLR2 agonist activity. Stimulation of RAW 264.7 macrophages by LPS, LAM, STF, and PIM rapidly activated nuclear factor (NF)-kappaB, activator protein-1 (AP-1), and mitogen-activated protein (MAP) kinases. These TLR agonists induced similar levels of NF-kappaB and AP-1 DNA-binding activity, as well as trans-activation function. Unexpectedly, these TLR agonists induced tumor necrosis factor alpha secretion, whereas only LPS was capable of inducing interleukin-1beta and nitric oxide secretion. Thus, different TLR proteins are still capable of activating distinct cellular responses, in spite of their shared capacities to activate NF-kappaB, AP-1, and MAP kinases.
Assuntos
Proteínas de Drosophila , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Glicoproteínas de Membrana/agonistas , Fosfatidilinositóis/farmacologia , Receptores de Superfície Celular/agonistas , Transdução de Sinais/efeitos dos fármacos , Animais , Células CHO , Cricetinae , Cricetulus , Ativação Enzimática/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-1/biossíntese , Interleucina-1/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Mycobacterium tuberculosis/química , NF-kappa B/metabolismo , Óxido Nítrico/biossíntese , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Fosfatidilinositóis/isolamento & purificação , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Receptores Toll-Like , Fator de Transcrição AP-1/metabolismo , Transfecção , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genéticaRESUMO
Beginning in 1997 with the identification of the first human homologue of the Drosophila protein Toll, a family of related molecules have been identified in both humans and other mammals. These Toll-like receptor (TLR) proteins appear to represent a conserved family of innate immune recognition receptors. TLR proteins share extended homology with receptors for the cytokines interleukin 1 (IL-1) and interleukin 18 (IL-18). These receptors are coupled to a signaling pathway that is conserved in mammals, insects, and plants, resulting in cellular activation, thereby stimulating innate immune defenses. A variety of bacterial and fungal products have been identified that serve as TLR ligands, and more recent studies have identified the first endogenous protein ligands for TLR proteins. While TLR signaling is likely to be a key feature of innate immune responses, these proteins may also regulate homeostasis via interaction with endogenous protein ligands.
Assuntos
Proteínas de Drosophila , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/fisiologia , Receptores de Superfície Celular/química , Receptores de Superfície Celular/fisiologia , Sequência de Aminoácidos , Animais , Drosophila , Humanos , Subunidade alfa de Receptor de Interleucina-18 , Camundongos , Dados de Sequência Molecular , Receptores de Interleucina/química , Receptores de Interleucina/fisiologia , Receptores de Interleucina-1/química , Receptores de Interleucina-1/fisiologia , Receptores de Interleucina-18 , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Receptor 5 Toll-Like , Receptores Toll-LikeRESUMO
BACKGROUND: Mammalian Toll-like receptor (TLR) proteins are pattern recognition receptors for a diverse array of bacterial and viral products. Gram negative bacterial lipopolysaccharide (LPS) activates cells through TLR4, whereas the mycobacterial cell wall glycolipids, lipoarabinomannan (LAM) and mannosylated phosphatidylinositol (PIM), activate cells through TLR2. Furthermore, short term culture filtrates of M. tuberculosis bacilli contain a TLR2 agonist activity, termed soluble tuberculosis factor (STF), that appears to be PIM. It was recently shown that stimulation of RAW264.7 murine macrophages by LPS, LAM, STF, and PIM rapidly activated NF-kappaB, AP1, and MAP kinases. RESULTS: This study shows that signalling by TLR2 and TLR4 also activates the protein kinase Akt, a downstream target of phosphatidylinositol-3'-kinase (PI-3-K). This finding suggests that activation of PI-3-K represents an additional signalling pathway induced by engagement of TLR2 and TLR4. Subsequently, the functional responses induced by the different TLR agonists were compared. LPS, the mycobacterial glycolipids, and the OspC lipoprotein (a TLR2 agonist) all induced macrophages to secrete tumour necrosis factor alpha (TNFalpha), whereas only LPS could induce nitric oxide (NO) secretion. Human alveolar macrophages also exhibited a distinct pattern of cellular response after stimulation with TLR2 and TLR4 agonists. Specifically, LPS induced TNFalpha, MIP-1beta, and RANTES production in these cells, whereas the TLR2 agonists induced only MIP-1beta production. CONCLUSION: Together, these data show that different TLR proteins mediate the activation of distinct cellular responses, despite their shared ability to activate NF-kappaB, AP1, MAP kinases, and PI-3-K.
Assuntos
Antígenos de Protozoários , Proteínas de Drosophila , Ativação de Macrófagos/fisiologia , Macrófagos/metabolismo , Glicoproteínas de Membrana/fisiologia , Receptores de Superfície Celular/fisiologia , Proteínas de Soja , Fator de Necrose Tumoral alfa/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica , Linhagem Celular , Quimiocina CCL4 , Quimiocina CCL5/metabolismo , Proteínas de Ligação a DNA/farmacologia , Humanos , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Proteínas Inflamatórias de Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Mycobacterium tuberculosis , Óxido Nítrico/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas de Protozoários/farmacologia , Receptores de Superfície Celular/genética , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Receptores Toll-LikeRESUMO
Recent studies have implicated a family of mammalian Toll-like receptors (TLR) in the activation of macrophages by Gram-negative and Gram-positive bacterial products. We have previously shown that different TLR proteins mediate cellular activation by the distinct CD14 ligands Gram-negative bacterial LPS and mycobacterial glycolipid lipoarabinomannan (LAM). Here we show that viable Mycobacterium tuberculosis bacilli activated both Chinese hamster ovary cells and murine macrophages that overexpressed either TLR2 or TLR4. This contrasted with Gram-positive bacteria and Mycobacterium avium, which activated cells via TLR2 but not TLR4. Both virulent and attenuated strains of M. tuberculosis could activate the cells in a TLR-dependent manner. Neither membrane-bound nor soluble CD14 was required for bacilli to activate cells in a TLR-dependent manner. We also assessed whether LAM was the mycobacterial cell wall component responsible for TLR-dependent cellular activation by M. tuberculosis. We found that TLR2, but not TLR4, could confer responsiveness to LAM isolated from rapidly growing mycobacteria. In contrast, LAM isolated from M. tuberculosis or Mycobacterium bovis bacillus Calmette-Guérin failed to induce TLR-dependent activation. Lastly, both soluble and cell wall-associated mycobacterial factors were capable of mediating activation via distinct TLR proteins. A soluble heat-stable and protease-resistant factor was found to mediate TLR2-dependent activation, whereas a heat-sensitive cell-associated mycobacterial factor mediated TLR4-dependent activation. Together, our data demonstrate that Toll-like receptors can mediate cellular activation by M. tuberculosis via CD14-independent ligands that are distinct from the mycobacterial cell wall glycolipid LAM.
Assuntos
Proteínas de Fase Aguda , Proteínas de Drosophila , Fibroblastos/metabolismo , Fibroblastos/microbiologia , Ativação de Macrófagos , Glicoproteínas de Membrana/fisiologia , Mycobacterium tuberculosis/fisiologia , Receptores de Superfície Celular/fisiologia , Animais , Proteínas de Bactérias/fisiologia , Células CHO , Proteínas de Transporte/fisiologia , Linhagem Celular , Cricetinae , Fibroblastos/imunologia , Humanos , Receptores de Lipopolissacarídeos/fisiologia , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/imunologia , Mananas/farmacologia , Camundongos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/imunologia , Solubilidade , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Receptores Toll-LikeRESUMO
Mammalian Toll-like receptor (TLR) proteins are new members of the IL-1 receptor family that participate in activation of cells by bacteria and bacterial products. Several recent reports indicate that TLR proteins mediate cellular activation by bacterial LPS via a signaling pathway that is largely shared by the type I IL-1 receptor. We previously showed that Chinese hamster ovary (CHO) fibroblasts engineered to express CD14 (CHO/CD14) were responsive to LPS, but not to a distinct CD14 ligand, mycobacterial lipoarabinomannan (LAM). These CHO/CD14 cells were subsequently found to possess a frame-shift mutation within the TLR2 gene which resulted in their inability to express functional TLR2 protein. Thus, we hypothesized that TLR2, but not TLR4, was necessary for LAM signaling. In this paper we show that CHO/CD14 cells engineered to express functional TLR2 protein acquired the ability to be activated by LAM. Similarly, overexpression of TLR2 in murine macrophages conferred enhanced LAM responsiveness. Together, our data demonstrate that the distinct CD14 ligands LAM and LPS utilize different TLR proteins to initiate intracellular signals. These findings suggest a novel receptor signaling paradigm in which the binding of distinct ligands is mediated by a common receptor chain, but cellular activation is initiated via distinct signal-transducing chains that confer ligand specificity. This paradigm contrasts with many cytokine receptor complexes in which receptor specificity is conferred by a unique ligand-binding chain but cellular activation is initiated via shared signal-transducing chains.
Assuntos
Proteínas de Drosophila , Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/metabolismo , Glicoproteínas de Membrana/fisiologia , Receptores de Superfície Celular/fisiologia , Animais , Células CHO , Linhagem Celular , Sequência Conservada , Cricetinae , Feminino , Genes Reporter , Humanos , Ligantes , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C3H , NF-kappa B/genética , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/fisiologia , Plasmídeos/síntese química , Plasmídeos/fisiologia , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Transdução de Sinais/imunologia , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Receptores Toll-Like , Ativação Transcricional/imunologia , Células Tumorais CultivadasRESUMO
We previously showed that viable Mycobacterium tuberculosis (Mtb) bacilli contain distinct ligands that activate cells via the mammalian Toll-like receptor (TLR) proteins TLR2 and TLR4. We now demonstrate that expression of a dominant negative TLR2 or TLR4 proteins in RAW 264.7 macrophages partially blocked Mtb-induced NF-kappa B activation. Coexpression of both dominant negative proteins blocked virtually all Mtb-induced NF-kappa B activation. The role of the TLR4 coreceptor MD-2 was also examined. Unlike LPS, Mtb-induced macrophage activation was not augmented by overexpression of ectopic MD-2. Moreover, cells expressing an LPS-unresponsive MD-2 mutant responded normally to Mtb. We also observed that the lipid A-like antagonist E5531 specifically inhibited TLR4-dependent Mtb-induced cellular responses. E5531 could substantially block LPS- and Mtb-induced TNF-alpha production in both RAW 264.7 cells and primary human alveolar macrophages (AM phi). E5531 inhibited Mtb-induced AM phi apoptosis in vitro, an effect that was a consequence of the inhibition of TNF-alpha production by E5531. In contrast, E5531 did not inhibit Mtb-induced NO production in RAW 264.7 cells and AM phi. Mtb-stimulated peritoneal macrophages from TLR2- and TLR4-deficient animals produced similar amounts of NO compared with control animals, demonstrating that these TLR proteins are not required for Mtb-induced NO production. Lastly, we demonstrated that a dominant negative MyD88 mutant could block Mtb-induced activation of the TNF-alpha promoter, but not the inducible NO synthase promoter, in murine macrophages. Together, these data suggest that Mtb-induced TNF-alpha production is largely dependent on TLR signaling. In contrast, Mtb-induced NO production may be either TLR independent or mediated by TLR proteins in a MyD88-independent manner.