RESUMO
Nod1 is a member of the NLR/Nod/CATERPILLER family. It acts as a sensor for intracellular bacteria by recognizing specific glycopeptides derived from peptidoglycan. Nod1 activation mediates distinct cellular responses including activation of MAP kinases, IL-8 release, apoptosis and suppression of several estrogen-dependent responses in MCF-7 cells. Here we have extended these studies by identifying key regulatory steps in Nod1-dependent signaling pathways. We provide multiple lines of data showing that Nod1-dependent apoptosis is a caspase 8-mediated event and that apoptosis requires RIP2. In contrast, several lines of evidence show that Nod1-dependent JNK activation and IL-8 production did not require the presence of caspase 8 but required activation of TAK1 as well as RIP2. Thus, we have identified several key control points that lie downstream of Nod1. This work provides the basis for further studies of the biological significance and regulation of the Nod1 pathway.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Apoptose/fisiologia , Neoplasias da Mama/genética , Caspase 8/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Interleucina-8/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Mutagênese Sítio-Dirigida , Mapeamento de Interação de Proteínas , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Transdução de Sinais/fisiologia , Células Tumorais CultivadasRESUMO
Macrophages and dendritic cells are in the front line of host defense. When they sense host invasion, they produce cytokines that alert other innate immune cells and also abet the development of an adaptive immune response. Although lipolysaccharide (LPS), peptidoglycan, unmethylated DNA, and other microbial products were long known to be the primary targets of innate immune recognition, there was puzzlement as to how each molecule triggered a response. It is now known that the Toll-like receptors (TLRs) are the principal signaling molecules through which mammals sense infection. Each TLR recognizes a restricted subset of molecules produced by microbes, and in some circumstances, only a single type of molecule is sensed (e.g., only LPS is sensed by TLR4). TLRs direct the activation of immune cells near to and far from the site of infection, mobilizing the comparatively vast immune resources of the host to confine and defeat an invasive organism before it has become widespread. The biochemical details of TLR signaling have been analyzed through forward and reverse genetic methods, and full elucidation of the molecular interactions that transpire within the first minutes following contact between host and pathogen will soon be at hand.
Assuntos
Imunidade Inata , Infecções/imunologia , Glicoproteínas de Membrana/fisiologia , Receptores de Superfície Celular/fisiologia , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal , Animais , Antígenos de Diferenciação/fisiologia , Humanos , Inflamação/imunologia , Lipopolissacarídeos/toxicidade , Fator 88 de Diferenciação Mieloide , Receptores Imunológicos/fisiologia , Sepse/imunologia , Receptor 4 Toll-Like , Receptores Toll-LikeRESUMO
OBJECTIVE: This review describes efforts to define new therapeutic targets by utilizing information derived from studies of the innate immune response to infection. These targets will provide new means to design novel therapeutics for the treatment of human disease caused by dysregulation of the innate immune response. DATA SOURCES AND ANALYSIS: The results were derived from model systems that reflect the cellular changes accompanying activation of the innate immune system. These model systems include the use of a rabbit model of endotoxin shock that requires multiple exposures to lipopolysaccharide to induce severe pathophysiological changes that result in death. The cellular systems range from primary explants of myeloid lineage cells to stably transfected lines bearing key receptors of the innate immune system, the latter of which includes the Toll-like receptors and CD14. Studies in animal models include blocking monoclonal antibodies to rabbit CD14 and rabbit tumor necrosis factor-alpha. Cellular studies use measurements of cell activation as defined by activation of nuclear factor-kappaB, MAP kinase, and gene expression. In some studies, direct measurements of secreted cytokine levels were performed. Standard approaches to data analysis have been used. CONCLUSIONS: Evidence demonstrates the utility of anti-CD14 monoclonal antibody therapy in septic shock and the potential value of targeting intracellular kinases to modulate harmful cellular responses during sepsis. The key element to each of these approaches is to blunt, but not eliminate, the dysregulated inflammatory response that may occur in sepsis.
Assuntos
Proteínas de Drosophila , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/imunologia , Síndrome de Resposta Inflamatória Sistêmica/imunologia , Síndrome de Resposta Inflamatória Sistêmica/terapia , Animais , Citocinas/efeitos dos fármacos , Citocinas/imunologia , Modelos Animais de Doenças , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/imunologia , Humanos , Imunidade Inata/genética , Receptores de Lipopolissacarídeos/efeitos dos fármacos , Receptores de Lipopolissacarídeos/imunologia , Glicoproteínas de Membrana/efeitos dos fármacos , Glicoproteínas de Membrana/imunologia , Quinases de Proteína Quinase Ativadas por Mitógeno/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/imunologia , NF-kappa B/efeitos dos fármacos , NF-kappa B/imunologia , Coelhos , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Superfície Celular/imunologia , Transdução de Sinais/imunologia , Receptores Toll-Like , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Cell signaling by coagulation factor Xa (Xa) contributes to pro-inflammatory responses in vivo. This study characterizes the signaling mechanism of Xa in a HeLa cell line that expresses protease-activated receptor 1 (PAR-1) but not PAR-2, -3, or -4. Xa induced NF-kappaB in HeLa cells efficiently but with delayed kinetics compared to thrombin. This delay caused no difference in gene expression patterns, as determined by high-density microarray analysis. Both proteases prominently induced the angiogenesis-promoting gene Cyr61 and connective tissue growth factor. Inhibition of PAR-1 cleavage abolished MAP kinase phosphorylation and gene induction by Xa, demonstrating that Xa signals through PAR-1 and not through a novel member of the PAR family. Activation of cell surface prothrombin with the snake venom enzyme Ecarin also produced PAR-1-dependent signaling. However, though the response to Ecarin was completely blocked by the thrombin inhibitor hirudin, the response to Xa was not. This suggests that the Xa response is not mediated by locally generated thrombin. The concentration dependence of Xa for PAR-1 activation is consistent with previously characterized Xa-mediated PAR-2 signaling, suggesting that local concentration of Xa on the cell surface, rather than sequence-specific recognition of the PAR scissile bond, determines receptor cleavage. This study demonstrates that PAR-1 cleavage by Xa can elicit the same cellular response as thrombin, but mechanistic differences in receptor recognition may be crucial for specific roles for Xa in signaling during spatial or temporal separation from thrombin generation.
Assuntos
Fator Xa/farmacologia , Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular , Receptores de Trombina/genética , Transdução de Sinais , Antitrombinas/farmacologia , Linhagem Celular , Fator de Crescimento do Tecido Conjuntivo , Proteína Rica em Cisteína 61 , Endotélio Vascular , Ativação Enzimática/efeitos dos fármacos , Substâncias de Crescimento/genética , Células HeLa , Hirudinas/farmacologia , Humanos , Proteínas Imediatamente Precoces/genética , Cinética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Receptor PAR-1 , Trombina/metabolismo , Trombina/farmacologia , Veias UmbilicaisRESUMO
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.
Assuntos
Proteínas de Drosophila , Leptospira interrogans/imunologia , Leptospira interrogans/patogenicidade , Lipopolissacarídeos/toxicidade , Ativação de Macrófagos/efeitos dos fármacos , Glicoproteínas de Membrana/imunologia , Receptores de Superfície Celular/imunologia , Animais , Células CHO , Linhagem Celular , Cricetinae , Humanos , Leptospirose/imunologia , Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/imunologia , Lipoproteínas/imunologia , Ativação de Macrófagos/imunologia , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Transdução de Sinais , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Receptores Toll-LikeRESUMO
The structural features of some proteins of the innate immune system involved in mediating responses to microbial pathogens are highly conserved throughout evolution. Examples include members of the Drosophila Toll (dToll) and the mammalian Toll-like receptor (TLR) protein families. Activation of Drosophila Toll is believed to occur via an endogenous peptide rather than through direct binding of microbial products to the Toll protein. In mammals there is a growing consensus that lipopolysaccharide (LPS) initiates its biological activities through a heteromeric receptor complex containing CD14, TLR4, and at least one other protein, MD-2. LPS binds directly to CD14 but whether LPS then binds to TLR4 and/or MD-2 is not known. We have used transient transfection to express human TLRs, MD-2, or CD14 alone or in different combinations in HEK 293 cells. Interactions between LPS and these proteins were studied using a chemically modified, radioiodinated LPS containing a covalently linked, UV light-activated cross-linking group ((125)I-ASD-Re595 LPS). Here we show that LPS is cross-linked specifically to TLR4 and MD-2 only when co-expressed with CD14. These data support the contention that LPS is in close proximity to the three known proteins of its membrane receptor complex. Thus, LPS binds directly to each of the members of the tripartite LPS receptor complex.
Assuntos
Antígenos de Superfície/metabolismo , Proteínas de Drosophila , Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Anticorpos Monoclonais , Antígenos de Superfície/química , Sítios de Ligação , Linhagem Celular , Reagentes de Ligações Cruzadas , Desoxiglucose/farmacologia , Drosophila , Humanos , Receptores de Lipopolissacarídeos/química , Lipopolissacarídeos/química , Antígeno 96 de Linfócito , Glicoproteínas de Membrana/química , Camundongos , Receptores de Superfície Celular/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Azida Sódica/farmacologia , Fluoreto de Sódio/farmacologia , Receptor 4 Toll-Like , Receptores Toll-Like , Transfecção , Raios UltravioletaRESUMO
Herein we describe the isolation of a cDNA encoding a novel human Toll-like receptor (hTLR) that we term hTLR10. Human TLR10 contains 811 amino acid residues. Deduced amino acid sequence analysis reveals that like the other known hTLRs (hTLR1-9) it is characterized by a signal peptide followed by multiple leucine-rich repeats (LRRs), a cysteine-rich domain, a transmembrane sequence and a cytoplasmic domain homologous to that of the human interleukin-1 receptor. Phylogenetic analysis indicates that among all the hTLRs, hTLR10 is most closely related to hTLR1 and hTLR6; the overall amino acid identity is 50% and 49%, respectively. hTLR10 mRNA is most highly expressed in lymphoid tissues such as spleen, lymph node, thymus, and tonsil. Expression analysis of cell lines indicates a predominance in a variety of immune cell types. Thus, hTLR10 is preferentially expressed in tissues and cells involved in immune responses.
Assuntos
Receptores de Superfície Celular/genética , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , DNA Complementar , Expressão Gênica , Humanos , Linfonodos/metabolismo , Dados de Sequência Molecular , Tonsila Palatina/metabolismo , Receptores de Superfície Celular/classificação , Receptores de Superfície Celular/metabolismo , Homologia de Sequência de Aminoácidos , Baço/metabolismo , Timo/metabolismo , Receptor 10 Toll-LikeRESUMO
During inflammation the balance between cell activation and cell death is determined by the tight regulation of multiple intracellular enzyme cascades. Key regulatory steps often involve protein kinases. We show that the prototypical pro-inflammatory molecule, bacterial lipopolysaccharide, activates multiple protein kinases such as p38, JNK, IKK-beta, and PKB/Akt via transforming growth factor beta-activated kinase-1 (TAK1). We also show that TAK1 plays an important role in similar activation pathways triggered by interleukin-1. Thus TAK1 must be considered as an important component of intracellular pathways in cells involved in host responses to physiological and/or environmental stress signals during inflammation.
Assuntos
Inflamação/enzimologia , Lipopolissacarídeos/farmacologia , MAP Quinase Quinase Quinases/fisiologia , Proteínas Quinases/metabolismo , Transdução de Sinais/fisiologia , Androstadienos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular/efeitos dos fármacos , Cromonas/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Humanos , Peróxido de Hidrogênio/farmacologia , Soluções Hipertônicas/farmacologia , Quinase I-kappa B , Imidazóis/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Interleucina-1/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Morfolinas/farmacologia , NF-kappa B/metabolismo , Ácido Okadáico/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Prostaglandinas A/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Piridinas/farmacologia , Proteínas Recombinantes de Fusão/fisiologia , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico/enzimologia , Transfecção , Células Tumorais Cultivadas/efeitos dos fármacos , Wortmanina , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Members of the Toll-like receptor family are essential components of the innate immune system. Herein we report the molecular cloning and characterization of three novel human Toll-like receptors (hTLRs) designated hTLR7, hTLR8, and hTLR9. Human TLR7-9, like the previously described members hTLR1-6 contain an ectodomain with multiple leucine-rich repeats (LRRs) and a cytoplasmic domain homologous to that of the human interleukin-1 (IL-1) receptor. When compared with hTLR1-6, the hTLR7-9 has a higher molecular weight largely as a result of a longer ectodomain. Phylogenetic analysis shows that hTLR7-9 belong to a new sub-family of the hTLRs. Analysis of mRNA expression at the tissue levels shows differential expression patterns; hTLR7 is predominantly expressed in lung, placenta and spleen, hTLR8 is more abundant in lung, peripheral blood leukocytes, and hTLR9 is preferentially expressed in immune cell rich tissues, such as spleen, lymph node, bone marrow and peripheral blood leukocytes. The hTLR7 and hTLR8 genes are located on the sex chromosome X, hTLR9 gene is located on chromosome 3. Expression of constitutively active hTLR7-9 stimulates an NF-kappaB signaling pathway indirectly supporting the contention that these receptors are involved in cellular responses to stimuli, which activate innate immunity.
Assuntos
Proteínas de Drosophila , Glicoproteínas de Membrana/genética , Receptores de Superfície Celular/genética , Sequência de Aminoácidos , Animais , Células Cultivadas , Clonagem Molecular , Evolução Molecular , Éxons , Regulação da Expressão Gênica , Humanos , Glicoproteínas de Membrana/química , Camundongos , Dados de Sequência Molecular , NF-kappa B/metabolismo , Especificidade de Órgãos , Filogenia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Receptores de Superfície Celular/química , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Receptor 7 Toll-Like , Receptor 8 Toll-Like , Receptor Toll-Like 9 , Receptores Toll-LikeRESUMO
The innate immune response is the first line of defence against infectious disease. The principal challenge for the host is to detect the pathogen and mount a rapid defensive response. A group of proteins that comprise the Toll or Toll-like family of receptors perform this role in vertebrate and invertebrate organisms. This reflects a remarkable conservation of function and it is therefore not surprising that studies of the mechanism by which they act has revealed new and important insights into host defence.
Assuntos
Proteínas de Drosophila , Imunidade/fisiologia , Glicoproteínas de Membrana/fisiologia , Receptores de Superfície Celular/fisiologia , Animais , Bactérias/imunologia , Humanos , Imunidade Inata/fisiologia , Transdução de Sinais , Receptores Toll-LikeRESUMO
All species require a rapid, systemic reply to pathogens in their environment. This response is known as the innate immune response and is characterized by de novo synthesis of mediators that directly or indirectly through phagocytosis remove and kill the pathogen. Innate immune responses have been preserved throughout evolution and have been studied in detail in organisms from the fruit fly Drosophila melanogaster to humans. In my laboratory, studies performed during the past 25 yr have focused on defining the molecular basis of innate immune responses to microbial pathogens. Specifically, we have used bacterial endotoxin (lipopolysaccharide) as a model stimulus to define how the innate immune system recognizes products of microbial pathogens and initiates responses to remove and/or kill such organisms. Such studies also serve as models to understand more fully the mechanisms underlying a serious human disease known as septic shock. This article discusses septic shock and its relationship to innate immunity.
Assuntos
Imunidade Inata , Animais , Humanos , Lipopolissacarídeos/imunologia , Choque Séptico/imunologiaRESUMO
The p38 family of mitogen-activated protein kinases is believed to mediate a variety of leukocyte responses to pro-inflammatory stimuli. There are four members of the p38 family, and although activation of the different members has been studied in transiently transfected cells much less is known about activation of the endogenous p38s, particularly in myeloid lineage cells. To investigate activation of endogenous p38s, we have made monoclonal antibodies specific for each p38 and have used these antibodies to study p38 activation by pro-inflammatory stimuli in several human monocytic cell lines. Without stimulation endogenous p38alpha kinase activity was readily detectable, whereas that of p38beta, gamma, and delta was barely measurable. In response to inflammatory stimuli, we observed a time- and dose-dependent activation of all four p38s. The kinetics of activation of each of the p38s were similar for each stimulus used, suggesting a common upstream activation pathway. Simultaneous activation of the p38s suggests that all four may be important in inflammation.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Anticorpos Monoclonais/farmacologia , Astrocitoma , Linhagem Celular , Ativação Enzimática , Escherichia coli , Humanos , Inflamação , Interferon gama/farmacologia , Cinética , Lipopolissacarídeos/farmacologia , Proteína Quinase 12 Ativada por Mitógeno , Proteínas Recombinantes/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Transfecção , Células Tumorais Cultivadas , Células U937 , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Shigella is a diarrheal pathogen that causes disease through invasion of the large intestinal mucosa. The endotoxin of the invading bacterium may play a key role in the disease process by causing inflammation and tissue injury during infection. Earlier studies have shown that various animal species lacking functional CD14 were protected against endotoxin-mediated shock. Rabbits experimentally infected with Shigella were used to test the hypothesis that blockade of endotoxin-induced cell activation with anti-CD14 mAb would diminish inflammation and thus disease severity. Unexpectedly, we observed that the intestinal mucosa of anti-CD14-treated animals exhibited a 50-fold increase in bacterial invasion and more severe tissue injury compared with controls. Despite higher bacterial loads in treated animals, the numbers of polymorphonuclear leukocytes that were recruited to the infection site were similar to those in controls. Furthermore, the phagocytic cells of CD14-blocked animals produced IL-1 and TNF-alpha. Moreover, in vitro blockade of CD14 did not impede bactericidal activity. Thus, anti-CD14 treatment interfered with host defense mechanisms involved with removal/eradication of Shigella.
Assuntos
Anticorpos Bloqueadores/farmacologia , Anticorpos Monoclonais/farmacologia , Disenteria Bacilar/imunologia , Disenteria Bacilar/patologia , Receptores de Lipopolissacarídeos/imunologia , Shigella flexneri/patogenicidade , Animais , Degranulação Celular/imunologia , Movimento Celular/imunologia , Citocinas/biossíntese , Disenteria Bacilar/microbiologia , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Coelhos , Shigella flexneri/crescimento & desenvolvimento , Shigella flexneri/imunologiaRESUMO
Mammalian Toll-like receptors (TLRs) are expressed on innate immune cells and respond to the membrane components of Gram-positive or Gram-negative bacteria. When activated, they convey signals to transcription factors that orchestrate the inflammatory response. However, the intracellular signaling events following TLR activation are largely unknown. Here we show that TLR2 stimulation by Staphylococcus aureus induces a fast and transient activation of the Rho GTPases Rac1 and Cdc42 in the human monocytic cell line THP-1 and in 293 cells expressing TLR2. Dominant-negative Rac1N17, but not dominant-negative Cdc42N17, block nuclear factor-kappa B (NF-kappa B) transactivation. S. aureus stimulation causes the recruitment of active Rac1 and phosphatidylinositol-3 kinase (PI3K) to the TLR2 cytosolic domain. Tyrosine phosphorylation of TLR2 is required for assembly of a multiprotein complex that is necessary for subsequent NF-kappa B transcriptional activity. A signaling cascade composed of Rac1, PI3K and Akt targets nuclear p65 transactivation independently of I kappa B alpha degradation. Thus Rac1 controls a second, I kappa B-independent, pathway to NF-kappa B activation and is essential in innate immune cell signaling via TLR2.
Assuntos
Proteínas de Drosophila , Glicoproteínas de Membrana/metabolismo , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases , Receptores de Superfície Celular/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Linhagem Celular , Expressão Gênica , Humanos , Glicoproteínas de Membrana/genética , Modelos Biológicos , Fosfatidilinositol 3-Quinases/metabolismo , Fosfotirosina/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Receptores de Superfície Celular/genética , Transdução de Sinais , Staphylococcus aureus/imunologia , Receptor 2 Toll-Like , Receptores Toll-Like , Transfecção , Proteína cdc42 de Ligação ao GTP/metabolismoAssuntos
Proteínas de Drosophila , Drosophila/imunologia , Glicoproteínas de Membrana/imunologia , Receptores de Superfície Celular/imunologia , Animais , Drosophila/microbiologia , Humanos , Imunidade Inata , Receptores de Lipopolissacarídeos/imunologia , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Glicoproteínas de Membrana/genética , Receptores de Superfície Celular/genética , Receptores Toll-LikeAssuntos
Anti-Inflamatórios/farmacologia , Citocinas/genética , Inflamação/fisiopatologia , Proteínas Serina-Treonina Quinases/metabolismo , Fator de Necrose Tumoral alfa/genética , Animais , Regulação da Expressão Gênica/imunologia , Humanos , Inflamação/genética , Peptídeos e Proteínas de Sinalização Intracelular , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Tissue factor (TF) is the major activator of the coagulation protease cascade and contributes to lethality in sepsis. Despite several studies analyzing TF expression in animal models of endotoxemia, there remains debate about the cell types that are induced to express TF in different tissues. In this study, we performed a detailed analysis of the induction of TF mRNA and protein expression in two rabbit models of endotoxemia to better understand the cell types that may contribute to local fibrin deposition and disseminated intravascular coagulation. Northern blot analysis demonstrated that lipopolysaccharide (LPS) increased TF expression in the brain, lung, and kidney. In situ hybridization showed that TF mRNA expression was increased in cells identified morphologically as epithelial cells in the lung and as astrocytes in the brain. In the kidney, in situ hybridization experiments and immunohistochemical analysis showed that TF mRNA and protein expression was increased in renal glomeruli and induced in tubular epithelium. Dual staining for TF and vWF failed to demonstrate TF expression in endothelial cells in LPS-treated animals. These results demonstrate that TF expression is induced in many different cell types in LPS-treated rabbits, which may contribute to local fibrin deposition and tissue injury during endotoxemia.
Assuntos
Lipopolissacarídeos/toxicidade , Tromboplastina/biossíntese , Tromboplastina/genética , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Endotoxemia/etiologia , Endotoxemia/genética , Endotoxemia/metabolismo , Expressão Gênica/efeitos dos fármacos , Imuno-Histoquímica , Hibridização In Situ , Rim/metabolismo , Pulmão/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Coelhos , Distribuição TecidualRESUMO
Until about 10 years ago the exact mechanisms controlling cellular responses to the endotoxin - or lipopolysaccharide (LPS) - of Gram-negative bacteria were unknown. Now a considerable body of evidence supports a model where LPS or LPS-containing particles (including intact bacteria) form complexes with a serum protein known as LPS-binding protein; the LPS in this complex is subsequently transferred to another protein which binds LPS, CD14. The latter is found on the plasma membrane of most cell types of the myeloid lineage as well as in the serum in its soluble form; LPS binding to these two forms of CD14 results in the activation of cell types of myeloid and nonmyeloid lineages, respectively.
Assuntos
Proteínas de Fase Aguda , Endotoxinas/imunologia , Bactérias Gram-Negativas/imunologia , Imunidade Inata/imunologia , Glicoproteínas de Membrana , Animais , Antígenos de Bactérias/imunologia , Proteínas de Transporte/imunologia , Infecções por Bactérias Gram-Negativas/imunologia , Humanos , Receptores de Lipopolissacarídeos/imunologiaRESUMO
Recently there have been some substantial progresses in signal transduction of cell activation by lipopolysaccharide (LPS). After binding with LBP in the plasma, LPS is transported to the cell membrane surface of monocytes, then binds with membrane CD14 to induce cell activation. Mitogen-activated protein kinases (MAPKs) take part in the signal transduction of LPS stimulated cell activation to produce bioactive substances including tumor necrosis factor. p38 MAPK is an important regulator for TNF-alpha expression. It is possible to provide new theory and idea for the treatment of septic shock from the studies of signal transduction of cell activation by LPS.