RESUMO
BACKGROUND & PURPOSE: Recent studies suggested a role of prostaglandin E2 (PGE2) in the expression of the chemokine IL-8 by monocytes. The function of EP4 receptor for TNFα-induced IL-8 expression was studied in monocytic cell lines. EXPERIMENTAL APPROACH: IL-8 mRNA and protein induction as well as IL-8 promoter activity and transcription factor activation were assessed in monocytic cell lines, primary blood mononuclear cells (PBMC) and transgenic HEK293 cells expressing the EP4 receptor. KEY RESULTS: In monocytic cell lines THP-1, MonoMac and U937 PGE2 had only a marginal impact on IL-8 induction but strongly enhanced TNFα-induced IL-8 mRNA and protein synthesis. Similarly, in PBMC IL-8 mRNA induction was larger by simultaneous stimulation with TNFα and PGE2 than by either stimulus alone. The EP4 receptor subtype was the most abundant EP receptor in all three cell lines and in PBMC. Stimulation of THP-1 cells with an EP4 specific agonist enhanced TNFα-induced IL-8 mRNA and protein formation to the same extent as PGE2. In HEK293 cells expressing EP4, but not in wild type HEK293 cells lacking EP4, PGE2 enhanced TNFα-induced IL-8 protein and mRNA synthesis. In THP-1 cells, the enhancement of TNFα-mediated IL-8 mRNA induction by PGE2 was mimicked by a PKA-activator. Furthermore in these cells PGE2 induced expression of transcription factor C/EBPß, enhanced NF-κB activation by TNFα and inhibited TNFα-mediated AP-1 activation. PGE2 and TNFα synergistically activated transcription factor CREB, induced C/EBPß expression and enhanced the activity of an IL-8 promoter fragment containing -223â¯bp upstream of the transcription start site. CONCLUSIONS AND IMPLICATIONS: These findings suggest that a combined stimulation of TNFα and PGE2/EP4 signal chains in monocytic cells leads to maximal IL-8 promoter activity, as well as IL-8 mRNA and protein induction, by activating the PKA/CREB/C/EBPß as well as NF-κB signal chains.
Assuntos
Dinoprostona/farmacologia , Interleucina-8/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Leucócitos Mononucleares/metabolismo , Monócitos/metabolismo , NF-kappa B/metabolismo , RNA Mensageiro/metabolismo , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células THP-1/efeitos dos fármacos , Células THP-1/metabolismo , Células U937RESUMO
BACKGROUND: Spleen tyrosine kinase (Syk) is an intracellular nonreceptor tyrosine kinase, which has been implicated as central immune modulator promoting allergic airway inflammation. Syk inhibition has been proposed as a new therapeutic approach in asthma. However, the direct effects of Syk inhibition on airway constriction independent of allergen sensitization remain elusive. METHODS: Spectral confocal microscopy of human and murine lung tissue was performed to localize Syk expression. The effects of prophylactic or therapeutic Syk inhibition on allergic airway inflammation, hyperresponsiveness, and airway remodeling were analyzed in allergen-sensitized and airway-challenged mice. The effects of Syk inhibitors BAY 61-3606 or BI 1002494 on airway function were investigated in isolated lungs of wild-type, PKCα-deficient, mast cell-deficient, or eNOS-deficient mice. RESULTS: Spleen tyrosine kinase expression was found in human and murine airway smooth muscle cells. Syk inhibition reduced allergic airway inflammation, airway hyperresponsiveness, and pulmonary collagen deposition. In naïve mice, Syk inhibition diminished airway responsiveness independently of mast cells, or PKCα or eNOS expression and rapidly reversed established bronchoconstriction independently of NO. Simultaneous inhibition of Syk and PKC revealed additive dilatory effects, whereas combined inhibition of Syk and rho kinase or Syk and p38 MAPK did not cause additive bronchodilation. CONCLUSIONS: Spleen tyrosine kinase inhibition directly attenuates airway smooth muscle cell contraction independent of its protective immunomodulatory effects on allergic airway inflammation, hyperresponsiveness, and airway remodeling. Syk mediates bronchoconstriction in a NO-independent manner, presumably via rho kinase and p38 MAPK, and Syk inhibition might present a promising therapeutic approach in chronic asthma as well as acute asthma attacks.
Assuntos
Remodelação das Vias Aéreas/efeitos dos fármacos , Remodelação das Vias Aéreas/imunologia , Hiper-Reatividade Brônquica/etiologia , Hiper-Reatividade Brônquica/metabolismo , Broncoconstrição/efeitos dos fármacos , Quinase Syk/antagonistas & inibidores , Células Th2/imunologia , Células Th2/metabolismo , Alérgenos/imunologia , Animais , Hiper-Reatividade Brônquica/tratamento farmacológico , Hiper-Reatividade Brônquica/patologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Modelos Animais de Doenças , Feminino , Receptor Quinase 1 Acoplada a Proteína G/metabolismo , Expressão Gênica , Humanos , Mediadores da Inflamação/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Naftiridinas/farmacologia , Niacinamida/análogos & derivados , Niacinamida/farmacologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Proteína Quinase C-alfa , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Pirrolidinonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Quinase Syk/genética , Quinase Syk/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
OBJECTIVES: Anti-inflammatory functions of antibiotics may counteract deleterious hyperinflammation in pneumonia. Moxifloxacin reportedly exhibits immunomodulatory properties, but experimental evidence in pneumonia is lacking. Therefore, we investigated moxifloxacin in comparison with ampicillin regarding pneumonia-associated pulmonary and systemic inflammation and lung injury. METHODS: Ex vivo infected human lung tissue and mice with pneumococcal pneumonia were examined regarding local inflammatory response and bacterial growth. In vivo, clinical course of the disease, leucocyte dynamics, pulmonary vascular permeability, lung pathology and systemic inflammation were investigated. In addition, transcellular electrical resistance of thrombin-stimulated endothelial cell monolayers was quantified. RESULTS: Moxifloxacin reduced cytokine production in TNF-α-stimulated, but not in pneumococci-infected, human lung tissue. In vivo, moxifloxacin treatment resulted in reduced bacterial load as compared with ampicillin, whereas inflammatory parameters and lung pathology were not different. Moxifloxacin-treated mice developed less pulmonary vascular permeability during pneumonia, but neither combination therapy with moxifloxacin and ampicillin in vivo nor examination of endothelial monolayer integrity in vitro supported direct barrier-stabilizing effects of moxifloxacin. CONCLUSIONS: The current experimental data do not support the hypothesis that moxifloxacin exhibits potent anti-inflammatory properties in pneumococcal pneumonia.
Assuntos
Anti-Inflamatórios/uso terapêutico , Fluoroquinolonas/uso terapêutico , Pneumonia Pneumocócica/tratamento farmacológico , Animais , Modelos Animais de Doenças , Feminino , Humanos , Pulmão/patologia , Camundongos Endogâmicos C57BL , Moxifloxacina , Pneumonia Pneumocócica/microbiologia , Pneumonia Pneumocócica/patologia , Streptococcus pneumoniae/crescimento & desenvolvimento , Resultado do TratamentoRESUMO
BACKGROUND AND PURPOSE: Recent studies suggested a role for PGE(2) in the expression of the chemokine IL-8. PGE(2) signals via four different GPCRs, EP(1) -EP(4) . The role of EP(1) and EP(4) receptors for IL-8 induction was studied in HEK293 cells, overexpressing EP(1) (HEK-EP(1) ), EP(4) (HEK-EP(4) ) or both receptors (HEK-EP(1) + EP(4) ). EXPERIMENTAL APPROACH: IL-8 mRNA and protein induction and IL-8 promoter and NF-κB activation were assessed in EP expressing HEK cells. KEY RESULTS: In HEK-EP(1) and HEK-EP(1) + EP(4) but not HEK or HEK-EP(4) cells, PGE(2) activated the IL-8 promoter and induced IL-8 mRNA and protein synthesis. Stimulation of HEK-EP(1) + EP(4) cells with an EP(1) -specific agonist activated IL-8 promoter and induced IL-8 mRNA and protein, whereas a specific EP(4) agonist neither activated the IL-8 promoter nor induced IL-8 mRNA and protein synthesis. Simultaneous stimulation of HEK- EP(1) + EP(4) cells with both agonists activated IL-8 promoter and induced IL-8 mRNA to the same extent as PGE(2) . In HEK-EP(1) + EP(4) cells, PGE(2) -mediated IL-8 promoter activation and IL-8 mRNA induction were blunted by inhibition of IκB kinase. PGE(2) activated NF-κB in HEK-EP(1) , HEK-EP(4) and HEK-EP(1) + EP(4) cells. In HEK-EP(1) + EP(4) cells, simultaneous activation of both receptors was needed for maximal PGE(2) -induced NF-κB activation. PGE(2) -stimulated NF-κB activation by EP(1) was blocked by inhibitors of PLC, calcium-signalling and Src-kinase, whereas that induced by EP(4) was only blunted by Src-kinase inhibition. CONCLUSIONS AND IMPLICATIONS: These findings suggest that PGE(2) -mediated NF-κB activation by simultaneous stimulation of EP(1) and EP(4) receptors induces maximal IL-8 promoter activation and IL-8 mRNA and protein induction.
Assuntos
Dinoprostona/farmacologia , Interleucina-8/metabolismo , NF-kappa B/metabolismo , Receptores de Prostaglandina E Subtipo EP1/metabolismo , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Células HEK293 , Humanos , Interleucina-8/genética , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Receptores de Prostaglandina E Subtipo EP1/genética , Receptores de Prostaglandina E Subtipo EP4/genética , Transdução de SinaisRESUMO
Severe community- and hospital-acquired pneumonia is caused by Legionella pneumophila. Lung airway and alveolar epithelial cells comprise an important sentinel system in airborne infections. Although interleukin (IL)-6 is known as a central regulator of the immune response in pneumonia, its regulation in the lung is widely unknown. Herein, we demonstrate that different L. pneumophila strains induce delayed expression of IL-6 in comparison with IL-8 by human lung epithelial cells. IL-6 expression depended, at early time points, on flagellin recognition by Toll-like receptor (TLR)5, activity of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)1 and p38 mitogen-activated protein (MAP) kinase, and, at later time points, on the type-IV secretion system. In the same manner, but more rapidly, the recently described transcription factor IκBζ was induced by Legionella infection and, binding to the nuclear factor (NF)-κB subunit p50 - recruited to the il6 promoter together with CCAAT-enhancer-binding protein ß and phosphorylated activator protein-1 subunit cJun. Similarly, histone modifications and NF-κB subunit p65/RelA appeared at the iκbζ and subsequently at the il6 gene promoter, thereby initiating gene expression. Gene silencing of IκBζ reduced Legionella-related IL-6 expression by 41%. Overall, these data indicate a sequence of flagellin/TLR5- and type IV-dependent IκBζ expression, recruitment of IκBζ/p50 to the il6 promoter, chromatin remodelling and subsequent IL-6 transcription in L. pneumophila-infected lung epithelial cells.
Assuntos
Células Epiteliais/microbiologia , Regulação da Expressão Gênica , Quinase I-kappa B/metabolismo , Legionella pneumophila/metabolismo , Legionelose/microbiologia , Pulmão/microbiologia , Linhagem Celular Tumoral , Cromatina/metabolismo , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Flagelina/metabolismo , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Legionelose/metabolismo , Pulmão/metabolismo , NF-kappa B/metabolismo , Pneumonia/metabolismo , Regiões Promotoras GenéticasRESUMO
Legionella pneumophila is an important causative agent of severe pneumonia in humans. The human alveolar epithelium is an effective barrier for inhaled microorganisms and actively participates in the initiation of innate host defense. Although secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF) is essential for the elimination of invading Legionella spp., mechanisms of Legionella pneumophila-induced release of this cytokine are widely unknown. In this study, we have demonstrated a toll-like receptor (TLR)2- and TLR5-dependent release of GM-CSF in L. pneumophila-infected human alveolar epithelial cells. GM-CSF secretion was not dependent on the bacteria type II or type IV secretion system. Furthermore, an increase in protein kinase C (PKC) activity, particularly PKC(alpha) and PKC(epsilon), was noted. Blocking of PKC(alpha) and PKC(epsilon) activity or expression, but not of PKC(beta), PKC(delta), PKC(eta), PKC(theta), and PKC(zeta), significantly reduced the synthesis of GM-CSF in infected cells. While PKC(alpha) was critical for the initiation of a nuclear factor-kappaB-mediated GM-CSF expression, PKC(epsilon) regulated GM-CSF production via activator protein 1. Thus, differential regulation of GM-CSF, production by PKC isoforms, contributes to the host response in Legionnaires' disease.
Assuntos
Epitélio/microbiologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Legionella pneumophila/metabolismo , Proteína Quinase C-alfa/metabolismo , Proteína Quinase C-épsilon/metabolismo , Alvéolos Pulmonares/microbiologia , Linhagem Celular Tumoral , Citocinas/metabolismo , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Humanos , Isoformas de Proteínas , Receptor 2 Toll-Like/metabolismo , Receptor 5 Toll-Like/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Moraxella catarrhalis is a major cause of infectious exacerbations of chronic obstructive lung disease. In pulmonary epithelial cells, M. catarrhalis induces release of the pro-inflammatory cytokine interleukin (IL)-8, which plays a pivotal role in orchestrating airway inflammation. The present study demonstrated that protein kinase (PK)C was activated by Moraxella infection and positively regulated M. catarrhalis-triggered nuclear factor (NF)-kappaB activation and subsequent IL-8 release. Activation of the PKC/NF-kappaB signalling pathway was found to be dependent on expression of the Moraxella-specific ubiquitous surface protein A2. In addition, it was shown that specific isoforms of PKC play differential roles in the fine-tuning of the M. catarrhalis-induced NF-kappaB-dependent gene expression through controlling il8 promoter activity. Inhibition of PKCalpha and epsilon with chemical inhibitors or using short interfering RNA-mediated gene silencing significantly suppressed, whereas inhibition of PKCtheta increased, the M. catarrhalis-induced IL-8 transcription and cytokine release. In conclusion, it was shown that Moraxella catarrhalis infection activates protein kinase C and its isoforms alpha, epsilon and theta, which differentially regulate interleukin-8 transcription in human pulmonary epithelial cells.
Assuntos
Brônquios/imunologia , Células Epiteliais/imunologia , Interleucina-8/metabolismo , Isoenzimas/imunologia , Infecções por Moraxellaceae/imunologia , Proteína Quinase C-alfa/imunologia , Proteína Quinase C-épsilon/imunologia , Proteína Quinase C/imunologia , Brônquios/citologia , Linhagem Celular , Regulação da Expressão Gênica/imunologia , Humanos , Moraxella catarrhalis/patogenicidade , Regiões Promotoras Genéticas , Proteína Quinase C-theta , Transdução de Sinais/imunologiaRESUMO
Porphyromonas gingivalis dihydroceramides are found in extracts of calculus-contaminated root surfaces, diseased gingival tissue, and atherosclerotic plaques. These ceramides have been shown to promote inflammatory secretory responses in gingival fibroblasts. Little is known about their effects on the vascular system. We tested the hypothesis that P. gingivalis lipids induce apoptosis of human endothelial cells, and investigated the effects of extracted and purified P. gingivalis lipids on human umbilical vein endothelial cells. P. gingivalis phosphoglycerol dihydroceramides induced apoptosis, but not necrosis, in endothelial cells. Early apoptotic cells showed exposure of phosphatidylserine on the cell surface, followed by the cleavage of procaspases 3, 6, and 9. The release of apoptosis-inducing factor was increased, suggesting mitochondrial involvement. Different caspase inhibitors and cAMP elevation blocked DNA fragmentation. Moreover, N-acetylcysteine significantly reduced apoptosis, suggesting a role for reactive oxygen species in this process. Analysis of these data indicates that dihydroceramides may be important virulence factors of P. gingivalis.
Assuntos
Apoptose/fisiologia , Ceramidas/fisiologia , Células Endoteliais/microbiologia , Porphyromonas gingivalis/fisiologia , Acetilcisteína/farmacologia , Apoptose/efeitos dos fármacos , Fator de Indução de Apoptose/metabolismo , Inibidores de Caspase , Células Cultivadas , AMP Cíclico/fisiologia , Células Endoteliais/fisiologia , Ativação Enzimática , Humanos , Veias Umbilicais/citologia , Fatores de VirulênciaRESUMO
Moraxella catarrhalis is a major cause of infectious exacerbations of chronic obstructive lung disease. Cyclooxygenase (COX)-derived prostaglandins, such as prostaglandin E(2) (PGE(2)), are considered to be important regulators of lung function. The present authors tested the hypothesis that M. catarrhalis induces COX-2-dependent PGE(2) production in pulmonary epithelial cells. In the present study, the authors demonstrate that M. catarrhalis specifically induces COX-2 expression and subsequent PGE(2) release in pulmonary epithelial cells. Furthermore, the prostanoid receptor subtypes EP2 and EP4 were also upregulated in these cells. The M. catarrhalis-specific ubiquitous cell surface protein A1 was important for the induction of COX-2 and PGE(2). Moreover, M. catarrhalis-induced COX-2 and PGE(2) expression was dependent on extracellular signal-regulated kinase 1/2-driven activation of nuclear factor-kappaB, but not on the activation of p38 mitogen-activated protein kinase. In conclusion, the present data suggest that ubiquitous cell surface protein A1 of Moraxella catarrhalis, extracellular signal-regulated kinase 1/2 and nuclear factor-kappaB control cyclooxygenase-2 expression and subsequent prostaglandin E(2) release by lung epithelial cells. Moraxella catarrhalis-induced prostaglandin E(2) expression might counteract lung inflammation promoting colonisation of the respiratory tract in chronic obstructive pulmonary disease patients.
Assuntos
Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Pulmão/imunologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Moraxella catarrhalis/imunologia , NF-kappa B/metabolismo , Mucosa Respiratória/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Linhagem Celular , Indução Enzimática/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Humanos , Técnicas In Vitro , Proteínas de Membrana/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaRESUMO
Pneumonia can lead to the critical impairment of gas exchange in the lung. Due to the great variability of pneumonia causing pathogens, a large variety of diverse virulence factors act on the lung. Besides stimulation of unspecific defense mechanisms, activation of receptor-dependent cell-mediated innate immune defense mechanisms are critical for the pulmonary immune defense. Pathogen-associated molecules are detected via transmembraneous and cytosolic receptors of the host. This interaction stimulates the expression of immunomodulatory molecules via signal cascades. Of particular importance, in addition to direct pathogen-caused lung damage, is the overwhelming activation of the inflammatory response which can result in lung barrier failure and impairment of pulmonary gas exchange. In addition to the design of new antibiotics, innovative therapeutic strategies should therefore concentrate on the enhancement of antimicrobial mechanisms by concurrent limitation of inflammation.
Assuntos
Pneumonia Bacteriana/imunologia , Toxinas Bacterianas/imunologia , Humanos , Imunidade Ativa/imunologia , Imunidade Celular/imunologia , Imunidade Inata/imunologia , Pulmão/imunologia , Macrófagos Alveolares/imunologia , Neutrófilos/imunologia , Pneumonia Pneumocócica/imunologia , Edema Pulmonar/imunologia , Troca Gasosa Pulmonar/fisiologia , Receptores Imunológicos/imunologia , Transdução de Sinais/imunologia , Síndrome de Resposta Inflamatória Sistêmica/imunologiaRESUMO
Legionella pneumophila causes community-acquired pneumonia with high mortality, but little is known about its interaction with the alveolar epithelium. The aim of this study was to investigate whether L. pneumophila infection of lung epithelial cells (A549) resulted in pro-inflammatory activation. L. pneumophila infection induced liberation of interleukin (IL)-2, -4, -6, -8 and -17, monocyte chemoattractant protein-1, tumour necrosis factor-alpha, IL-1beta, interferon-gamma and granulocyte colony-stimulating factor, but not of IL-5, -7, -10, -12 (p70) or -13 or granulocyte-macrophage colony-stimulating factor. The present study focused on IL-8 and found induction by L. pneumophila strains 130b, Philadelphia 1, Corby and, to a lesser extent, JR32. Knockout of dotA, a central gene involved in type IVB secretion, did not alter IL-8 induction, whereas lack of flagellin significantly reduced IL-8 release by Legionella. Moreover, p38 mitogen-activated protein kinase (MAPK) was activated and kinase inhibition reduced secretion of induced cytokines, with the exception of IL-2 and granulocyte colony-stimulating factor. In contrast, inhibition of the MAPK kinase 1/extracellular signal-regulated kinase pathway only reduced the expression of a few cytokines. L. pneumophila also induced binding of nuclear factor-kappaB subunit RelA/p65 and RNA polymerase II to the il8 promoter, and a specific inhibitor of the inhibitor of nuclear factor-kappaB complex dose-dependently lowered IL-8 expression. Taken together, Legionella pneumophila activated p38 mitogen-activated protein kinase- and nuclear factor-kappaB/RelA pathway-dependent expression of a complex pattern of cytokines by human alveolar epithelial cells, presumably contributing to the immune response in legionellosis.
Assuntos
Citocinas/metabolismo , Células Epiteliais/metabolismo , Legionella pneumophila/fisiologia , Pulmão/patologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Técnicas de Cultura de Células , Células Cultivadas , Citocinas/genética , Gliceraldeído-3-Fosfato Desidrogenases/genética , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Humanos , Pulmão/metabolismo , RNA Mensageiro/metabolismo , Quinase Induzida por NF-kappaBRESUMO
Seroepidemiological studies and demonstration of viable bacteria in atherosclerotic plaques have linked Chlamydophila pneumoniae infection to the development of chronic vascular lesions and coronary heart disease. In this study, we characterized C. pneumoniae-mediated effects on human endothelial cells and demonstrated enhanced phosphorylation and activation of the endothelial mitogen-activated protein kinase (MAPK) family members extracellular receptor kinase (ERK1/2), p38-MAPK, and c-Jun-NH2 kinase (JNK). Subsequent interleukin-8 (IL-8) expression was dependent on p38-MAPK and ERK1/2 activation as demonstrated by preincubation of endothelial cells with specific inhibitors for the p38-MAPK (SB202190) or ERK (U0126) pathway. Inhibition of either MAPK had almost no effect on intercellular cell adhesion molecule 1 (ICAM-1) expression. While Chlamydia trachomatis was also able to infect endothelial cells, it did not induce the expression of endothelial IL-8 or ICAM-1. These effects were specific for a direct stimulation with viable C. pneumoniae and independent of paracrine release of endothelial cell-derived mediators like platelet-activating factor, NO, prostaglandins, or leukotrienes. Thus, C. pneumoniae triggers an early signal transduction cascade in target cells that could lead to endothelial cell activation, inflammation, and thrombosis, which in turn may result in or promote atherosclerosis.
Assuntos
Chlamydia trachomatis/patogenicidade , Chlamydophila pneumoniae/patogenicidade , Células Endoteliais/microbiologia , Endotélio Vascular/microbiologia , Células Endoteliais/enzimologia , Endotélio Vascular/citologia , Endotélio Vascular/enzimologia , Ativação Enzimática , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-8/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Fosforilação , Transdução de Sinais , Veias Umbilicais/citologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The endothelium is a specific target for Bartonella henselae, and endothelial cell infection represents an important step in the pathogenesis of cat scratch disease and bacillary angiomatosis. Mechanisms of Bartonella-endothelial cell interaction as well as signaling pathways involved in target cell activation were analyzed. B. henselae strain Berlin-1, isolated from bacillary angiomatosis lesions of a human immunodeficiency virus-infected patient, potently stimulated human umbilical cord vein endothelial cells (HUVEC), as determined by NF-kappaB activation and enhanced adhesion molecule expression. These effects were accompanied by increased PMN rolling on and adhesion to infected endothelial cell monolayers, as measured in a parallel-plate flow chamber assay. Monoclonal antibodies against E-selectin significantly reduced PMN rolling and adhesion. In our hands, B. henselae Berlin-1 was substantially more active than the typing strain B. henselae ATCC 49882. E-selectin and ICAM-1 upregulation occurred for up to 9 days, as verified by Northern blotting and cell surface enzyme-linked immunosorbent assay. Induction of adhesion molecules was mediated via NF-kappaB activation and could be blocked by a specific NF-kappaB inhibitor. Additional studies indicated that B. henselae-induced effects did not require living bacteria or Bartonella lipopolysaccharides. Exposure of HUVEC to purified B. henselae outer membrane proteins (OMPs), however, reproduced all aspects of endothelial cell activation. In conclusion, B. henselae, the causative agent of cat scratch disease and bacillary angiomatosis, infects and activates endothelial cells. B. henselae OMPs are sufficient to induce NF-kappaB activation and adhesion molecule expression followed by enhanced rolling and adhesion of leukocytes. These observations identify important new properties of B. henselae, demonstrating its capacity to initiate a cascade of events culminating in a proinflammatory phenotype of infected endothelial cells.
Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Bartonella henselae/imunologia , Selectina E/genética , Molécula 1 de Adesão Intercelular/genética , NF-kappa B/imunologia , Regulação para Cima/imunologia , Animais , Adesão Celular/imunologia , Movimento Celular/imunologia , Endotélio Vascular/citologia , Endotélio Vascular/imunologia , Expressão Gênica , Humanos , Neutrófilos/imunologia , CoelhosRESUMO
Vascular endothelial growth factor (VEGF) induces both angiogenesis and an increase in vascular permeability, 2 processes that are considered to be important for both tumor growth and the delivery of drugs to the site of tumors. This study demonstrates that transmembrane expression of tumor necrosis factor (tmTNF) is up-regulated in the endothelium of a murine methylcholanthrene (meth A)-induced sarcoma in comparison to the adjacent normal dermal vasculature and is also present on cultivated human endothelial cells. It is further shown that tmTNF is required for VEGF-mediated endothelial hyperpermeability in vitro and in vivo. This permissive activity of TNF appears to be selective, because anti-TNF antibodies ablated the VEGF-induced permeability but not proliferation of cultivated human endothelial cells. Furthermore, tnf gene-deficient mice show no obvious defects in vascularization and develop normally but failed to respond to administration of VEGF with an increase in vascular permeability. Subsequent studies indicated that the tmTNF and VEGF signaling pathways converge at the level of a secondary messenger, the "stress-activated protein kinase-2" (SAPK-2)/p38: (1) up-regulated endothelial expression of tmTNF resulted in the continuous activation of SAPK-2/p38 in vitro, and (2) an inhibitor of SAPK-2/p38 activation abolished the vascular permeability activity of VEGF in vivo. In conclusion, the study's finding that continuous autocrine signaling by tmTNF sensitizes endothelial cells to respond to VEGF by increasing their vascular permeability provides new therapeutic concepts for manipulating vascular hyperpermeability.
Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Fatores de Crescimento Endotelial/farmacologia , Linfocinas/farmacologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Comunicação Autócrina/efeitos dos fármacos , Dipeptídeos/farmacologia , Fatores de Crescimento Endotelial/metabolismo , Endotélio Vascular/química , Endotélio Vascular/citologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Imuno-Histoquímica , Linfocinas/metabolismo , Glicoproteínas de Membrana/farmacologia , Glicoproteínas de Membrana/fisiologia , Metilcolantreno , Camundongos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/farmacologia , Proteínas de Neoplasias/fisiologia , Sarcoma Experimental/irrigação sanguínea , Sarcoma Experimental/induzido quimicamente , Tromboplastina/efeitos dos fármacos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/farmacologia , Cordão Umbilical/citologia , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Bacterial endotoxin (lipopolysaccharide, or LPS) has potent proinflammatory properties by acting on many cell types, including endothelial cells. Secretion of the CXC-chemokine interleukin-8 (IL-8) by LPS-activated endothelial cells contributes substantially to the inflammatory response. Using human umbilical vein endothelial cells (HUVECs), we analyzed the role of small GTP-binding Rho proteins and p38 mitogen-activated protein kinase (MAPK) for LPS-dependent IL-8 expression in endothelial cells. Specific inactivation of RhoA/Cdc42/Rac1 by Clostridium difficile toxin B-10463 (TcdB-10463) reduced LPS-induced tyrosine phosphorylation, nuclear factor (NF)-kappaB-dependent gene expression, IL-8 messenger RNA, and IL-8 protein accumulation but showed no effect on LPS-dependent p38 MAPK activation. Inhibition of p38 MAPK by SB 202190 also blocked LPS-induced NF-kappaB activation and IL-8 synthesis. Furthermore, selective activation of the p38 MAPK pathway by transient expression of a constitutively active form of MAPK kinase (MKK)6, the upstream activator of p38, was as effective as LPS with respect to IL-8 expression in HUVECs. In summary, our data suggest that LPS-induced NF-kappaB activation and IL-8 synthesis in HUVECs are regulated by both a Rho-dependent signaling pathway and the MKK6/p38 kinase cascade.
Assuntos
Endotélio Vascular/metabolismo , Interleucina-8/biossíntese , Lipopolissacarídeos/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Células Cultivadas , Humanos , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Chlamydia pneumoniae is an important respiratory pathogen. Recently, its presence has been demonstrated in atherosclerotic lesions. In this study, we characterized C. pneumoniae-mediated activation of endothelial cells and demonstrated an enhanced expression of endothelial adhesion molecules followed by subsequent rolling, adhesion, and transmigration of leukocytes (monocytes, granulocytes). These effects were blocked by mAbs against endothelial and/or leukocyte adhesion molecules (beta1 and beta2 integrins). Additionally, activation of different signal transduction pathways in C. pneumoniae-infected endothelial cells was shown: protein tyrosine phosphorylation, up-regulation of phosphorylated p42/p44 mitogen-activated protein kinase, and NF-kappaB activation/translocation occurred within 10-15 min. Increased mRNA and surface expression of E-selectin, ICAM-1, and VCAM-1 were noted within hours. Thus, C. pneumoniae triggers a cascade of events that could lead to endothelial activation, inflammation, and thrombosis, which in turn may result in or may promote atherosclerosis.
Assuntos
Chlamydophila pneumoniae/imunologia , Endotélio Vascular/imunologia , Endotélio Vascular/microbiologia , Proteínas Quinases Ativadas por Mitógeno , Transdução de Sinais/imunologia , Northern Blotting , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Comunicação Celular/imunologia , Células Cultivadas , Selectina E/biossíntese , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Leucócitos/imunologia , Leucócitos/microbiologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , NF-kappa B/fisiologia , Fosforilação , Fosfotirosina/metabolismo , Veias Umbilicais , Molécula 1 de Adesão de Célula Vascular/biossínteseRESUMO
Epithelial cells actively participate in inflammatory airway disease by liberating mediators such as arachidonate metabolites and cytokines. Inhibition of phosphodiesterases (PDEs) may be a useful anti-inflammatory approach. The PDE isoenzyme pattern and the effects of PDE inhibition on mediator generation were analyzed in primary cultures of human and porcine airway epithelial cells (AEC) and in the bronchial epithelial cell line BEAS-2B. PDE4 and PDE5 were detected in lysates of all cell types studied. In primary cultures of human AEC, the PDE4 variants PDE4A5, PDE4C1, PDE4D2, and PDE4D3 were identified by polymerase chain reaction analysis. Evidence of the recently described PDE7 was obtained by rolipram- insensitive cyclic adenosine monophosphate (cAMP) degradation, and its presence was verified by the demonstration of PDE7 messenger RNA. Primary cultures of human airway epithelium also expressed PDE1. Enhanced epithelial cAMP levels, induced by forskolin and PDE4 inhibition, increased formation of prostaglandin E2 (PGE2), but not of interleukin (IL)-8 or 15-hydroxyeicosatetraenoic acid (15-HETE) in airway epithelial cells. Increased cyclic guanosine monophosphate levels in these cells provoked by sodium nitroprusside and the PDE5 inhibitor zaprinast reduced the PGE2 synthesis, whereas 15-HETE and IL-8 formation were unchanged. The data suggest that PDE isoenzymes are important in airway inflammation and that PDE inhibitors exert anti-inflammatory effects by acting on AEC.
Assuntos
Brônquios/enzimologia , Glicoproteínas/metabolismo , Isoenzimas/metabolismo , Traqueia/enzimologia , Animais , Sequência de Bases , Brônquios/citologia , Células Cultivadas , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Primers do DNA , Dinoprostona/metabolismo , Células Epiteliais/enzimologia , Humanos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Interleucina-8/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Suínos , Traqueia/citologiaRESUMO
Infection of endothelial cells by Listeria monocytogenes is an essential step in the pathogenesis of listeriosis. We recently reported that L. monocytogenes induces up-regulation of E-selectin and other endothelial adhesion molecules and subsequent polymorphonuclear leukocyte (PMN) adhesion into cultured human endothelial cells. In the present study, we characterized the mechanisms of enhanced E-selectin expression using L. monocytogenes wild type (EGD), the isogenic in-frame deletion mutants for phosphatidylcholine (PC)- and phosphatidylinositol (PI)-specific phospholipases EGD delta plcA and EGD delta plcB, as well as the nonvirulent control strain Listeria innocua. Infection of endothelial cells with EGD delta plcA or EGD delta plcB for 6 h induced, as compared with EGD wild type, intermediate levels of E-selectin mRNA and protein as well as PMN rolling and adhesion at a shear rate of 1 dyne/cm2, indicating that both bacterial phospholipases are required for a maximal effect. Similarly, ceramide content and NF-kappa B activity were increased in L. monocytogenes-exposed endothelial cells, but only to intermediate levels for PC- or PI-phospholipase C (PLC)-deficient listerial mutants. Phospholipase effects could be mimicked by exogenously added ceramides or bacterial sphingomyelinase. The data presented indicate that PI-PLC and PC-PLC are important virulence factors for L. monocytogenes infections that induce accumulation of ceramides that in turn may act as second messengers to control host cell signal-transduction pathways leading to persistent NF-kappa B activation, increased E-selectin expression, and enhanced PMN rolling/adhesion. The ability of L. monocytogenes to stimulate PMN adhesion to endothelial cells may be an important mechanism in the pathogenesis of severe listeriosis.
Assuntos
Ceramidas/metabolismo , Selectina E/biossíntese , Endotélio Vascular/enzimologia , Listeria monocytogenes/enzimologia , NF-kappa B/metabolismo , Fosfolipases Tipo C/fisiologia , Células Cultivadas , Ceramidas/fisiologia , Selectina E/genética , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Endotélio Vascular/microbiologia , Ativação Enzimática/imunologia , Humanos , Listeriose/enzimologia , Listeriose/imunologia , Listeriose/metabolismo , Fosfatidilinositol Diacilglicerol-Liase , Fosfoinositídeo Fosfolipase C , RNA Mensageiro/biossíntese , Sistemas do Segundo Mensageiro/imunologia , Veias Umbilicais , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
Vascular endothelial growth factor (VEGF) is a key regulator of vasculo- and angiogenesis. Earlier studies demonstrated a permeability-increasing effect of VEGF in skin tests, leading to its other name, vascular permeability factor. We wondered whether VEGF-induced hyperpermeability was a direct effect of VEGF on endothelial cells and studied the permeability of human and porcine endothelial cell monolayers in a well-characterized in vitro system. VEGF increased the hydraulic conductivity up to 20-fold and simultaneously decreased the albumin reflection coefficient. This effect occurred after a delay of 150 min, although VEGF-induced early endothelial cell activation was verified by enhanced inositol phosphate accumulation within 5 min and increased P-selectin expression within 15 min. Platelet-derived growth factor and granulocyte-macrophage colony-stimulating factor, two endothelial cell nonspecific mitogens, also stimulated phosphatidylinositol metabolism and P-selectin expression; however, they had no effect on endothelial permeability. The increase in intracellular cyclic nucleotide levels of human endothelial monolayers abolished VEGF-induced endothelial hyperpermeability. In summary, VEGF increased endothelial permeability by a direct action on endothelial cells. Based on the pattern of endothelial cell activation by growth factors, VEGF appears to be a unique stimulus.
Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Fatores de Crescimento Endotelial/farmacologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Linfocinas/farmacologia , Células Cultivadas , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Selectina-P/metabolismo , Fosfatidilinositóis/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Small GTP-binding proteins of the Ras and Rho family participate in various important signalling pathways. Large clostridial cytotoxins inactivate GTPases by UDP-glucosylation. Using Clostridium difficile toxin B-10463 (TcdB) for inactivation of Rho proteins (RhoA/Rac/Cdc42) and Clostridium sordellii lethal toxin-1522 (TcsL) for inactivation of Ras-proteins (Ras/Rac/Ral, Rap) the role of these GTPases in protein kinase C (PKC) stimulation was studied. Phorbol-myristate-acetate (PMA) induced a rapid PKC translocation to and activation in the particulate cell fraction as determined by PKC-activity measurements and Western blots for PKC alpha. These effects were blocked by TcdB inhibiting Rho proteins in endothelial cells, but not in TcsL-treated cells (i.e., cells without Ras activity), suggesting that Rho GTPases (RhoA and/or Cdc42) are the most likely GTP-binding proteins responsible for PKC activation. The Rho requirement for PKC activation/translocation was also verified for human epithelial cells and for lipopolysaccharide-stimulated endothelial cells. In summary, the data presented indicate that Rho protein inhibition blocked PKC translocation/activation in endothelial and epithelial cells.