RESUMO
BACKGROUND: Disruption of the epithelial barrier might be a risk factor for allergen sensitization and asthma. Viral respiratory tract infections are strongly associated with asthma exacerbation, but the effects of respiratory viruses on airway epithelial barrier function are not well understood. Many viruses generate double-stranded RNA, which can lead to airway inflammation and initiate an antiviral immune response. OBJECTIVES: We investigated the effects of the synthetic double-stranded RNA polyinosinic:polycytidylic acid (polyI:C) on the structure and function of the airway epithelial barrier in vitro. METHODS: 16HBE14o- human bronchial epithelial cells and primary airway epithelial cells at an air-liquid interface were grown to confluence on Transwell inserts and exposed to polyI:C. We studied epithelial barrier function by measuring transepithelial electrical resistance and paracellular flux of fluorescent markers and structure of epithelial apical junctions by means of immunofluorescence microscopy. RESULTS: PolyI:C induced a profound decrease in transepithelial electrical resistance and increase in paracellular permeability. Immunofluorescence microscopy revealed markedly reduced junctional localization of zonula occludens-1, occludin, E-cadherin, ß-catenin, and disorganization of junction-associated actin filaments. PolyI:C induced protein kinase D (PKD) phosphorylation, and a PKD antagonist attenuated polyI:C-induced disassembly of apical junctions and barrier dysfunction. CONCLUSIONS: PolyI:C has a powerful and previously unsuspected disruptive effect on the airway epithelial barrier. PolyI:C-dependent barrier disruption is mediated by disassembly of epithelial apical junctions, which is dependent on PKD signaling. These findings suggest a new mechanism potentially underlying the associations between viral respiratory tract infections, airway inflammation, and allergen sensitization.
Assuntos
Células Epiteliais/patologia , Poli I-C/imunologia , Proteína Quinase C/metabolismo , Mucosa Respiratória/patologia , Transdução de Sinais/imunologia , Asma/imunologia , Asma/metabolismo , Asma/patologia , Linhagem Celular , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Humanos , Immunoblotting , Indutores de Interferon/imunologia , Indutores de Interferon/metabolismo , Microscopia de Fluorescência , Permeabilidade , Poli I-C/metabolismo , Proteína Quinase C/imunologia , Interferência de RNA , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Infecções Respiratórias/imunologia , Infecções Respiratórias/metabolismo , Infecções Respiratórias/patologia , Junções Íntimas/imunologia , Junções Íntimas/metabolismo , Junções Íntimas/patologiaRESUMO
Pulmonary ossification (PO) is a rare metastatic disease characterized by the formation of diffuse heterotopic bone units in the lung parenchyma. Herein, we describe a 45-year-old Filipino male with dendriform pulmonary ossification in combination with gastroesophageal reflux disease and chronic dust exposure, a notably unique association. Radiographic imaging and pathology findings are examined with discussion of various pulmonary disease entities from current literature. Further recognition of PO will facilitate appropriate treatment and better outcomes for patients diagnosed with this enigmatic condition.
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Sphingosine kinase 1 (SK1) is one of the two known kinases, which generates sphingosine-1-phosphate (S1P), a potent endogenous lipid mediator involved in cell survival, proliferation, and cell-cell interactions. Activation of SK1 and intracellular generation of S1P were suggested to be part of the growth and survival factor-induced signaling, and overexpression of SK1 provoked cell tumorigenic transformation. Using a highly selective and sensitive LC-MS/MS approach, here we show that SK1 overexpression, but not SK2, in different primary cells and cultured cell lines results in predominant upregulation of the synthesis of dihydrosphingosine-1-phosphate (DHS1P) compared to S1P. Stable isotope pulse-labeling experiments in conjunction with LC-MS/MS quantitation of different sphingolipids demonstrated strong interference of overexpressed SK1 with the de novo sphingolipid biosynthesis by deviating metabolic flow of newly formed sphingoid bases from ceramide formation toward the synthesis of DHS1P. On the contrary, S1P biosynthesis was not directly linked to the de novo sphingoid bases transformations and was dependent on catabolic generation of sphingosine from complex sphingolipids. As a result of SK1 overexpression, migration and Ca2+-response of human pulmonary artery endothelial cells (HPAEC) to stimulation with external S1P, but not thrombin, was strongly impaired. In contrast, selective increase in intracellular content of DHS1P or S1P through the uptake and phosphorylation of corresponding sphingoid bases had no effect on S1P-induced signaling or facilitation of wound healing. Furthermore, infection of human bronchial epithelial cells (HBEpC) with RSV A-2 virus increased SK1-mediated synthesis of DHS1P and S1P, whereas TNF-alpha enhanced only S1P production in HPAEC. These findings uncover a new functional role for SK1, which can control survival/death (DHS1P-S1P/ceramides) balance by targeting sphingolipid de novo biosynthesis and selectively generating DHS1P at a metabolic step preceding ceramide formation.
Assuntos
Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Esfingosina/análogos & derivados , Vírus do Sarcoma Aviário/fisiologia , Cálcio/metabolismo , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Humanos , Lisofosfolipídeos/análise , Lisofosfolipídeos/química , Lisofosfolipídeos/farmacologia , Fosforilação/efeitos dos fármacos , RNA Interferente Pequeno , Serina C-Palmitoiltransferase/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esfingosina/análise , Esfingosina/biossíntese , Esfingosina/química , Esfingosina/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
HBEpCs (human bronchial epithelial cells) contribute to airway inflammation by secreting a variety of cytokines and chemokines in response to allergens, pathogens, viruses and environmental toxins and pollutants. The potent neutrophil chemoattractant, IL-8 (interleukin-8), is a major cytokine secreted by HBEpCs. We have recently demonstrated that LPA (lysophosphatidic acid) stimulated IL-8 production in HBEpCs via protein kinase C delta dependent signal transduction. However, mechanisms of IL-8 expression and secretion are complex and involve multiple protein kinases and transcriptional factors. The present study was undertaken to investigate MAPK (mitogen-activated protein kinase) signalling in the transcriptional regulation of IL-8 expression and secretion in HBEpCs. Exposure of HBEpCs to LPA (1 microM) enhanced expression and secretion of IL-8 by 5-8-fold and stimulated threonine/tyrosine phosphorylation of ERK (extracellular-signal-regulated kinase), p38 MAPK and JNK (c-Jun N-terminal kinase). The LPA-induced secretion of IL-8 was blocked by the p38 MAPK inhibitor SB203580, by p38 MAPK siRNA (small interfering RNA), and by the JNK inhibitor JNK(i) II, but not by the MEK (MAPK/ERK kinase) inhibitor, PD98059. LPA enhanced the transcriptional activity of the IL-8 gene; that effect relied on activation of the transcriptional factors NF-kappaB (nuclear factor kappaB) and AP-1 (activator protein-1). Furthermore, SB203580 attenuated LPA-dependent phosphorylation of IkappaB (inhibitory kappaB), NF-kappaB and phospho-p38 translocation to the nucleus, NF-kappaB transcription and IL-8 promoter-mediated luciferase reporter activity, without affecting the JNK pathway and AP-1 transcription. Similarly, JNK(i) II only blocked LPA-mediated phosphorylation of JNK and c-Jun, AP-1 transcription and IL-8 promoter-mediated luciferase reporter activity, without blocking p38 MAPK-dependent NF-kappaB transcription. Additionally, siRNA for LPA(1-3) receptors partially blocked LPA-induced IL-8 production and activation of MAPKs. The LPA1 and LPA3 receptors, as compared with LPA2, were most efficient in transducing LPA-mediated IL-8 production. These results show an independent role for p38 MAPK and JNK in LPA-induced IL-8 expression and secretion via NF-kappaB and AP-1 transcription respectively in HBEpCs.
Assuntos
Brônquios/citologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-8/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Lisofosfolipídeos/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Humanos , Interleucina-8/genética , NF-kappa B/metabolismo , Fosforilação , Transporte Proteico , Mucosa Respiratória/citologia , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/enzimologia , Fator de Transcrição AP-1/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
We show that LPA1 (lysophosphatidic acid receptor-1) is constitutively localized in the nucleus of mammalian cells. LPA1 also traffics from cell membranes to the nucleus in response to LPA (lysophosphatidic acid). Several lines of evidence suggest an important role for cell-matrix interaction in regulating the constitutive nuclear localization of LPA1. First, the RGDS peptide, which blocks cell matrix-induced integrin clustering and cytoskeletal rearrangement, reduced the number of cells containing LPA1 in the nucleus. Secondly, a higher proportion of cells contained nuclear LPA1 when adhesion on fibronectin-coated glass was compared with adherence to polylysine-coated glass. Thirdly, pre-treatment of cells with the Rho kinase inhibitor (Y27632) or the myosin light chain kinase inhibitor (ML9) reduced the number of cells containing nuclear LPA1. The addition of LPA and/or Ki16425 (which binds to LPA1) to isolated nuclei containing LPA1 induced the phosphorylation of several proteins with molecular masses of 34, 32, 14 and 11 kDa. These findings demonstrate that trafficking of LPA1 to the nucleus is influenced by cell-matrix interactions and that nuclear LPA1 may be involved in regulating intranuclear protein phosphorylation and signalling.
Assuntos
Núcleo Celular/metabolismo , Integrinas/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Transdução de Sinais , Animais , Células CHO , Contagem de Células , Células Cultivadas , Cricetinae , Cricetulus , Células Epiteliais/citologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Isoxazóis/farmacologia , Lisofosfolipídeos/farmacologia , Células PC12 , Toxina Pertussis/farmacologia , Fosforilação/efeitos dos fármacos , Propionatos/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico/efeitos dos fármacos , Ratos , Quinases Associadas a rhoRESUMO
LPA (lysophosphatidic acid), a potent bioactive phospholipid, elicits diverse cellular responses through activation of the G-protein-coupled receptors LPA1-LPA4. LPA-mediated signalling is partially regulated by LPPs (lipid phosphate phosphatases; LPP-1, -2 and -3) that belong to the phosphatase superfamily. This study addresses the role of LPPs in regulating LPA-mediated cell signalling and IL-8 (interleukin-8) secretion in HBEpCs (human bronchial epithelial cells). Reverse transcription-PCR and Western blotting revealed the presence and expression of LPP-1-3 in HBEpCs. Exogenous [3H]oleoyl LPA was hydrolysed to [3H]-mono-oleoylglycerol. Infection of HBEpCs with an adenoviral construct of human LPP-1 for 48 h enhanced the dephosphorylation of exogenous LPA by 2-3-fold compared with vector controls. Furthermore, overexpression of LPP-1 partially attenuated LPA-induced increases in the intracellular Ca2+ concentration, phosphorylation of IkappaB (inhibitory kappaB) and translocation of NF-kappaB (nuclear factor-kappaB) to the nucleus, and almost completely prevented IL-8 secretion. Infection of cells with an adenoviral construct of the mouse LPP-1 (R217K) mutant partially attenuated LPA-induced IL-8 secretion without altering LPA-induced changes in intracellular Ca2+ concentration, phosphorylation of IkappaB, NF-kappaB activation or IL-8 gene expression. Our results identify LPP-1 as a key regulator of LPA signalling and IL-8 secretion in HBEpCs. Thus LPPs could represent potential targets in regulating leucocyte infiltration and airway inflammation.
Assuntos
Brônquios/citologia , Cálcio/metabolismo , Células Epiteliais/enzimologia , Células Epiteliais/metabolismo , Interleucina-8/metabolismo , Lisofosfolipídeos/metabolismo , NF-kappa B/metabolismo , Fosfatidato Fosfatase/fisiologia , Adenoviridae , Arginina/genética , Arginina/fisiologia , Extratos Celulares/química , Células Cultivadas , Células Epiteliais/virologia , Humanos , Transplante de Pulmão , Lisina/genética , Lisina/fisiologia , Mutação de Sentido Incorreto/genética , Mutação de Sentido Incorreto/fisiologia , Fosfatidato Fosfatase/biossíntese , Fosfatidato Fosfatase/genética , Receptores de Ácidos Lisofosfatídicos/agonistas , Doadores de TecidosRESUMO
BACKGROUND: Melioidosis, an infection caused by the gram-negative bacterium Burkholderia pseudomallei, is an important cause of pneumonia, skin infection, sepsis, and death in Southeast Asia and Australia, but is exceedingly rare in North America. Pulmonary melioidosis typically presents as acute bacterial pneumonia or cavitary lung lesions resembling tuberculosis. CASE REPORT: We report melioidosis in a 70-year-old active smoker from Mexico with no history of travel to disease-endemic areas. The patient presented with a left supraclavicular abscess and a non-cavitary, left lung mass encasing a pulmonary vein. Incision and drainage of the patient's subcutaneous abscess isolated B. pseudomallei, and fine-needle aspiration of enlarged mediastinal lymph nodes revealed the presence of intracellular gram-negative bacilli with no evidence of malignancy. Biochemical tests determined that the strain the patient acquired from Mexico is identical to only 1 other isolate from Thailand. CONCLUSIONS: This report highlights the blurring epidemiological borders of this organism, its rare presentation mimicking lung malignancy, and an aggressive antimicrobial treatment that resulted in resolution of the patient's symptoms.
Assuntos
Antibacterianos/uso terapêutico , Burkholderia pseudomallei/isolamento & purificação , Drenagem/métodos , Melioidose/diagnóstico , Idoso , Biópsia por Agulha Fina , Diagnóstico Diferencial , Feminino , Humanos , Melioidose/microbiologia , Melioidose/terapia , México , Tomografia Computadorizada por Raios XRESUMO
Of the several factors believed to exacerbate asthmatic symptoms, air pollution and viral infections are considered to be particularly important. Although evidence indicates that each of these respiratory insults individually can increase asthma severity in susceptible individuals, we know little about the extent to which exposure to environmental oxidant pollutants can influence the course of respiratory viral infection and its associated inflammation. To investigate the interaction of these two stimuli within their common epithelial cell targets in the upper and lower respiratory tracks, we infected primary human nasal epithelial cells and cells of the BEAS-2B line grown at the air-liquid interface with human rhinovirus type 16 (RV16) and exposed them to NO2 (2.0 ppm) or O3 (0.2 ppm) for 3 hr. Independently, RV16, NO2, and O3 rapidly increased release of the inflammatory cytokine interleukin-8 through oxidant-dependent mechanisms. The combined effect of RV16 and oxidant ranged from 42% to 250% greater than additive for NO2 and from 41% to 67% for O3. We abrogated these effects by treating the cells with the antioxidant N-acetylcysteine. Surface expression of intercellular adhesion molecule 1 (ICAM-1) underwent additive enhancement in response to combined stimulation. These data indicate that oxidant pollutants can amplify the generation of proinflammatory cytokines by RV16-infected cells and suggest that virus-induced inflammation in upper and lower airways may be exacerbated by concurrent exposure to ambient levels of oxidants commonly encountered the indoor and outdoor environments.
Assuntos
Citocinas/biossíntese , Oxidantes Fotoquímicos/efeitos adversos , Ozônio/efeitos adversos , Infecções por Picornaviridae/imunologia , Técnicas de Cultura de Células , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Humanos , Inflamação , Dióxido de Nitrogênio , Nariz/citologia , Infecções por Picornaviridae/fisiopatologia , Rhinovirus/patogenicidadeRESUMO
OBJECTIVES/HYPOTHESIS: Although the mechanisms underlying the initiation and maintenance of inflammation in chronic rhinosinusitis are poorly understood, the activation of memory T cells within the nasal mucosa is thought to play an important role. T-cell activation requires specialized antigen processing and presentation of antigen by immunocompetent cells in the context of cell surface immune molecules. The purpose of this study was to investigate the expression of such molecules by human sinonasal epithelial cells grown in culture at the air-liquid interface (ALI). METHODS: Middle meatal epithelium was obtained from six patients undergoing endoscopic sinus surgery. Dissociated epithelial cells were grown to confluence in serum-free, defined medium and transferred to filter inserts for culture at the ALI. Cells were harvested at 2 and 21 days of growth at the ALI and processed for real-time polymerase chain reaction (PCR). The presence and relative abundance of constitutively expressed mRNA for human leukocyte antigen (HLA)-B, HLA-DR, B7 to 1, B7 to 2, B7-H2, B7-H3, and cathepsin D were assessed. RESULTS: After 2 days at the ALI, middle meatal epithelial cells demonstrated expression of genes for each of the antigen processing associated genes tested. The expression of HLA-B and HLA-DR increased significantly with cellular maturation at the ALI. Expression of HLA-DR and B7 to 1 increased with cytokine stimulation. CONCLUSIONS: Primary human epithelial cells obtained from the middle meatus express genes associated with antigen presentation function. The pattern of gene expression is modulated by cytokine stimulation and changes as the cells differentiate at the ALI. These findings suggest that mature middle meatal epithelial cells have the cellular machinery to interact with T cells and therefore may be direct participants in the modulation of T-cell activity in chronic sinusitis.
Assuntos
Apresentação de Antígeno/imunologia , Genes MHC da Classe II/imunologia , Mucosa Nasal/imunologia , RNA Mensageiro/imunologia , Sinusite/imunologia , Apresentação de Antígeno/genética , Citocinas/imunologia , Células Epiteliais/imunologia , Feminino , Expressão Gênica/genética , Genes MHC da Classe II/genética , Humanos , Masculino , RNA Mensageiro/genética , Sinusite/genéticaRESUMO
Emerging evidence indicates that airway epithelial barrier function is compromised in asthma, a disease characterized by Th2-skewed immune response against inhaled allergens, but the mechanisms involved are not well understood. The purpose of this study was to investigate the effects of Th2-type cytokines on airway epithelial barrier function. 16HBE14o- human bronchial epithelial cells monolayers were grown on collagen coated Transwell inserts. The basolateral or apical surfaces of airway epithelia were exposed to human interleukin-4 (IL-4), IL-13, IL-25, IL-33, thymic stromal lymphopoietin (TSLP) alone or in combination at various concentrations and time points. We analyzed epithelial apical junctional complex (AJC) function by measuring transepithelial electrical resistance (TEER) and permeability to FITC-conjugated dextran over time. We analyzed AJC structure using immunofluorescence with antibodies directed against key junctional components including occludin, ZO-1, ß-catenin and E-cadherin. Transepithelial resistance was significantly decreased after both basolateral and apical exposure to IL-4. Permeability to 3 kDa dextran was also increased in IL-4-exposed cells. Similar results were obtained with IL-13, but none of the innate type 2 cytokines examined (TSLP, IL-25 or IL-33) significantly affected barrier function. IL-4 and IL-13-induced barrier dysfunction was accompanied by reduced expression of membrane AJC components but not by induction of claudin- 2. Enhanced permeability caused by IL-4 was not affected by wortmannin, an inhibitor of PI3 kinase signaling, but was attenuated by a broad spectrum inhibitor of janus associated kinases. Our study indicates that IL-4 and IL-13 have disruptive effect on airway epithelial barrier function. Th2-cytokine induced epithelial barrier dysfunction may contribute to airway inflammation in allergic asthma.
RESUMO
OBJECTIVE: 6-mercaptopurine (6-MP) is efficacious in the treatment of inflammatory bowel disease (IBD). However, about one-third of patients respond poorly to therapy. This study aimed to characterize the inherent differences in 6-MP transport that may cotribute to the differences in treatment responses. METHODS: Intracellular 6-MP accumulation was assayed in Epstein-Barr virus (EBV)-transformed lymphocytes from IBD patients, using (14) C-radiolabeled 6-MP. Cell proliferation was determined by methyl thiazolyl tetrazolium (MTT) assay. Apoptosis was assayed based on the activation of caspase 3. The expressions of 15 potential 6-MP transporters were evaluated by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Intracellular 6-MP accumulation, varying significantly among patients, was carrier-dependent and partially sodium-dependent. 6-MP cytotoxicity was, at least in part, due to apoptosis and correlated with intracellular drug accumulation. The efflux transporters did not appear to contribute to the variability of intracellular drug accumulation between patients, since none correlated with drug accumulation or cytotoxicity. Rather, differential expression of five influx/uptake transporters might be a key contributor to the difference in the accumulation of and susceptibility to the drug. CONCLUSIONS: The heterogeneity of the drug transporters may be the reason for the therapeutic sensitivity of 6-MP in IBD patients. As the 6-MP uptake is a carrier-mediated and partially sodium-dependent process, future studies are necessary to evaluate the role of the putative transporters and their correlation with drug sensitivity in patients.
Assuntos
Doenças Inflamatórias Intestinais/tratamento farmacológico , Linfócitos/metabolismo , Mercaptopurina/efeitos adversos , Mercaptopurina/farmacocinética , Adolescente , Adulto , Idoso , Apoptose/efeitos dos fármacos , Ligação Competitiva/fisiologia , Radioisótopos de Carbono , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Caspase 3/metabolismo , Linhagem Celular Transformada , Sobrevivência Celular/efeitos dos fármacos , Citotoxinas/farmacocinética , Resistência a Medicamentos/fisiologia , Feminino , Expressão Gênica/fisiologia , Gliceraldeído 3-Fosfato Desidrogenase (NADP+)/metabolismo , Humanos , Imunossupressores/efeitos adversos , Imunossupressores/farmacocinética , Doenças Inflamatórias Intestinais/metabolismo , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Sódio/farmacologia , Adulto JovemRESUMO
We have demonstrated earlier that lysophosphatidic acid (LPA)-induced interleukin-8 (IL-8) secretion is regulated by protein kinase Cdelta (PKCdelta)-dependent NF-kappaB activation in human bronchial epithelial cells (HBEpCs). Here we provide evidence for signaling pathways that regulate LPA-mediated transactivation of epidermal growth factor receptor (EGFR) and the role of cross-talk between G-protein-coupled receptors and receptor-tyrosine kinases in IL-8 secretion in HBEpCs. Treatment of HBEpCs with LPA stimulated tyrosine phosphorylation of EGFR, which was attenuated by matrix metalloproteinase (MMP) inhibitor (GM6001), heparin binding (HB)-EGF inhibitor (CRM 197), and HB-EGF neutralizing antibody. Overexpression of dominant negative PKCdelta or pretreatment with a PKCdelta inhibitor (rottlerin) or Src kinase family inhibitor (PP2) partially blocked LPA-induced MMP activation, proHB-EGF shedding, and EGFR tyrosine phosphorylation. Down-regulation of Lyn kinase, but not Src kinase, by specific small interfering RNA mitigated LPA-induced MMP activation, proHB-EGF shedding, and EGFR phosphorylation. In addition, overexpression of dominant negative PKCdelta blocked LPA-induced phosphorylation and translocation of Lyn kinase to the plasma membrane. Furthermore, down-regulation of EGFR by EGFR small interfering RNA or pretreatment of cells with EGFR inhibitors AG1478 and PD158780 almost completely blocked LPA-dependent EGFR phosphorylation and partially attenuated IL-8 secretion, respectively. These results demonstrate that LPA-induced IL-8 secretion is partly dependent on EGFR transactivation regulated by PKCdelta-dependent activation of Lyn kinase and MMPs and proHB-EGF shedding, suggesting a novel mechanism of cross-talk and interaction between G-protein-coupled receptors and receptor-tyrosine kinases in HBEpCs.
Assuntos
Brônquios/citologia , Fator de Crescimento Epidérmico/metabolismo , Células Epiteliais/citologia , Receptores ErbB/metabolismo , Regulação da Expressão Gênica , Interleucina-8/metabolismo , Lisofosfolipídeos/farmacologia , Metaloproteinases da Matriz/metabolismo , Proteína Quinase C-delta/metabolismo , Ativação Transcricional , Quinases da Família src/metabolismo , Inibidores Enzimáticos/farmacologia , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Peptídeos e Proteínas de Sinalização IntercelularRESUMO
Superoxide (O(2)(-)) production by nonphagocytes, similar to phagocytes, is by activation of the NADPH oxidase multicomponent system. Although activation of neutrophil NADPH oxidase involves extensive serine phosphorylation of p47(phox), the role of tyrosine phosphorylation of p47(phox) in NADPH oxidase-dependent O(2)(-) production is unclear. We have shown recently that hyperoxia-induced NADPH oxidase activation in human pulmonary artery endothelial cells (HPAECs) is regulated by mitogen-activated protein kinase signal transduction. Here we provided evidence on the role of nonreceptor tyrosine kinase, Src, in hyperoxia-induced tyrosine phosphorylation of p47(phox) and NADPH oxidase activation in HPAECs. Exposure of HPAECs to hyperoxia for 1 h resulted in increased O(2)(-) and reactive oxygen species (ROS) production and enhanced tyrosine phosphorylation of Src as determined by Western blotting with phospho-Src antibodies. Pretreatment of HPAECs with the Src kinase inhibitor PP2 (1 mum) or transient expression of a dominant-negative mutant of Src attenuated hyperoxia-induced tyrosine phosphorylation of Src and ROS production. Furthermore, exposure of cells to hyperoxia enhanced tyrosine phosphorylation of p47(phox) and its translocation to cell peripheries that were attenuated by PP2. In vitro, Src phosphorylated recombinant p47(phox) in a time-dependent manner. Src immunoprecipitates of cell lysates from control cells revealed the presence of immunodetectable p47(phox) and p67(phox), suggesting the association of oxidase components with Src under basal conditions. Moreover, exposure of HPAECs to hyperoxia for 1 h enhanced the association of p47(phox), but not p67(phox), with Src. These results indicated that Src-dependent tyrosine phosphorylation of p47(phox) regulates hyperoxia-induced NADPH oxidase activation and ROS production in HPAECs.
Assuntos
NADPH Oxidases/metabolismo , Oxigênio/administração & dosagem , Fosfoproteínas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Tirosina/metabolismo , Quinases da Família src/metabolismo , Western Blotting , Proteína Tirosina Quinase CSK , Linhagem Celular , Células Endoteliais/enzimologia , Células Endoteliais/metabolismo , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Proteínas de Fluorescência Verde/genética , Fosfoproteínas/genética , Fosforilação , Fosfotirosina/análise , Proteínas Tirosina Quinases/análise , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Artéria Pulmonar , Pirimidinas/farmacologia , RNA Interferente Pequeno/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Superóxidos/metabolismo , Transfecção , Quinases da Família src/antagonistas & inibidoresRESUMO
Epithelial cells of the human respiratory tract express human leukocyte antigen (HLA) and the costimulatory molecules B7-1 and B7-2. Little is known, however, about the constitutive expression of genes encoding for the more recently identified members of the B7 homolog family of costimulatory molecules or about the influence of cellular differentiation and cytokines on their activity or on that of HLA or B7-1 and B7-2. Human nasal epithelial (HNE) cells were grown at the air-liquid interface (ALI) for 2 or 21 days to model in vivo conditions. Expression of genes for HLA-B and HLA-DR1 increased during mucociliary differentiation during this period and became more similar to HNE cells obtained fresh by brush biopsy from nasal turbinates. Gene transcripts for B7-H3 and B7-H2 were abundantly expressed in cells cultured at the ALI, but neither their activities nor that of B7-2 was significantly altered during differentiation. IFN-gamma and TNF-alpha upregulated mRNA encoding for both HLA molecules, but not for the B7 molecules. This study describes, for the first time, the expression of B7-H3 and B7-H2 by HNE cells and thus expands the range of potential costimulatory signals through which these cells may interact with activated mucosal T lymphocytes. In addition, the results suggest that the extent of mucociliary differentiation of cultured cells may influence this capability.
Assuntos
Proteínas de Transporte/metabolismo , Mucosa Nasal/metabolismo , Mucosa Nasal/patologia , Proteínas , Linfócitos T/metabolismo , Antígenos CD , Biópsia/métodos , Proteínas de Transporte/genética , Diferenciação Celular/genética , Células Cultivadas , Feminino , Expressão Gênica/efeitos dos fármacos , Antígenos HLA-B/genética , Antígeno HLA-DR1/genética , Humanos , Ligante Coestimulador de Linfócitos T Induzíveis , Interferon gama/farmacologia , Ligantes , Masculino , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Lysophosphatidic acid (LPA), a potent bioactive lipid, elicits many of its biological actions via the specific G-protein-coupled receptors LPA1, LPA2, LPA3, and LPA4. Recently, we have shown that LPA-induced transactivation of platelet-derived growth factor receptor-beta is regulated by phospholipase D2 in human bronchial epithelial cells (HBEpCs) (Wang, L., Cummings, R. J., Zhao, Y., Kazlauskas, A., Sham, J., Morris, A., Brindley, D. N., Georas, S., and Natarajan, V. (2003) J. Biol. Chem. 278, 39931-39940). Here, we report that protein kinase Cdelta (PKCdelta) mediates LPA-induced NF-kappaB transcription and interleukin-8 (IL-8) secretion in HBEpCs. Treatment of HBEpCs with LPA increased both IL-8 gene and protein expression, which was coupled to Gi and G(12/13) proteins. LPA caused a marked activation of NF-kappaB in HBEpCs as determined by IkappaB phosphorylation and of NF-kappaB nuclear translocation and a strong induction of NF-kappaB promoter-mediated luciferase activity. Furthermore, LPA-activated PKCdelta and the LPA-mediated activation of NF-kappaB and IL-8 production were attenuated by overexpression of dominant-negative PKCdelta and rottlerin. Intratracheal administration of LPA in mice resulted in elevated levels of macrophage inflammatory protein-2, a murine homolog of IL-8, and an influx of neutrophils in the bronchoalveolar lavage fluid. These results demonstrate for the first time that LPA is a potent stimulator of IL-8 production in HBEpCs, which involves PKCdelta/NF-kappaB signaling pathways.