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1.
Thorax ; 74(7): 675-683, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31036772

RESUMO

RATIONALE: Associations between urban (outdoor) airborne particulate matter (PM) exposure and TB and potential biological mechanisms are poorly explored. OBJECTIVES: To examine whether in vivo exposure to urban outdoor PM in Mexico City and in vitro exposure to urban outdoor PM2.5 (< 2.5 µm median aerodynamic diameter) alters human host immune cell responses to Mycobacterium tuberculosis. METHODS: Cellular toxicity (flow cytometry, proliferation assay (MTS assay)), M. tuberculosis and PM2.5 phagocytosis (microscopy), cytokine-producing cells (Enzyme-linked immune absorbent spot (ELISPOT)), and signalling pathway markers (western blot) were examined in bronchoalveolar cells (BAC) and peripheral blood mononuclear cells (PBMC) from healthy, non-smoking, residents of Mexico City (n=35; 13 female, 22 male). In vivo-acquired PM burden in alveolar macrophages (AM) was measured by digital image analysis. MEASUREMENTS AND MAIN RESULTS: In vitro exposure of AM to PM2.5 did not affect M. tuberculosis phagocytosis. High in vivo-acquired AM PM burden reduced constitutive, M. tuberculosis and PM-induced interleukin-1ß production in freshly isolated BAC but not in autologous PBMC while it reduced constitutive production of tumour necrosis factor-alpha in both BAC and PBMC. Further, PM burden was positively correlated with constitutive, PM, M. tuberculosis and purified protein derivative (PPD)-induced interferon gamma (IFN-γ) in BAC, and negatively correlated with PPD-induced IFN-γ in PBMC. CONCLUSIONS: Inhalation exposure to urban air pollution PM impairs important components of the protective human lung and systemic immune response against M. tuberculosis. PM load in AM is correlated with altered M. tuberculosis-induced cytokine production in the lung and systemic compartments. Chronic PM exposure with high constitutive expression of proinflammatory cytokines results in relative cellular unresponsiveness.

2.
Acta Biomater ; 57: 85-94, 2017 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-28522412

RESUMO

Enhanced bioactive anti-oxidant formulations are critical for treatment of inflammatory diseases, such as atherosclerosis. A hallmark of early atherosclerosis is the uptake of oxidized low density lipoprotein (oxLDL) by macrophages, which results in foam cell and plaque formation in the arterial wall. The hypolipidemic, anti-inflammatory, and antioxidative properties of polyphenol compounds make them attractive targets for treatment of atherosclerosis. However, high concentrations of antioxidants can reverse their anti-atheroprotective properties and cause oxidative stress within the artery. Here, we designed a new class of nanoparticles with anti-oxidant polymer cores and shells comprised of scavenger receptor targeting amphiphilic macromolecules (AMs). Specifically, we designed ferulic acid-based poly(anhydride-ester) nanoparticles to counteract the uptake of high levels of oxLDL and regulate reactive oxygen species generation (ROS) in human monocyte derived macrophages (HMDMs). Compared to all compositions examined, nanoparticles with core ferulic acid-based polymers linked by diglycolic acid (PFAG) showed the greatest inhibition of oxLDL uptake. At high oxLDL concentrations, the ferulic acid diacids and polymer nanoparticles displayed similar oxLDL uptake. Treatment with the PFAG nanoparticles downregulated the expression of macrophage scavenger receptors, CD-36, MSR-1, and LOX-1 by about 20-50%, one of the causal factors for the decrease in oxLDL uptake. The PFAG nanoparticle lowered ROS production by HMDMs, which is important for maintaining macrophage growth and prevention of apoptosis. Based on these results, we propose that ferulic acid-based poly(anhydride ester) nanoparticles may offer an integrative strategy for the localized passivation of the early stages of the atheroinflammatory cascade in cardiovascular disease. STATEMENT OF SIGNIFICANCE: Future development of anti-oxidant formulations for atherosclerosis applications is essential to deliver an efficacious dose while limiting localized concentrations of pro-oxidants. In this study, we illustrate the potential of degradable ferulic acid-based polymer nanoparticles to control macrophage foam cell formation by significantly reducing oxLDL uptake through downregulation of scavenger receptors, CD-36, MSR-1, and LOX-1. Another critical finding is the ability of the degradable ferulate-based polymer nanoparticles to lower macrophage reactive oxygen species (ROS) levels, a precursor to apoptosis and plaque escalation. The degradable ferulic acid-based polymer nanoparticles hold significant promise as a means to alter the treatment and progression of atherosclerosis.


Assuntos
Anti-Inflamatórios , Aterosclerose , Ácidos Cumáricos , Células Espumosas/metabolismo , Lipogênese/efeitos dos fármacos , Nanopartículas , Polianidridos , Espécies Reativas de Oxigênio/metabolismo , Anti-Inflamatórios/farmacologia , Aterosclerose/tratamento farmacológico , Aterosclerose/metabolismo , Aterosclerose/patologia , Ácidos Cumáricos/química , Ácidos Cumáricos/farmacologia , Células Espumosas/patologia , Humanos , Nanopartículas/química , Nanopartículas/uso terapêutico , Polianidridos/química , Polianidridos/farmacologia
3.
Toxicol Appl Pharmacol ; 304: 110-20, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27163765

RESUMO

Macrophages have been shown to play a role in acetaminophen (APAP)-induced hepatotoxicity, contributing to both pro- and anti-inflammatory processes. In these studies, we analyzed the role of the spleen as an extramedullary source of hepatic macrophages. APAP administration (300mg/kg, i.p.) to control mice resulted in an increase in CD11b(+) infiltrating Ly6G(+) granulocytic and Ly6G(-) monocytic cells in the spleen and the liver. The majority of the Ly6G(+) cells were also positive for the monocyte/macrophage activation marker, Ly6C, suggesting a myeloid derived suppressor cell (MDSC) phenotype. By comparison, Ly6G(-) cells consisted of 3 subpopulations expressing high, intermediate, and low levels of Ly6C. Splenectomy was associated with increases in mature (F4/80(+)) and immature (F4/80(-)) pro-inflammatory Ly6C(hi) macrophages and mature anti-inflammatory (Ly6C(lo)) macrophages in the liver after APAP; increases in MDSCs were also noted in the livers of splenectomized (SPX) mice after APAP. This was associated with increases in APAP-induced expression of chemokine receptors regulating pro-inflammatory (CCR2) and anti-inflammatory (CX3CR1) macrophage trafficking. In contrast, APAP-induced increases in pro-inflammatory galectin-3(+) macrophages were blunted in livers of SPX mice relative to control mice, along with hepatic expression of TNF-α, as well as the anti-inflammatory macrophage markers, FIZZ-1 and YM-1. These data demonstrate that multiple subpopulations of pro- and anti-inflammatory cells respond to APAP-induced injury, and that these cells originate from distinct hematopoietic reservoirs.


Assuntos
Acetaminofen/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/fisiopatologia , Mediadores da Inflamação/metabolismo , Células Mieloides/efeitos dos fármacos , Baço/metabolismo , Animais , Receptor 1 de Quimiocina CX3C , Quimiocinas/biossíntese , Galectina 3/metabolismo , Ativação de Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microssomos Hepáticos/efeitos dos fármacos , Fenótipo , Receptores CCR2/biossíntese , Receptores de Quimiocinas/biossíntese , Esplenectomia
4.
Chem Res Toxicol ; 27(5): 882-94, 2014 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-24661219

RESUMO

Acetaminophen (APAP) is metabolized in the liver to N-acetyl-p-benzoquinone imine (NAPQI), an electrophilic metabolite known to bind liver proteins resulting in hepatotoxicity. Mammalian thioredoxin reductase (TrxR) is a cellular antioxidant containing selenocysteine (Sec) in its C-terminal redox center, a highly accessible target for electrophilic modification. In the present study, we determined if NAPQI targets TrxR. Hepatotoxicity induced by APAP treatment of mice (300 mg/kg, i.p.) was associated with a marked inhibition of both cytosolic TrxR1 and mitochondrial TrxR2 activity. Maximal inhibition was detected at 1 and 6 h post-APAP for TrxR1 and TrxR2, respectively. In purified rat liver TrxR1, enzyme inactivation was correlated with the metabolic activation of APAP by cytochrome P450, indicating that enzyme inhibition was due to APAP-reactive metabolites. NAPQI was also found to inhibit TrxR1. NADPH-reduced TrxR1 was significantly more sensitive to NAPQI (IC50 = 0.023 µM) than the oxidized enzyme (IC50 = 1.0 µM) or a human TrxR1 Sec498Cys mutant enzyme (IC50 = 17 µM), indicating that cysteine and selenocysteine residues in the redox motifs of TrxR are critical for enzyme inactivation. This is supported by our findings that alkylation of reduced TrxR with biotin-conjugated iodoacetamide, which selectively reacts with selenol or thiol groups on proteins, was inhibited by NAPQI. LC-MS/MS analysis confirmed that NAPQI modified cysteine 59, cysteine 497, and selenocysteine 498 residues in the redox centers of TrxR, resulting in enzyme inhibition. In addition to disulfide reduction, TrxR is also known to mediate chemical redox cycling. We found that menadione redox cycling by TrxR was markedly less sensitive to NAPQI than disulfide reduction, suggesting that TrxR mediates these reactions via distinct mechanisms. These data demonstrate that APAP-reactive metabolites target TrxR, suggesting an additional mechanism by which APAP induces oxidative stress and hepatotoxicity.


Assuntos
Acetaminofen/metabolismo , Analgésicos não Entorpecentes/metabolismo , Benzoquinonas/toxicidade , Iminas/toxicidade , Fígado/enzimologia , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Tiorredoxina Dissulfeto Redutase/metabolismo , Sequência de Aminoácidos/efeitos dos fármacos , Animais , Benzoquinonas/metabolismo , Humanos , Iminas/metabolismo , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução , Ratos , Selenocisteína/metabolismo , Tiorredoxina Dissulfeto Redutase/química , Tiorredoxinas/metabolismo
5.
Exp Mol Pathol ; 94(1): 160-7, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23103612

RESUMO

The ability of rat hepatic sinusoidal endothelial cells (HSEC) to become activated in response to diverse inflammatory stimuli was analyzed. Whereas the classical macrophage activators, IFNγ and/or LPS upregulated expression of iNOS in HSEC, the alternative macrophage activators, IL-10 or IL-4+IL-13 upregulated arginase-1 and mannose receptor. Similar upregulation of iNOS and arginase-1 was observed in classically and alternatively activated Kupffer cells, respectively. Removal of inducing stimuli from the cells had no effect on expression of these markers, demonstrating that activation is persistent. Washing and incubation of IFNγ treated cells with IL-4+IL-13 resulted in decreased iNOS and increased arginase-1 expression, while washing and incubation of IL-4+IL-13 treated cells with IFNγ resulted in decreased arginase-1 and increased iNOS, indicating that classical and alternative activation of the cells is reversible. HSEC were more sensitive to phenotypic switching than Kupffer cells, suggesting greater functional plasticity. Hepatocyte viability and expression of PCNA, ß-catenin and MMP-9 increased in the presence of alternatively activated HSEC. In contrast, the viability of hepatocytes pretreated for 2 h with 5 mM acetaminophen decreased in the presence of classically activated HSEC. These data demonstrate that activated HSEC can modulate hepatocyte responses following injury. The ability of hepatocytes to activate HSEC was also investigated. Co-culture of HSEC with acetaminophen-injured hepatocytes, but not control hepatocytes, increased the sensitivity of HSEC to classical and alternative activating stimuli. The capacity of HSEC to respond to phenotypic activators may represent an important mechanism by which they participate in inflammatory responses associated with hepatotoxicity.


Assuntos
Células Endoteliais/fisiologia , Hepatócitos/fisiologia , Macrófagos do Fígado/fisiologia , Fígado/citologia , Fígado/metabolismo , Ativação de Macrófagos , Acetaminofen/farmacologia , Animais , Arginase/biossíntese , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Inflamação , Interferon gama/farmacologia , Interleucina-10/farmacologia , Interleucina-13/farmacologia , Interleucina-4/farmacologia , Macrófagos do Fígado/efeitos dos fármacos , Lectinas Tipo C/biossíntese , Lipopolissacarídeos/farmacologia , Fígado/efeitos dos fármacos , Masculino , Lectinas de Ligação a Manose/biossíntese , Metaloproteinase 9 da Matriz/biossíntese , Óxido Nítrico Sintase Tipo II/biossíntese , Antígeno Nuclear de Célula em Proliferação/biossíntese , Ratos , Ratos Sprague-Dawley , Receptores de Superfície Celular/biossíntese , beta Catenina/biossíntese
6.
Toxicol Appl Pharmacol ; 262(2): 139-48, 2012 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-22575169

RESUMO

Stem cell-derived tyrosine kinase (STK) is a transmembrane receptor reported to play a role in macrophage switching from a classically activated/proinflammatory phenotype to an alternatively activated/wound repair phenotype. In the present studies, STK⁻/⁻ mice were used to assess the role of STK in acetaminophen-induced hepatotoxicity as evidence suggests that the pathogenic process involves both of these macrophage subpopulations. In wild type mice, centrilobular hepatic necrosis and increases in serum transaminase levels were observed within 6h of acetaminophen administration (300 mg/kg, i.p.). Loss of STK resulted in a significant increase in sensitivity of mice to the hepatotoxic effects of acetaminophen and increased mortality, effects independent of its metabolism. This was associated with reduced levels of hepatic glutathione, rapid upregulation of inducible nitric oxide synthase, and prolonged induction of heme oxygenase-1, suggesting excessive oxidative stress in STK⁻/⁻ mice. F4/80, a marker of mature macrophages, was highly expressed on subpopulations of Kupffer cells in livers of wild type, but not STK⁻/⁻ mice. Whereas F4/80⁺ macrophages rapidly declined in the livers of wild type mice following acetaminophen intoxication, they increased in STK⁻/⁻ mice. In wild type mice hepatic expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-12, products of classically activated macrophages, increased after acetaminophen administration. Monocyte chemotactic protein-1 (MCP-1) and its receptor, CCR2, as well as IL-10, mediators involved in recruiting and activating anti-inflammatory/wound repair macrophages, also increased in wild type mice after acetaminophen. Loss of STK blunted the effects of acetaminophen on expression of TNFα, IL-1ß, IL-12, MCP-1 and CCR2, while expression of IL-10 increased. Hepatic expression of CX3CL1, and its receptor, CX3CR1 also increased in STK⁻/⁻ mice treated with acetaminophen. These data demonstrate that STK plays a role in regulating macrophage recruitment and activation in the liver following acetaminophen administration, and in hepatotoxicity.


Assuntos
Acetaminofen/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Ativação de Macrófagos/efeitos dos fármacos , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/imunologia , Citocinas/genética , Citocinas/metabolismo , Glutationa/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Fígado/efeitos dos fármacos , Fígado/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/metabolismo , RNA/química , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
7.
J Immunol ; 188(6): 2778-93, 2012 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-22345648

RESUMO

Epidemiological studies suggest that chronic exposure to air pollution increases susceptibility to respiratory infections, including tuberculosis in humans. A possible link between particulate air pollutant exposure and antimycobacterial immunity has not been explored in human primary immune cells. We hypothesized that exposure to diesel exhaust particles (DEP), a major component of urban fine particulate matter, suppresses antimycobacterial human immune effector cell functions by modulating TLR-signaling pathways and NF-κB activation. We show that DEP and H37Ra, an avirulent laboratory strain of Mycobacterium tuberculosis, were both taken up by the same peripheral human blood monocytes. To examine the effects of DEP on M. tuberculosis-induced production of cytokines, PBMC were stimulated with DEP and M. tuberculosis or purified protein derivative. The production of M. tuberculosis and purified protein derivative-induced IFN-γ, TNF-α, IL-1ß, and IL-6 was reduced in a DEP dose-dependent manner. In contrast, the production of anti-inflammatory IL-10 remained unchanged. Furthermore, DEP stimulation prior to M. tuberculosis infection altered the expression of TLR3, -4, -7, and -10 mRNAs and of a subset of M. tuberculosis-induced host genes including inhibition of expression of many NF-κB (e.g., CSF3, IFNG, IFNA, IFNB, IL1A, IL6, and NFKBIA) and IFN regulatory factor (e.g., IFNG, IFNA1, IFNB1, and CXCL10) pathway target genes. We propose that DEP downregulate M. tuberculosis-induced host gene expression via MyD88-dependent (IL6, IL1A, and PTGS2) as well as MyD88-independent (IFNA, IFNB) pathways. Prestimulation of PBMC with DEP suppressed the expression of proinflammatory mediators upon M. tuberculosis infection, inducing a hyporesponsive cellular state. Therefore, DEP alters crucial components of antimycobacterial host immune responses, providing a possible mechanism by which air pollutants alter antimicrobial immunity.


Assuntos
Monócitos/imunologia , Monócitos/microbiologia , NF-kappa B , Material Particulado/efeitos adversos , Tuberculose/imunologia , Emissões de Veículos/toxicidade , Adulto , Apoptose , Sobrevivência Celular , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Mycobacterium tuberculosis , NF-kappa B/imunologia , NF-kappa B/metabolismo , Material Particulado/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/imunologia , Adulto Jovem
8.
Toxicol Sci ; 125(2): 607-12, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22048645

RESUMO

Fenbendazole is a broad-spectrum anthelmintic drug widely used to prevent or treat nematode infections in laboratory rodent colonies. Potential interactions between fenbendazole and hepatotoxicants such as acetaminophen are unknown, and this was investigated in this study. Mice were fed a control diet or a diet containing fenbendazole (8-12 mg/kg/day) for 7 days prior to treatment with acetaminophen (300 mg/kg) or phosphate buffered saline. In mice fed a control diet, acetaminophen administration resulted in centrilobular hepatic necrosis and increases in serum transaminases, which were evident within 12 h. Acetaminophen-induced hepatotoxicity was markedly increased in mice fed the fenbendazole-containing diet, as measured histologically and by significant increases in serum transaminase levels. Moreover, in mice fed the fenbendazole-containing diet, but not the control diet, 63% mortality was observed within 24 h of acetaminophen administration. Fenbendazole by itself had no effect on liver histology or serum transaminases. To determine if exaggerated hepatotoxicity was due to alterations in acetaminophen metabolism, we analyzed sera for the presence of free acetaminophen and acetaminophen-glucuronide. We found that there were no differences in acetaminophen turnover. We also measured cytochrome P450 (cyp) 2e1, cyp3a, and cyp1a2 activity. Whereas fenbendazole had no effect on the activity of cyp2e1 or cyp3a, cyp1a2 was suppressed. A prolonged suppression of hepatic glutathione (GSH) was also observed in acetaminophen-treated mice fed the fenbendazole-containing diet when compared with the control diet. These data demonstrate that fenbendazole exacerbates the hepatotoxicity of acetaminophen, an effect that is related to persistent GSH depletion. These findings are novel and suggest a potential drug-drug interaction that should be considered in experimental protocols evaluating mechanisms of hepatotoxicity in rodent colonies treated with fenbendazole.


Assuntos
Acetaminofen/toxicidade , Anti-Helmínticos/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Fenbendazol/toxicidade , Fígado/efeitos dos fármacos , Acetaminofen/metabolismo , Animais , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Citocromo P-450 CYP1A2/metabolismo , Inibidores do Citocromo P-450 CYP1A2 , Citocromo P-450 CYP2E1/metabolismo , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/metabolismo , Modelos Animais de Doenças , Interações de Medicamentos , Inibidores Enzimáticos/farmacologia , Glutationa/metabolismo , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Tempo
9.
Annu Rev Pharmacol Toxicol ; 51: 267-88, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-20887196

RESUMO

The past several years have seen the accumulation of evidence demonstrating that tissue injury induced by diverse toxicants is due not only to their direct effects on target tissues but also indirectly to the actions of resident and infiltrating macrophages. These cells release an array of mediators with cytotoxic, pro- and anti-inflammatory, angiogenic, fibrogenic, and mitogenic activity, which function to fight infections, limit tissue injury, and promote wound healing. However, following exposure to toxicants, macrophages can become hyperresponsive, resulting in uncontrolled or dysregulated release of mediators that exacerbate acute tissue injury and/or promote the development of chronic diseases such as fibrosis and cancer. Evidence suggests that the diverse activity of macrophages is mediated by distinct subpopulations that develop in response to signals within their microenvironment. Understanding the precise roles of these different macrophage populations in the pathogenic response to toxicants is key to designing effective treatments for minimizing tissue damage and chronic disease and for facilitating wound repair.


Assuntos
Mediadores da Inflamação/metabolismo , Macrófagos/metabolismo , Xenobióticos/toxicidade , Animais , Fibrose/fisiopatologia , Humanos , Inflamação/etiologia , Inflamação/fisiopatologia , Macrófagos/efeitos dos fármacos , Neoplasias/fisiopatologia , Cicatrização/fisiologia
10.
Toxicol Appl Pharmacol ; 245(1): 36-46, 2010 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-20100502

RESUMO

Caveolin-1 (Cav-1) is a membrane scaffolding protein, which functions to regulate intracellular compartmentalization of various signaling molecules. In the present studies, transgenic mice with a targeted disruption of the Cav-1 gene (Cav-1(-/-)) were used to assess the role of Cav-1 in acetaminophen-induced hepatotoxicity. Treatment of wild-type mice with acetaminophen (300 mg/kg) resulted in centrilobular hepatic necrosis and increases in serum transaminases. This was correlated with decreased expression of Cav-1 in the liver. Acetaminophen-induced hepatotoxicity was significantly attenuated in Cav-1(-/-) mice, an effect that was independent of acetaminophen metabolism. Acetaminophen administration resulted in increased hepatic expression of the oxidative stress marker, lipocalin 24p3, as well as hemeoxygenase-1, but decreased glutathione and superoxide dismutase-1; no differences were noted between the genotypes suggesting that reduced toxicity in Cav-1(-/-) mice is not due to alterations in antioxidant defense. In wild-type mice, acetaminophen increased mRNA expression of the pro-inflammatory cytokines, interleukin-1beta, and monocyte chemoattractant protein-1 (MCP-1), as well as cyclooxygenase-2, while 15-lipoxygenase (15-LOX), which generates anti-inflammatory lipoxins, decreased. Acetaminophen-induced changes in MCP-1 and 15-LOX expression were greater in Cav-1(-/-) mice. Although expression of tumor necrosis factor-alpha, a potent hepatocyte mitogen, was up-regulated in the liver of Cav-1(-/-) mice after acetaminophen, expression of proliferating cell nuclear antigen and survivin, markers of cellular proliferation, were delayed, which may reflect the reduced need for tissue repair. Taken together, these data demonstrate that Cav-1 plays a role in promoting inflammation and toxicity during the pathogenesis of acetaminophen-induced injury.


Assuntos
Acetaminofen/toxicidade , Caveolina 1/fisiologia , Fígado/efeitos dos fármacos , Animais , Antioxidantes/metabolismo , Caveolina 1/genética , Proliferação de Células/efeitos dos fármacos , Quimiocina CCL2/metabolismo , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Interleucina-1/metabolismo , Interleucina-10/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout
11.
J Occup Environ Med ; 47(11): 1182-9, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16282880

RESUMO

OBJECTIVE: Our objective was to determine if low levels of a mixture of volatile organic compounds (VOCs) and their ozone (O3) oxidation products, similar to what might be found in "sick buildings," cause nasal irritation and inflammation under controlled exposure conditions. METHODS: Healthy, nonsmoking women (n=130) completed 2-hour controlled exposures to VOCs, VOCs and O3, and a masked air "MA" control in random order at least 1 week apart. VOCs and O3 concentrations were approximately 25 mg/m and approximately 40 ppb, respectively. Nasal symptoms were rated before, during, and after exposure. Nasal lavage fluid was analyzed for polymorphonuclear cells, total protein, interleukin-6, and interleukin-8. RESULTS: We found no significant differences in symptoms or markers of nasal inflammation between exposure conditions. CONCLUSIONS: Results suggest that VOCs and their oxidation products may not cause acute nasal effects at low concentrations.


Assuntos
Poluição do Ar em Ambientes Fechados/efeitos adversos , Mucosa Nasal/fisiopatologia , Compostos Orgânicos/efeitos adversos , Oxidantes Fotoquímicos/efeitos adversos , Adulto , Feminino , Humanos , Líquido da Lavagem Nasal , Razão de Chances , Compostos Orgânicos/química , Ozônio/efeitos adversos , Ozônio/química , Volatilização
12.
Mediators Inflamm ; 2005(1): 31-8, 2005 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-15770064

RESUMO

The accumulation of neutrophils at sites of tissue injury or infection is mediated by chemotactic factors released as part of the inflammatory process. Some of these factors are generated as a direct consequence of tissue injury or infection, including degradation fragments of connective tissue collagen and bacterial- or viral-derived peptides containing collagen-related structural motifs. In these studies, we examined biochemical mechanisms mediating the biologic activity of synthetic polypeptides consisting of repeated units of proline (Pro), glycine (Gly), and hydroxyproline (Hyp), major amino acids found within mammalian and bacterial collagens. We found that the peptides were chemoattractants for neutrophils. Moreover, their chemotactic potency was directly related to their size and composition. Thus, the pentameric peptides (Pro-Pro-Gly)5 and (Pro-Hyp-Gly)5 were more active in inducing chemotaxis than the corresponding decameric peptides (Pro-Pro-Gly)10 and (Pro-Hyp-Gly)10. In addition, the presence of Hyp in peptides reduced chemotactic activity. The synthetic peptides were also found to reduce neutrophil apoptosis. In contrast to chemotaxis, this activity was independent of peptide size or composition. The effects of the peptides on both chemotaxis and apoptosis were blocked by inhibitors of phosphatidylinositol 3-kinase (PI3-K) and p38 mitogen-activated protein (MAP) kinase. However, only (Pro-Pro-Gly)5 and (Pro-Pro-Gly)10 induced expression of PI3-K and phosphorylation of p38 MAP kinase, suggesting a potential mechanism underlying reduced chemotactic activity of Hyp-containing peptides. Although none of the synthetic peptides tested had any effect on intracellular calcium mobilization, each induced nuclear binding activity of the transcription factor NF-kappa B. These findings indicate that polymeric polypeptides containing Gly-X-Y collagen-related structural motifs promote inflammation by inducing chemotaxis and blocking apoptosis. However, distinct calcium-independent signaling pathways appear to be involved in these activities.


Assuntos
Glicina , Hidroxiprolina , Neutrófilos/metabolismo , Peptídeos/farmacologia , Prolina , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Células Cultivadas , Quimiotaxia de Leucócito/efeitos dos fármacos , Cromonas/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Glicina/química , Glicina/metabolismo , Humanos , Hidroxiprolina/química , Hidroxiprolina/metabolismo , Morfolinas/farmacologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , NF-kappa B/efeitos dos fármacos , NF-kappa B/metabolismo , Neutrófilos/efeitos dos fármacos , Peptídeos/síntese química , Peptídeos/metabolismo , Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Prolina/química , Prolina/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
13.
Mol Pharm ; 1(2): 145-55, 2004 Mar-Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15832511

RESUMO

Penetration of epithelial cells represents the rate-determining step for the absorption of many drugs and pharmaceutical macromolecules such as proteins and nucleic acid therapeutics. While the potential of using cell-penetrating peptides (CPPs) to facilitate absorption has been increasingly recognized, the mechanism of cell penetration and the uptake into certain cells have recently been called into question due to methodological artifacts. Therefore, the objective of this study was to quantitatively assess the ability of RI-Tat-9, a proteolytically stable CPP, to penetrate epithelial cell monolayers. The permeability of RI-Tat-9 with two epithelial cell lines, Madin-Darby canine kidney (MDCK) and Caco-2 cells, was comparable to the leakiness of the respective intact monolayers. Microscopic imaging showed that fluorescence-tagged RI-Tat-9 did not enter these cells, further supporting a paracellular transport mechanism. Although insufficient data were generated in these studies to generalize the observed phenomenon, the entry of RI-Tat-9 into nonepithelial T lymphocytic MT2 cells, possibly by endocytosis, suggested that a cell type-specific barrier might exist that controlled uptake of RI-Tat-9 by cells. Compared to that in MT2 and HeLa cells, the active uptake of the peptide into MDCK monolayers was much slower and showed no dependence of cell energy. Furthermore, the equilibrium binding of RI-Tat-9 to MDCK cells at 0 degrees C was indicative of an interaction with a nonspecific receptor. A correlation between binding density and concentration difference across a leaky separation barrier suggested that repulsion of free peptide molecules by bound peptide molecules at the MDCK monolayer surface may be significant at micromolar concentrations. The results of this study quantitatively show that Tat CPP uptake into two commonly used epithelial cell types is minimal and possibly cell type-specific. Implications for Tat CPP-assisted drug delivery are discussed.


Assuntos
Membrana Celular/metabolismo , Peptídeos/farmacocinética , Urotélio/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Cães , Células HeLa , Humanos , Rim , Microscopia Confocal
14.
Am J Respir Cell Mol Biol ; 30(3): 280-7, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12855403

RESUMO

Reactive oxygen intermediates have been implicated in lung injury induced by inhaled irritants. The present studies used mice overexpressing Cu/Zn-superoxide dismutase (SOD+/+) to analyze their role in ozone-induced lung inflammation and cytotoxicity. Treatment of wild-type mice with ozone (0.8 ppm, 3 h) resulted in increased bronchoalveolar lavage fluid protein, which was maximal after 24-48 h. Significant increases in lung macrophages and 4-hydroxyalkenals were also observed. In contrast, bronchoalveolar lavage fluid protein and macrophage content and 4-hydroxyalkenals were at control levels in ozone-treated SOD+/+ mice. There was also no evidence of peroxynitrite-mediated lung damage, demonstrating that SOD+/+ mice are resistant to ozone toxicity. Whereas alveolar macrophages from wild-type mice produced increased amounts of nitric oxide and expressed more inducible nitric oxide synthase, phospholipase A(2), and tumor necrosis factor-alpha after ozone inhalation, this was not evident in cells from SOD+/+ mice. Ozone-induced decreases in interleukin-10 were also not observed. In wild-type mice, ozone inhalation resulted in activation of nuclear factor-kappaB, which regulates proinflammatory gene activity. This response was significantly reduced in SOD+/+ mice. These data demonstrate that antioxidant enzymes play a critical role in ozone-induced tissue injury and in inflammatory mediator production.


Assuntos
Resistência a Medicamentos , Lesão Pulmonar , Óxido Nítrico/metabolismo , Ozônio/toxicidade , Superóxido Dismutase/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Administração por Inalação , Animais , Líquido da Lavagem Broncoalveolar/química , Sobrevivência Celular , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Glutationa/metabolismo , Interleucina-10/metabolismo , Isoenzimas/metabolismo , Pulmão/enzimologia , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patologia , Malondialdeído/metabolismo , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , NF-kappa B/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Ozônio/administração & dosagem , Ácido Peroxinitroso/metabolismo , Fosfolipases A/metabolismo , Pneumonia/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Espécies Reativas de Oxigênio , Superóxido Dismutase/genética
15.
Toxicol Appl Pharmacol ; 193(2): 218-27, 2003 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-14644624

RESUMO

Hepatocyte proliferation represents an important part of tissue repair. In these studies, TNF receptor 1 (TNFR1) knockout mice were used to analyze the role of TNF-alpha in hepatocyte proliferation during acetaminophen-induced hepatotoxicity. Treatment of wild-type (WT) mice with acetaminophen (300 mg/kg) resulted in centrilobular hepatic necrosis. This was associated with proliferation of hepatocytes surrounding the damaged areas, which was evident at 24 h. The cell cycle regulatory proteins, cyclin D1 and cyclin A, were also up regulated in hepatocytes. In contrast, in TNFR1-/- mice, which exhibit exaggerated acetaminophen hepatotoxicity, hepatocyte proliferation, and expression of cyclin D1 and cyclin A, as well as the cyclin dependent kinases, Cdk4 and Cdk2, were reduced. The cyclin-dependent kinase inhibitor p21 was also induced in the liver following acetaminophen administration. This was greater in TNFR1-/- mice compared to WT mice. To investigate mechanisms mediating the reduced hepatic proliferative response of TNFR1-/- mice, we analyzed phosphatidyl inositol-3-kinase (PI-3K) signaling. In both WT and TNFR1-/- mice, acetaminophen caused a rapid increase in total PI-3K within 3 h. Acetaminophen also increased phosphorylated PI-3K, but this was delayed 6-12 h in TNFR1-/- mice. Expression of Akt, a downstream target of PI-3K, was increased in both WT and TNFR1-/- mice in response to acetaminophen. However, the increase was greater in WT mice. Acetaminophen-induced expression of phosphorylated STAT3, a key regulator of cytokine-induced hepatocyte proliferation, was also delayed in TNFR1-/- mice relative to WT. These data suggest that TNF-alpha signaling through TNFR1 is important in regulating hepatocyte proliferation following acetaminophen-induced tissue injury. Delayed cytokine signaling may account for reduced hepatocyte proliferation and contribute to exaggerated acetaminophen-induced hepatotoxicity in TNFR1-/- mice.


Assuntos
Acetaminofen/toxicidade , Analgésicos não Entorpecentes/toxicidade , Antígenos CD/metabolismo , Hepatócitos/metabolismo , Proteínas Proto-Oncogênicas , Receptores do Fator de Necrose Tumoral/metabolismo , Animais , Antígenos CD/genética , CDC2-CDC28 Quinases/metabolismo , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Ciclina A/metabolismo , Ciclina D1/metabolismo , Quinase 2 Dependente de Ciclina , Quinase 4 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/biossíntese , Proteínas de Ligação a DNA/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Imuno-Histoquímica , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosfatidilinositol 3-Quinases/metabolismo , Receptores do Fator de Necrose Tumoral/deficiência , Receptores do Fator de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral , Fator de Transcrição STAT3 , Transdução de Sinais , Transativadores/metabolismo , Regulação para Cima
16.
Toxicol Appl Pharmacol ; 192(2): 119-30, 2003 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-14550746

RESUMO

Transgenic mice with a targeted disruption of the tumor necrosis factor receptor 1 (TNFR1) gene were used to analyze the role of TNF-alpha in pro- and anti-inflammatory mediator production and liver injury induced by acetaminophen. Treatment of wild-type mice with acetaminophen (300 mg/kg) resulted in centrilobular hepatic necrosis. This was correlated with expression of inducible nitric oxide synthase (NOS II) and nitrotyrosine staining of the liver. Expression of macrophage chemotactic protein-1 (MCP-1), KC/gro, interleukin-1beta (IL-1beta), matrix metalloproteinase-9 (MMP-9), and connective tissue growth factor (CTGF), inflammatory mediators known to participate in tissue repair, as well as the anti-inflammatory cytokine, interleukin-10 (IL-10), also increased in the liver following acetaminophen administration. TNFR1(-/-) mice were found to be significantly more sensitive to the hepatotoxic effects of acetaminophen than wild-type mice. This was correlated with more rapid and prolonged induction of NOS II in the liver and changes in the pattern of nitrotyrosine staining. Acetaminophen-induced expression of MCP-1, IL-1beta, CTGF, and MMP-9 mRNA was also delayed or reduced in TNFR1(-/-) mice relative to wild-type mice. In contrast, increases in IL-10 were more rapid and more pronounced. These data demonstrate that signaling through TNFR1 is important in inflammatory mediator production and toxicity induced by acetaminophen.


Assuntos
Acetaminofen/toxicidade , Analgésicos não Entorpecentes/toxicidade , Antígenos CD/genética , Doença Hepática Induzida por Substâncias e Drogas , Fígado , Receptores do Fator de Necrose Tumoral/genética , Transdução de Sinais , Animais , Antígenos CD/fisiologia , Western Blotting , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Citocinas/biossíntese , Relação Dose-Resposta a Droga , Indução Enzimática , Injeções Intraperitoneais , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase Tipo II , RNA Mensageiro/análise , Receptores do Fator de Necrose Tumoral/fisiologia , Receptores Tipo I de Fatores de Necrose Tumoral , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia , Fatores de Tempo
17.
Am J Physiol Gastrointest Liver Physiol ; 285(5): G959-66, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12842828

RESUMO

Tumor necrosis factor (TNF)-alpha is a macrophage-derived proinflammatory cytokine implicated in hepatotoxicity. In the present studies, p55 TNF receptor 1 (TNFR1) -/- mice were used to assess the role of TNF-alpha in acetaminophen-induced antioxidant defense. Treatment of wild-type (WT) mice with acetaminophen (300 mg/kg) resulted in centrilobular hepatic necrosis and increased serum alanine transaminases. This was correlated with a rapid depletion of hepatic glutathione (GSH). Whereas in WT mice GSH levels returned to control after 6-12 h, in TNFR1-/- mice recovery was delayed for 48 h. Delayed induction of heme oxygenase-1 and reduced expression of CuZn superoxide dismutase were also observed in TNFR1-/- compared with WT mice. This was associated with exaggerated hepatotoxicity. In WT mice, acetaminophen caused a time-dependent increase in activator protein-1 nuclear binding activity and in c-Jun expression. This response was significantly attenuated in TNFR1-/- mice. Constitutive NF-kappaB binding activity was detectable in livers of both WT and TNFR1-/- mice. A transient decrease in this activity was observed 3 h after acetaminophen in WT mice, followed by an increase that was maximal after 6-12 h. In contrast, in TNFR1-/- mice, acetaminophen-induced decreases in NF-kappaB activity were prolonged and did not return to control levels for 24 h. These data indicate that TNF-alpha signaling through TNFR1 plays an important role in regulating the expression of antioxidants in this model. Reduced generation of antioxidants may contribute to the increased sensitivity of TNFR1-/- mice to acetaminophen.


Assuntos
Acetaminofen/farmacologia , Antígenos CD/fisiologia , Antioxidantes/metabolismo , Receptores do Fator de Necrose Tumoral/fisiologia , Acetaminofen/envenenamento , Alanina Transaminase/sangue , Animais , Indução Enzimática/efeitos dos fármacos , Glutationa/antagonistas & inibidores , Glutationa/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Heme Oxigenase-1 , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Necrose , Receptores Tipo I de Fatores de Necrose Tumoral , Superóxido Dismutase/antagonistas & inibidores , Fator de Transcrição AP-1/metabolismo
18.
Toxicol Appl Pharmacol ; 184(1): 27-36, 2002 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-12392966

RESUMO

Macrophage-derived inflammatory mediators have been implicated in tissue injury induced by a number of hepatotoxicants. In the present studies, we used transgenic mice with a targeted disruption of the gene for inducible nitric oxide synthase (NOS II) to analyze the role of nitric oxide in inflammatory mediator production in the liver and in tissue injury induced by acetaminophen. Treatment of wild-type mice with acetaminophen (300 mg/kg) resulted in centrilobular hepatic necrosis, which was evident within 3 h and reached a maximum at 18 h. This was correlated with NOS II expression and nitrotyrosine staining of the liver, which was most prominent after 6 h. Expression of mRNA for tumor necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10), matrix metalloproteinase-9, and connective tissue growth factor (CTGF) also increased in the liver following acetaminophen treatment of wild-type mice. NOS II knockout mice were found to be less sensitive to the hepatotoxic effects of acetaminophen than wild-type mice. This did not appear to be due to differences in acetaminophen-induced glutathione depletion or adduct formation. In NOS II knockout mice treated with acetaminophen, hepatic expression of TNF-alpha, as well as CTGF, was significantly increased compared to wild-type mice. In contrast, IL-10 expression was reduced. These data demonstrate that nitric oxide is important in hepatotoxicity induced by acetaminophen. Moreover, some of its effects may be mediated by altering production of pro- and antiinflammatory cytokines and proteins important in tissue repair.


Assuntos
Acetaminofen/toxicidade , Doença Hepática Induzida por Substâncias e Drogas , Interleucina-10/fisiologia , Hepatopatias/enzimologia , Óxido Nítrico Sintase/deficiência , Fator de Necrose Tumoral alfa/fisiologia , Animais , Fator de Crescimento do Tecido Conjuntivo , Deleção de Genes , Expressão Gênica/efeitos dos fármacos , Guanidinas/farmacologia , Proteínas Imediatamente Precoces/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Interleucina-10/genética , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/metabolismo , Fígado/patologia , Hepatopatias/metabolismo , Hepatopatias/patologia , Metaloproteinase 9 da Matriz/genética , Camundongos , Camundongos Knockout , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/genética
19.
Am J Physiol Cell Physiol ; 283(4): C1267-77, 2002 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12225989

RESUMO

The 60-kDa heat shock protein (HSP60), an endogenous ligand for the toll-like 4 receptor, is generated in response to inflammation, tissue injury, and/or stress and stimulates macrophages to produce cytotoxic and proinflammatory mediators including nitric oxide, tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-12. In the present studies we report that HSP60 is an effective inducer of cyclooxygenase-2 (COX-2) in macrophages, as well as endothelial cells. In both cell types, the synthesis of COX-2 was coordinate with induction of nitric oxide synthase (NOS)-2 and with nitric oxide production. With the use of promoter constructs in transient transfection assays, optimal expression of COX-2 in macrophages was found to require nuclear factor (NF)-kappaB, the cAMP-response element (CRE), and NF-IL-6, but not the E-box. Mobility shift assays revealed that HSP60 induced NF-kappaB and CRE binding activity, while CCAAT/enhancer binding protein (C/EBP), which binds to NF-IL-6, was constitutively active in the cells. Both c-Jun and CRE binding protein (CREB) bound to the CRE, while C/EBP-beta bound to NF-IL-6. These data indicate that NF-kappaB, C/EBP-beta, c-Jun, and CREB are important in HSP60-induced expression of COX-2. The c-Jun-NH(2)-terminal kinase (JNK), p44/42 mitogen-activated protein (MAP) kinase [extracellular signal-regulated kinase 1/2 (ERK1/2)], and p38 MAP kinase were rapidly activated by HSP60 in the macrophages. PD-98059, an inhibitor of phosphorylation of ERK1/2, caused a marked inhibition of HSP60-induced COX-2 and NOS-2 expression. Unexpectedly, SB-203580, a p38 kinase antagonist, was found to block HSP60-induced expression of COX-2, but not NOS-2. These data indicate that both ERK1/2 kinase and p38 kinase play a role in regulating HSP60-induced expression of COX-2.


Assuntos
Chaperonina 60/farmacologia , Endotélio/enzimologia , Indução Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Isoenzimas/metabolismo , Macrófagos/enzimologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Animais , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Ciclo-Oxigenase 2 , Endotélio/citologia , Endotélio/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Genes Reporter , Interferon gama/farmacologia , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutagênese Sítio-Dirigida , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Regiões Promotoras Genéticas , Prostaglandina-Endoperóxido Sintases/genética , RNA Mensageiro/biossíntese , Ratos , Sequências Reguladoras de Ácido Nucleico/fisiologia , Fatores de Transcrição/metabolismo , Transfecção
20.
J Cell Physiol ; 190(3): 382-9, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11857454

RESUMO

Acute endotoxemia is associated with prolonged survival of adherent neutrophils in the lung vasculature. In the present studies, the effects of inflammatory mediators on signaling pathways regulating neutrophil survival were examined. We found that the protein kinase C activator, 12-O-tetradecanoyl-phorbol 13-acetate (TPA), but not interferon-gamma (IFN-gamma), prolonged the survival of adherent vasculature lung neutrophils from endotoxemic rats, a response that was correlated with reduced apoptosis. Although endotoxin administration to rats induced the expression of the anti-apoptotic protein Mcl-1 in lung neutrophils, TPA had no effect on this response. Endotoxin administration also induced expression of total p38 and p44/42 mitogen activated protein kinases (MAPK) in neutrophils, as well as phosphatidyl inositol 3 kinase (PI3K) and its downstream target protein kinase B (PKB). Treatment of the cells with TPA increased p38 MAPK expression in cells from both control and endotoxin treated animals. Cells from endotoxin treated, but not control animals, were found to exhibit constitutive binding activity of nuclear factor kappa B (NF-kappaB) which was blocked by TPA. In contrast, constitutive CCAAT/enhancer binding protein (C/EBP) nuclear binding activity evident in neutrophils from control animals was reduced following endotoxin administration. Moreover, this response was independent of TPA. These data suggest that NF-kappaB plays a role in TPA-induced signaling leading to prolonged survival of adherent vascular neutrophils in the lung during acute endotoxemia.


Assuntos
Endotoxemia/fisiopatologia , Neutrófilos/fisiologia , Proteínas Serina-Treonina Quinases , Circulação Pulmonar , Acetato de Tetradecanoilforbol/farmacologia , Doença Aguda , Animais , Apoptose/efeitos dos fármacos , Proteínas Estimuladoras de Ligação a CCAAT/antagonistas & inibidores , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Endotoxemia/patologia , Feminino , Interferon gama/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Ratos , Ratos Sprague-Dawley
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