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1.
Exp Lung Res ; 35(9): 770-80, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19916860

RESUMEN

Both hyperoxia-induced proapoptotic sensitization of alveolar type II cells (TII cells) and high-stretch mechanical ventilation induced pulmonary inflammation are tumor necrosis factor alpha (TNFalpha) mediated. Therefore, binding of free TNFalpha should protect from TNFalpha-mediated acute lung injury and ameliorate the subsequently developing chronic lung disease. Here, the authors show that a single subcutaneous pretreatment of rat with etanercept, a recombinant p75 TNF receptor 2 human immunoglobulin G1 (IgG1) construct, inhibits the hyperoxia-induced and TNFalpha-mediated increase in the expression of TNFalpha receptor, the activation of caspase 3 in TII cells, and, as an early indicator of lung injury, the capillary-alveolar leakage and granulocyte number in lung lavage. The authors assume that subcutaneous administration of etanercept might be suitable to prevent acute lung injury and its sequelae induced by hyperoxic ventilation of premature neonates and critically ill patients.


Asunto(s)
Hiperoxia/complicaciones , Inmunoglobulina G/farmacología , Lesión Pulmonar/prevención & control , Animales , Antiinflamatorios no Esteroideos , Permeabilidad Capilar , Caspasa 3/metabolismo , Etanercept , Granulocitos , Inmunoglobulina G/administración & dosificación , Inmunoglobulina G/uso terapéutico , Alveolos Pulmonares/irrigación sanguínea , Ratas , Receptores del Factor de Necrosis Tumoral/administración & dosificación , Receptores del Factor de Necrosis Tumoral/antagonistas & inhibidores , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/uso terapéutico , Activación Transcripcional/efectos de los fármacos , Factor de Necrosis Tumoral alfa
2.
Biochim Biophys Acta ; 1743(1-2): 37-48, 2005 Mar 22.
Artículo en Inglés | MEDLINE | ID: mdl-15777838

RESUMEN

Nuclear factor-kappa B (NF-kappa B) plays an important role in inflammation, proliferation and regulation of apoptosis. The purpose of the present study on type II cells was to investigate whether Chlamydophila pneumoniae contact induces (I) a Ca2+ release, that (II) disrupts F-actin/beta-tubulin cytoskeletal association with NF-kappa B/I kappa B alpha, leading to (III) a subsequent NF-kappa B activation. Incubation of rat type II pneumocytes with C. pneumoniae caused an intracellular calcium release within seconds. Confocal laser scanning microscopy (CLSM) revealed that bacterial contact with cell surface leads to a disappearance of the microvilli and disturbs the co-localization between F-actin and NF-kappa B (p65). Using semi-quantitative CLSM, we show that at 10-30 min I kappa B alpha was decreased and p65 or p50 was simultaneously translocated from cytoplasm to the nucleus, resulting in a 19-fold and 17-fold increase versus control cells. During this time no bacteria were internalized into type II cells. The pre-treatment of cells with BAPTA-AM inhibited C. pneumoniae-mediated calcium release. BAPTA-AM or SN50 prevented the C. pneumoniae-induced changes in F-actin cytoskeleton and inhibited NF-kappa B activation. Paclitaxel reduced C. pneumoniae-mediated changes of beta-tubulin cytoskeleton and activation of NF-kappa B. These results suggest that calcium-mediated cytoskeleton reorganization is involved in C. pneumoniae-induced NF-kappa B activation in type II cells.


Asunto(s)
Chlamydophila pneumoniae/metabolismo , Ácido Egtácico/análogos & derivados , Pulmón/citología , Pulmón/microbiología , FN-kappa B/metabolismo , Actinas/metabolismo , Animales , Western Blotting , Calcio/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Citoesqueleto/metabolismo , Ácido Egtácico/farmacología , Inmunohistoquímica , L-Lactato Deshidrogenasa/metabolismo , Masculino , Microscopía Confocal , Unión Proteica , Transporte de Proteínas , Ratas , Ratas Wistar , Factores de Tiempo , Tubulina (Proteína)/metabolismo
3.
Biochim Biophys Acta ; 1636(2-3): 196-204, 2004 Mar 22.
Artículo en Inglés | MEDLINE | ID: mdl-15164767

RESUMEN

Based on the assumption that fatty-acid-binding proteins (FABPs) of the epidermal-type (E-FABP) and heart-type (H-FABP) in murine alveolar type II (TII) cells mediate the synthesis of dipalmitoyl phosphatidylcholine (DPPC), the main surfactant phospholipid, we analysed TII cells isolated from wild-type (wt) and E/H-FABP double-knockout (double-ko) mice. Application of labelled palmitic acid to these cells revealed a drop in uptake, beta-oxidation, and incorporation into neutral lipids and total phosphatidylcholine (PC) of TII cells from double-ko mice. Whereas incorporation of labelled palmitic acid into DPPC remained unchanged, degradation studies demonstrated a substantial shift in DPPC synthesis from de novo to reacylation. In addition, increased expression of mRNAs and proteins of caveolin-1 and PPARgamma, and an increase of the mRNA encoding fatty acid translocase (FAT) was observed in the double-ko phenotype. As caveolin-1 interacted with PPARgamma, we assumed that FAT, caveolin-1, and PPARgamma form a signalling chain for fatty acid or drug. Consequently, PPARgamma-selective pioglitazone was added to the diet of double-ko mice. We found that further activation of PPARgamma could 'heal' the E/H-FABP double-ko effect in these TII cells as transport and utilisation of labelled palmitic acid restored a wt phenocopy. This indicated that E-FABP and/or H-FABP are involved in the mediation of DPPC synthesis in wt TII cells.


Asunto(s)
Proteínas Portadoras/fisiología , Caveolinas/fisiología , Proteínas del Tejido Nervioso , Ácido Palmítico/metabolismo , Alveolos Pulmonares/metabolismo , Receptores Citoplasmáticos y Nucleares/fisiología , Factores de Transcripción/fisiología , Animales , Secuencia de Bases , Transporte Biológico , Proteínas Portadoras/genética , Caveolina 1 , Cartilla de ADN , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos , Ratones , Ratones Noqueados , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética
4.
Respir Res ; 6: 51, 2005 Jun 03.
Artículo en Inglés | MEDLINE | ID: mdl-15935092

RESUMEN

BACKGROUND: The role of alveolar type II cells in the regulation of innate and adaptive immunity is unclear. Toll-like receptors (TLRs) have been implicated in host defense. The purpose of the present study was to investigate whether Chlamydophila pneumoniae (I) alters the expression of TLR2 and/orTLR4 in type II cells in a (II) Rho-GTPase- and (III) NF-kappaB-dependent pathway, subsequently (IV) leading to the production of (IV) pro-inflammatory TNF-alpha and MIP-2. METHODS: Isolated rat type II pneumocytes were incubated with C. pneumoniae after pre-treatment with calcium chelator BAPTA-AM, inhibitors of NF-kappaB (parthenolide, SN50) or with a specific inhibitor of the Rho-GTPase (mevastatin). TLR2 and TLR4 mRNA expressions were analyzed by PCR. Activation of TLR4, Rac1, RhoA protein and NF-kappaB was determined by Western blotting and confocal laser scan microscopy (CLSM) and TNF-alpha and MIP-2 release by ELISA. RESULTS: Type II cells constitutively expressed TLR4 and TLR2 mRNA. A prominent induction of TLR4 but not TLR2 mRNA was detected after 2 hours of incubation with C. pneumoniae. The TLR4 protein expression reached a peak at 30 min, began to decrease within 1-2 hours and peaked again at 3 hours. Incubation of cells with heat-inactivated bacteria (56 degrees C for 30 min) significantly reduced the TLR4 expression. Treated bacteria with polymyxin B (2 mug/ml) did not alter TLR4 expression. C. pneumoniae-induced NF-kappaB activity was blocked by TLR4 blocking antibodies. TLR4 mRNA and protein expression were inhibited in the presence of BAPTA-AM, SN50 or parthenolide. TNF-alpha and MIP-2 release was increased in type II cells in response to C. pneumoniae, whereas BAPTA-AM, SN50 or parthenolide decreased the C. pneumoniae-induced TNF-alpha and MIP-2 release. Mevastatin inhibited C. pneumoniae-mediated Rac1, RhoA and TLR4 expression. CONCLUSION: The TLR4 protein expression in rat type II cells is likely to be mediated by a heat-sensitive C. pneumoniae protein that induces a fast Ca2+-mediated NF-kappaB activity, necessary for maintenance of TLR4 expression and TNF-alpha and MIP-2 release through possibly Rac and Rho protein-dependent mechanism. These results indicate that type II pneumocytes play an important role in the innate pulmonary immune system and in inflammatory response mechanism of the alveolus.


Asunto(s)
Quimiocinas CXC/metabolismo , Chlamydophila pneumoniae/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , FN-kappa B/metabolismo , Alveolos Pulmonares/metabolismo , Receptor Toll-Like 4/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Células Cultivadas , Quimiocina CXCL2 , Regulación de la Expresión Génica , Ratas , Transducción de Señal
5.
Respir Res ; 6: 10, 2005 Jan 21.
Artículo en Inglés | MEDLINE | ID: mdl-15663790

RESUMEN

BACKGROUND: The mechanisms during the initial phase of oxygen toxicity leading to pulmonary tissue damage are incompletely known. Increase of tumour necrosis factor alpha (TNFalpha) represents one of the first pulmonary responses to hyperoxia. We hypothesised that, in the initial phase of hyperoxia, TNFalpha activates the caspase cascade in type II pneumocytes (TIIcells). METHODS: Lung sections or freshly isolated TIIcells of control and hyperoxic treated rats (48 hrs) were used for the determination of TNFalpha (ELISA), TNF-receptor 1 (Western blot) and activity of caspases 8, 3, and 9 (colorimetrically). NF-kappaB activation was determined by EMSA, by increase of the p65 subunit in the nuclear fraction, and by immunocytochemistry using a monoclonal anti-NF-kappaB-antibody which selectively stained the activated, nuclear form of NF-kappa B. Apoptotic markers in lung tissue sections (TUNEL) and in TIIcells (cell death detection ELISA, Bax, Bcl-2, mitochondrial membrane potential, and late and early apoptotic cells) were measured using commercially available kits. RESULTS: In vivo, hyperoxia activated NF-kappaB and increased the expression of TNFalpha, TNF-receptor 1 and the activity of caspase 8 and 3 in freshly isolated TIIcells. Intratracheal application of anti-TNFalpha antibodies prevented the increase of TNFRI and of caspase 3 activity. Under hyperoxia, there was neither a significant change of cytosolic cytochrome C or of caspase 9 activity, nor an increase in apoptosis of TIIcells. Hyperoxia-induced activation of caspase 3 gradually decreased over two days of normoxia without increasing apoptosis. Therefore, activation of caspase 3 is a temporary effect in sublethal hyperoxia and did not mark the "point of no return" in TIIcells. CONCLUSION: In the initiation phase of pulmonary oxygen toxicity, an increase of TNFalpha and its receptor TNFR1 leads to the activation of caspase 8 and 3 in TIIcells. Together with the hyperoxic induced increase of Bax and the decrease of the mitochondrial membrane potential, activation of caspase 3 can be seen as sensitisation for apoptosis. Eliminating the TNFalpha effect in vivo by anti-TNFalpha antibodies prevents the pro-apoptotic sensitisation of TIIcells.


Asunto(s)
Caspasas/metabolismo , Hiperoxia/metabolismo , Pulmón/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Caspasa 3 , Caspasa 8 , Células Cultivadas , Citocinas/metabolismo , Activación Enzimática , Estrés Oxidativo , Ratas , Ratas Wistar
6.
Am J Physiol Lung Cell Mol Physiol ; 294(6): L1043-8, 2008 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-18359887

RESUMEN

Perfluorocarbons (PFC) reduce the production of various inflammatory cytokines, including TNF-alpha. The anti-inflammatory effect is not entirely understood. If anti-inflammatory properties are caused by a mechanical barrier, PFC in the alveoli should have no effect on the inflammatory response to intravenous LPS administration. To test that hypothesis, rats (n=31) were administered LPS intravenously and were either spontaneously breathing (Spont), conventionally ventilated (CMV), or receiving partial liquid ventilation (PLV). Serum concentration of TNF-alpha was measured. The pulmonary expressions of TNF-alpha and TNF-alpha receptor 1 protein and of TNF-alpha and ICAM-1 mRNA were determined. LPS caused a significant (P<0.001) increase in serum TNF-alpha. Serum TNF-alpha concentration was similar in LPS/Spont (525+/-180 pg/ml) and LPS/CMV (504+/-154 pg/ml) but was significantly (P<0.001) lower in animals of the LPS/PLV group (274+/-101 pg/ml). Immunohistochemical data on TNF-alpha protein expression showed a LPS-induced increase in TNF-alpha and TNF-alpha receptor 1 expression that was diminished by partial liquid ventilation. PCR measurements revealed a lower expression of TNF-alpha and ICAM-1 mRNA in LPS/PLV than in LPS/CMV or LPS/Spont animals. Semiquantitative histological evaluation revealed only minor alveolar inflammation with no significant differences between the groups. Low serum TNF-alpha concentration in PFC-treated animals is most likely explained by a decreased production of TNF-alpha in the lung.


Asunto(s)
Antiinflamatorios/farmacología , Fluorocarburos/farmacología , Pulmón/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Animales , Antiinflamatorios/administración & dosificación , Fluorocarburos/administración & dosificación , Interleucina-10/sangre , Ventilación con Presión Positiva Intermitente , Lipopolisacáridos/efectos adversos , Ventilación Liquida , Masculino , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Volumen de Ventilación Pulmonar/efectos de los fármacos , Tráquea , Factor de Necrosis Tumoral alfa/efectos de los fármacos
7.
Pediatr Res ; 60(3): 264-9, 2006 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-16857767

RESUMEN

Chlamydophila pneumoniae alter the expression of Toll-like receptor (TLR) 4 in alveolar type II (ATII)-cells. Subsequently nuclear factor kappaB (NF-kappaB) is activated and tumor necrosis factor-alpha (TNF-alpha) and macrophage inflammatory protein 2 (MIP-2) are produced. Perfluorocarbons (PFC) are beneficial in animals with bacterial pneumonia and reduce production of TNF-alpha. Using isolated ATII-cells, it was studied whether PFC prevent C. pneumoniae-induced TNF-alpha and MIP-2 release and what the underlying pathway is. PF5080 preincubation prevented C. pneumoniae-induced secretion of TNF-alpha (43 +/- 10 versus 661 +/- 41 pg/mL) and MIP-2 (573 +/- 41 versus 4786 +/- 502 pg/mL). The C. pneumoniae-induced 2.2-fold increase of TNF-alpha Receptor 1 expression was reduced by PF5080. C. pneumoniae reduced cytoplasmatic IkappaBalpha (3.7 +/- 0.3 versus 14 +/- 1) and increased NF-kappaB p65 (31 +/- 7.5 versus 3.6 +/- 1.1) compared with control. PF5080 prevented NF-kappaB activation. TLR4 expression was 1.5-fold higher after C. pneumoniae incubation, but remained at control levels after PF5080 pretreatment. After 24 h of C. pneumoniae incubation, in 88 +/- 6% of cells bacteria were found in the perinuclear region and in 50% of these cells bacteria adhered to cellular surface. After PF5080 preincubation, C. pneumoniae were in 32 +/- 4% attached to and in 5 +/- 1% internalized in ATII-cells. Since PF5080 was found in ATII-cell membranes, PF5080 effect could be explained by an alteration of the cellular membrane, preventing activation of the inflammatory cascade.


Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Chlamydophila pneumoniae/fisiología , Fluorocarburos/farmacología , Pulmón/microbiología , Pulmón/patología , Animales , Células Cultivadas , Chlamydophila pneumoniae/efectos de los fármacos , Pulmón/citología , Pulmón/metabolismo , Ratas , Factor de Necrosis Tumoral alfa/metabolismo
8.
Am J Respir Cell Mol Biol ; 27(1): 57-63, 2002 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-12091246

RESUMEN

Alveolar type II cells accumulate vitamin E preferentially from high-density lipoproteins (HDL) and express at least three receptors that are specific for HDL. The expression of these receptors increases in response to vitamin E deficiency. Beside receptors for specific lipid transfer from HDL, cubilin and megalin, several other receptors that mediate HDL-particle uptake were found in the lung. We hypothesize that alveolar type II cells also exhibit the HDL-particle uptake and that this process can be regulated by the vitamin E status. By confocal laser microscopy and flow cytometry we showed that type II cells accumulate protein-labeled HDL-particle. Vitamin E depletion in rats increased HDL-particle uptake in alveolar type II cells and the expression of megalin. The expression of cubilin did not change. Refeeding with vitamin E reversed HDL-particle uptake and megalin expression. Long-time incubation of type II cells with phorbol myristyl acetate (PMA) reduced HDL-holoparticle uptake and megalin expression. We assume that alveolar type II cells exhibit HDL-holoparticle uptake mediated by megalin and cubilin. Megalin represents the regulated element of the megalin/cubilin receptor-cooperation and can be modulated by protein kinase C.


Asunto(s)
Lipoproteínas HDL/metabolismo , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Alveolos Pulmonares/metabolismo , Receptores de Superficie Celular/metabolismo , Vitamina E/metabolismo , Animales , Suplementos Dietéticos , Citometría de Flujo , Colorantes Fluorescentes , Cinética , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Microscopía Confocal , Tamaño de la Partícula , Alveolos Pulmonares/citología , ARN Mensajero/biosíntesis , Ratas , Ratas Wistar , Receptores de Superficie Celular/genética , Coloración y Etiquetado , Deficiencia de Vitamina E/metabolismo
9.
Am J Respir Cell Mol Biol ; 29(3 Pt 1): 303-13, 2003 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-12676805

RESUMEN

Understanding the surfactant dysfunction by gram-negative bacteria pulmonary infection, the intracellular fate of Chlamydia pneumoniae (Cpn), its interaction with uptake, recycling, and secretion of surfactant and with the cytoskeleton of type II pneumocytes was investigated. Bacteria colocalized with surfactant protein (SP)-A-mediated endocytosed lipid and early endosomes (EEA1- and Rab5-positive) after 3 and 6 h of infection. No specific contact with late endosomes (Rab7- and M6PR-positive), lysosomal, or lamellar body markers (CD63, 3C9) was found after 12 h of infection. In Cpn-infected cells, SP-A-mediated lipid uptake was significantly increased. After SP-A-mediated lipid uptake followed by "re-secretion," 90% of the internalized lipid remained intracellularly. SP-A and lipid did strongly colocalize with early endosomes. Internalized SP-A cannot be resecreted rapidly to plasma membrane, and lipid is not transported toward late endosomes (Rab7- and M6PR-positive) or lamellar bodies (CD63- and 3C9-positive). These results indicate that increased surfactant internalization is caused by an inhibition in intracellular surfactant transport. Accumulation of SP-A-mediated lipid was associated with changes in beta-tubulin. Increases in surfactant secretion were associated with changes in F-actin. We postulate that Cpn infection of type II cells causes changes of the cytoskeleton, and that these effects are associated with alterations in intracellular transport and secretion of surfactant.


Asunto(s)
Chlamydophila pneumoniae/metabolismo , Citoesqueleto/metabolismo , Pulmón/microbiología , Tensoactivos/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Antígenos CD/biosíntesis , Antineoplásicos Fitogénicos/farmacología , Cromatografía en Capa Delgada , Endocitosis , Endosomas/metabolismo , Inmunohistoquímica , Metabolismo de los Lípidos , Pulmón/inmunología , Masculino , Microtúbulos/metabolismo , Modelos Biológicos , Paclitaxel/farmacología , Faloidina/farmacología , Glicoproteínas de Membrana Plaquetaria/biosíntesis , Ratas , Ratas Wistar , Tetraspanina 30 , Factores de Tiempo , Tubulina (Proteína)/metabolismo
10.
Crit Care Med ; 31(4): 1190-6, 2003 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-12682492

RESUMEN

OBJECTIVE: Because alveoli fill with perfluorocarbons during liquid ventilation, an uptake of perfluorocarbons by type II pneumocytes can be postulated that might affect synthesis and secretion of pulmonary surfactant. The study was performed to answer the following questions: Do isolated type II pneumocytes take up perfluorocarbons? Do perfluorocarbons affect lipid synthesis of type II cells? Do perfluorocarbons change surfactant secretion of type II pneumocytes? DESIGN: Controlled experiments that used isolated type II pneumocytes. SETTING: Experimental laboratory of a university hospital. SUBJECTS: Male Wistar rats. INTERVENTIONS: To study perfluorocarbon uptake, isolated type II cells were incubated with fluorescence-labeled perfluorocarbons and examined with a laser scanning microscope. The effect of perfluorocarbons on biosynthesis of phospholipids and triglycerides was measured by incubating cells that were pulse-labeled with [H]-palmitic acid for 30 secs, with two different perfluorocarbons (PF 5080 or RM 101) for 10 mins. The effect of perfluorocarbon incubation on lipid secretion was studied by transmission electron microscopy. To quantify secretion, adherent type II pneumocytes (containing radioactively labeled phospholipids) were incubated with perfluorocarbons, and extra- and intracellular radioactivity was measured. MEASUREMENTS AND MAIN RESULTS: We found a significant uptake of labeled perfluorocarbons into lamellar bodies within 10 mins. Both perfluorocarbon species significantly (p <.05) reduced the biosynthesis of phospholipids when compared with control. Perfluorocarbon incubation did not affect mitochondrial activity, tested by MitoTracker staining. Transmission electron microscopy revealed changes that suggest an increased secretion of surfactant by type II cells. Studies with radioactively labeled surfactant revealed a significantly (p <.01) higher amount of extracellular lipids after RM 101 and PF 5080 treatment (RM 101, 17 +/- 7.9%; PF 5080, 9 +/- 1.9%) compared with control (5.3 +/- 1.9%). CONCLUSIONS: Our results suggest that perfluorocarbons are taken up by type II pneumocytes and cause an increased secretion of surfactant, despite a relative reduction in the synthesis of phospholipids.


Asunto(s)
Fluorocarburos/farmacocinética , Ventilación Liquida , Fosfolípidos/biosíntesis , Alveolos Pulmonares/metabolismo , Surfactantes Pulmonares/metabolismo , Triglicéridos/biosíntesis , Animales , Fluorocarburos/farmacología , Técnicas In Vitro , Masculino , Microscopía Confocal , Microscopía Electrónica , Alveolos Pulmonares/citología , Alveolos Pulmonares/ultraestructura , Ratas , Ratas Wistar
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