RESUMEN
Chronic airway inflammation induces numerous structural changes of the airways involving hypertrophy and hyperplasia of airway smooth muscle (ASM). Thrombin has been identified in the bronchoalveolar lavage fluid of asthmatic subjects and displays potent bronchoconstrictor and mitogenic activity towards ASM. This study has addressed which proteinase-activated receptors (PARs) and signalling pathways are involved in mediating distinct effects of thrombin. Using cultured bovine tracheal smooth muscle (BTSM) cells as a model system, thrombin stimulated a marked increase in [3H]inositol phosphate ([3H]InsPs) accumulation, which was fully mimicked by a selective PAR1 activating peptide. In contrast, PAR1, PAR2, PAR3 and PAR4 activating peptides were unable to replicate the ability of thrombin to stimulate DNA synthesis as assessed by [3H]thymidine incorporation. Further investigation demonstrated that the mitogenic effect of thrombin did not involve stimulation of PDGF secretion but did involve activation of PDGF or EGF receptors and a G(i/o)-dependent activation of phosphoinositide 3-kinase. Thrombin, but not the PAR1, PAR2, PAR3 or PAR4 activating peptides was able to stimulate PtdIns(3,4,5)P3 mass accumulation. PAR3 antisense oligonucleotides substantially inhibit thrombin-stimulated [3H]thymidine incorporation and PtdIns(3,4,5)P3 generation but had no effect on thrombin-induced phosphoinositide hydrolysis. These data indicate that while PI hydrolysis and Ca2+ mobilisation induced by thrombin operates via PAR1-dependent activation of phospholipase C, phosphoinositide 3-kinase activation and DNA synthesis occurs via a distinct proteinase-activated receptor pathway, possibly involving PAR3.
Asunto(s)
Replicación del ADN/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Fosfatidilinositoles/metabolismo , Receptores Proteinasa-Activados/agonistas , Trombina/farmacología , Tráquea/efectos de los fármacos , Secuencia de Bases , Células Cultivadas , Cartilla de ADN , Activación Enzimática , Hidrólisis , Músculo Liso/citología , Músculo Liso/enzimología , Músculo Liso/metabolismo , Oligonucleótidos Antisentido/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Tráquea/citología , Tráquea/enzimología , Tráquea/metabolismo , Fosfolipasas de Tipo C/metabolismoRESUMEN
The production of reactive oxygen species (ROS) by human neutrophils is imperative for their bactericidal activity. Proinflammatory agents such as granulocyte macrophage-colony stimulating factor (GM-CSF) can prime ROS production in response to chemoattractants such as N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). In neutrophils from patients suffering from Myelodysplastic syndromes (MDS), a clonal, hematological disorder characterized by recurrent bacterial infections, this GM-CSF priming is severely impaired. In this study, we set out to further delineate the defects in neutrophils from MDS patients. We examined the effect of GM-CSF priming on fMLP-triggered activation of Rac, a small GTPase implicated in neutrophil ROS production. In contrast to healthy neutrophils, activation of Rac in response to fMLP was not enhanced by GM-CSF pretreatment in MDS neutrophils. Furthermore, activation of Rac was attenuated by pretreatment of neutrophils with the phosphatidylinositol 3-kinase (PI-3K) inhibitor LY294002. Unlike healthy neutrophils, fMLP-induced accumulation of the PI-3K lipid product PI(3,4,5)trisphosphate was not increased by GM-CSF pretreatment in MDS neutrophils. The disturbed Rac and PI-3K activation observed in MDS neutrophils did not appear to reflect a general GM-CSF or fMLP receptor-signaling defect, as fMLP-triggered Ras activation could be primed by GM-CSF in MDS and healthy neutrophils. Moreover, fMLP-induced activation of the GTPase Ral was also normal in neutrophils from MDS patients. Taken together, our data suggest that in neutrophils from MDS patients, a defect in priming of the PI-3K-Rac signaling pathway, located at the level of PI-3K, results in a decreased GM-CSF priming of ROS production.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Inhibidores Enzimáticos/farmacología , Humanos , Síndromes Mielodisplásicos , Activación Neutrófila/efectos de los fármacos , Activación Neutrófila/fisiología , Neutrófilos/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas de Unión al GTP rac/efectos de los fármacos , Proteínas de Unión al GTP ral/efectos de los fármacos , Proteínas de Unión al GTP ral/metabolismo , Proteínas ras/efectos de los fármacos , Proteínas ras/metabolismoRESUMEN
UNLABELLED: Neutrophil granulocytes play a key role in the pathogenesis of a wide variety of pulmonary diseases. In many such conditions, the injury observed reflects the activation status rather than the total number of inflammatory cells present. The metabolic activity of neutrophils can now be assessed noninvasively using PET to measure the regional uptake of (18)F-FDG after intravenous injection. METHODS: To understand the mechanism responsible for the increased (18)F-FDG signal, we have measured the uptake of tritiated deoxyglucose (DG) in neutrophils isolated from human peripheral blood and sought to determine which aspects of neutrophil function correlate with an increase in DG uptake. RESULTS: Our results show that formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated respiratory burst activity and (3)H-DG uptake are temporally dissociated, that neutrophil-priming agents such as tumor necrosis factor-alpha (TNFalpha) cause an identical increase in (3)H-DG uptake compared with fMLP without affecting respiratory burst activity, and that fMLP stimulation of TNFalpha-primed cells causes major upregulation of superoxide anion generation (O(2)(-)) yet no incremental increase in (3)H-DG uptake. Furthermore, direct activation of reduced nicotinamide adenine dinucleotide phosphate oxidase activity with the ester phorbol 12-myristate 13-acetate resulted in a concentration-dependent loss of cell-associated (3)H-DG, and preincubation of neutrophils with the phosphoinositide 3-kinase inhibitor wortmannin, which abolished both agonist-stimulated superoxide anion generation and degranulation, had no effect on TNFalpha- or fMLP-stimulated (3)H-DG uptake. In contrast, the fMLP-stimulated change in neutrophil shape was not influenced by priming or wortmannin, and of the functional responses examined, this appeared to correlate most closely with (3)H-DG uptake. CONCLUSION: DG uptake occurs in both primed and activated neutrophils. It does not correlate with respiratory burst or secretory activity but may reflect the polarization and migrational status of these cells. These data have important implications for the analysis of (18)F-FDG signals in vivo.
Asunto(s)
Desoxiglucosa/metabolismo , Fluorodesoxiglucosa F18 , Neutrófilos/metabolismo , Estallido Respiratorio , Tomografía Computarizada de Emisión , Humanos , N-Formilmetionina Leucil-Fenilalanina/farmacología , Peroxidasa/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Apoptosis through the TRAIL receptor pathway can be induced via agonistic IgG to either TRAIL-R1 or TRAIL-R2. Here we describe the use of phage display to isolate a substantive panel of fully human anti-TRAIL receptor single chain Fv fragments (scFvs); 234 and 269 different scFvs specific for TRAIL-R1 and TRAIL-R2 respectively. In addition, 134 different scFvs that were cross-reactive for both receptors were isolated. To facilitate screening of all 637 scFvs for potential agonistic activity in vitro, a novel high-throughput surrogate apoptosis assay was developed. Ten TRAIL-R1 specific scFv and 6 TRAIL-R2 specific scFv were shown to inhibit growth of tumor cells in vitro in the absence of any cross-linking agents. These scFv were all highly specific for either TRAIL-R1 or TRAIL-R2, potently inhibited tumor cell proliferation, and were antagonists of TRAIL binding. Moreover, further characterization of TRAIL-R1 agonistic scFv demonstrated significant anti-tumor activity when expressed and purified as a monomeric Fab fragment. Thus, scFv and Fab fragments, in addition to whole IgG, can be agonistic and induce tumor cell death through specific binding to either TRAIL-R1 or TRAIL-R2. These potent agonistic scFv were all isolated directly from the starting phage antibody library and demonstrated significant tumor cell killing properties without any requirement for affinity maturation. Some of these selected scFv have been converted to IgG format and are being studied extensively in clinical trials to investigate their potential utility as human monoclonal antibody therapeutics for the treatment of human cancer.
Asunto(s)
Fragmentos Fab de Inmunoglobulinas/farmacología , Inmunoterapia , Neoplasias/terapia , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/agonistas , Anticuerpos de Cadena Única/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos/genética , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Unión Competitiva , Caspasa 3/metabolismo , Proliferación Celular/efectos de los fármacos , Ingeniería Genética , Células HeLa , Ensayos Analíticos de Alto Rendimiento , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/aislamiento & purificación , Fragmentos Fab de Inmunoglobulinas/metabolismo , Neoplasias/inmunología , Neoplasias/patología , Anticuerpos de Cadena Única/biosíntesis , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/aislamiento & purificaciónRESUMEN
Neutrophil apoptosis plays a central role in the resolution of granulocytic inflammation. We have shown previously that tumor necrosis factor-alpha (TNFalpha) enhances the rate of neutrophil apoptosis at early time points via a mechanism involving both TNF receptor (TNFR) I and TNFRII. Here we reveal a marked but consistent variation in the magnitude of the pro-apoptotic effect of TNFalpha in neutrophils isolated from healthy donors, and we show that inhibition of cell surface aminopeptidase N (APN) using actinonin, bestatin, or inhibitory peptides significantly enhanced the efficacy of TNFalpha-induced killing. Notably, an inverse correlation is shown to exist between neutrophil APN activity and the sensitivity of donor cells to TNFalpha-induced apoptosis. Inhibition of cell surface APN appears to interfere with the shedding of TNFRI, and as a consequence results in augmented TNFalpha-induced apoptosis, cell polarization, and TNFalpha-primed, formyl-methionyl-leucyl-phenylalanine-stimulated respiratory burst. Of note, actinonin and bestatin had no effect on TNFRII expression under resting or TNFalpha-stimulated conditions and did not alter CXCRI or CXCRII expression. These data suggest significant variation in the activity of APN/CD13 on the cell surface of neutrophils in normal individuals and reveal a novel mechanism whereby APN/CD13 regulates TNFalpha-induced apoptosis via inhibition of TNFRI shedding. This has therapeutic relevance for driving neutrophil apoptosis in vivo.
Asunto(s)
Apoptosis , Antígenos CD13/biosíntesis , Regulación Enzimológica de la Expresión Génica , Neutrófilos/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Antibacterianos/farmacología , Antígenos CD13/metabolismo , Caspasa 3 , Caspasas/metabolismo , Humanos , Ácidos Hidroxámicos/farmacología , Técnicas In Vitro , Leucina/análogos & derivados , Leucina/farmacología , Neutrófilos/patología , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismoRESUMEN
Myeloid cells are key effectors of the innate immune response, and as such are often required to migrate to, and function within, sites that are markedly hypoxic. To adapt to such oxygen deplete environments they have developed functional and survival responses that are regulated by the hypoxia-inducible factor (HIF) oxygen-sensing pathway. In this review, we describe three key aspects of HIF-dependent regulation of myeloid cell function: (i) the maintenance of ATP pools and the subsequent regulation of proinflammatory responses, (ii) the HIF-dependent inhibition of neutrophil apoptosis and (iii) the HIF-mediated regulation beta2 integrin expression.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Inflamación/metabolismo , Inflamación/patología , Células Mieloides/metabolismo , Células Mieloides/patología , Animales , Apoptosis , Humanos , Oxígeno/metabolismoRESUMEN
Phosphoinositide 3-kinase (PI3-kinase)-dependent phosphorylation of the proapoptotic Bcl-2 family member Bad has been proposed as an important regulator of apoptotic cell death. To understand the importance of this pathway in nontransformed hematopoietic cells, we have examined the effect of survival cytokines on PI3-kinase activity and Bad expression and phosphorylation status in human neutrophils. Granulocyte macrophage-colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF-alpha) both reduced the rate of apoptosis in neutrophils cultured in vitro for 20 hours. Coincubation with the PI3-kinase inhibitor LY294002, which in parallel experiments abolished GM-CSF-primed, fMLP-stimulated superoxide anion production and GM-CSF-stimulated PtdIns(3,4,5)P(3) accumulation, inhibited the GM-CSF and TNF-alpha survival effect. In contrast, the MAP kinase kinase (MEK1/2) inhibitor PD98059 and the protein kinase A inhibitor H-89 had only a marginal effect on GM-CSF-mediated neutrophil survival. GM-CSF substantially increased Bad phosphorylation at Ser112 and Ser136 and increased the cytosolic accumulation of Bad. GM-CSF also regulated Bad at a transcription level with a marked decrease in mRNA levels at 4 hours. TNF-alpha caused a biphasic effect on the rate of morphologic apoptosis, which corresponded to an early increase, and a late inhibition, of Bad mRNA levels. LY294002 inhibited GM-CSF- and TNF-alpha-mediated changes in Bad phosphorylation and mRNA levels. These data suggest that the survival effect of GM-CSF and TNF-alpha in neutrophils is caused by a PI3-kinase-dependent phosphorylation and cytosolic translocation of Bad, together with an inhibition of Bad mRNA levels. This has important implications for the regulation of neutrophil apoptosis in vivo.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Supervivencia Celular/fisiología , Citocinas/sangre , Neutrófilos/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Transcripción Genética/fisiología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Secuencia de Bases , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cromonas/farmacología , Cartilla de ADN , Inhibidores Enzimáticos/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Cinética , Morfolinas/farmacología , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/farmacología , Proteína Letal Asociada a bclRESUMEN
Neutrophil priming by agents such as TNF-alpha and GM-CSF causes a dramatic increase in the response of these cells to secretagogue agonists and affects the capacity of neutrophils to induce tissue injury. In view of the central role of phosphatidylinositol 3-kinase (PI3-kinase) in regulating NADPH oxidase activity we examined the influence of priming agents on agonist-stimulated phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) accumulation in human neutrophils. Pretreatment of neutrophils with TNF-alpha or GM-CSF, while not influencing fMLP-stimulated PtdIns(3,4,5)P3 accumulation at 5 s, caused a major increase in PtdIns(3,4,5)P3 at later times (10-60 s), which paralleled the augmented superoxide anion (O2-) response. The intimate relationship between PtdIns(3,4,5)P3 accumulation and O2- release was confirmed using platelet-activating factor, which caused full but transient priming of both responses. Likewise, LY294002, a PI3-kinase inhibitor, and genistein, a tyrosine kinase inhibitor, caused parallel inhibition of O2- generation and PtdIns(3,4,5)P3 accumulation; in contrast, radicicol, which inhibits receptor-mediated activation of p85 PI3-kinase, had no effect on either response. Despite major increases in PI3-kinase activity observed in p85 and anti-phosphotyrosine immunoprecipitates in growth factor-stimulated smooth muscle cells, no such increase was observed in primed/stimulated neutrophils. In contrast, both fMLP and TNF-alpha alone caused a 3-fold increase in PI3-kinase activity in p110gamma PI3-kinase immunoprecipitates. p21(ras) activation (an upstream regulator of PI3-kinase) was unaffected by priming. These data demonstrate that timing and magnitude of PtdIns(3,4,5)P3 accumulation in neutrophils correlate closely with O2- generation, that PI3-kinase-gamma is responsible for the enhanced PtdIns(3,4,5)P3 production seen in primed cells, and that factors other than activation of p21(ras) underlie this response.
Asunto(s)
Activación Neutrófila/fisiología , Neutrófilos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Animales , Células COS , Bovinos , Línea Celular , Citocalasina B/farmacología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Inhibidores Enzimáticos/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Fosfatos de Inositol/metabolismo , Isoenzimas/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacología , Activación Neutrófila/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Neutrófilos/enzimología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Factor de Activación Plaquetaria/farmacología , Inhibidores de Proteínas Quinasas , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Superóxidos/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Plasma deficiency of alpha(1)-antitrypsin is most commonly due to the Z mutation ((342)Glu--> Lys) and is associated with early-onset panlobular emphysema. The lung disease in these patients is attributed to the relative deficiency of circulating alpha(1)-antitrypsin resulting in uncontrolled neutrophil-derived proteolytic activity. We have previously demonstrated that the local deficiency of Z alpha(1)-antitrypsin is exacerbated by the formation of polymers within the lung and now show that this polymerization not only inactivates alpha(1)-antitrypsin but also converts the molecule to a chemoattractant for human neutrophils. The chemotactic action of polymeric alpha(1)-antitrypsin was substantially greater than that seen with other conformers, was of similar magnitude to C5a, and was apparent over a range of physiologically relevant concentrations (EC(50) 0.0045 +/- 0.002 mg/ml). The biologic activity of polymeric alpha(1)-antitrypsin was confirmed by the demonstration that polymers, but not native alpha(1)-antitrypsin, induced neutrophil shape change and stimulated myeloperoxidase release and neutrophil adhesion. Polymeric alpha(1)-antitrypsin had no effect on basal or N-formyl-Met-Leu-Phe- stimulated superoxide anion release or constitutive apoptosis. The chemotactic properties of polymeric alpha(1)-antitrypsin may provide an explanation for the excessive neutrophils found in the lungs of Z alpha(1)-antitrypsin homozygotes and suggests a new paradigm for the pathogenesis of emphysema in these patients.
Asunto(s)
Biopolímeros/metabolismo , Quimiotaxis de Leucocito , Enfisema/etiología , Neutrófilos/citología , alfa 1-Antitripsina/metabolismo , Apoptosis , Adhesión Celular , HumanosRESUMEN
Phospholipase D (PLD) plays a major role in the activation of the neutrophil respiratory burst. However, the repertoire of PLD isoforms present in these primary cells, the precise mechanism of activation, and the impact of cell priming on PLD activity and localization remain poorly defined. RT-PCR analysis showed that both PLD1 and PLD2 isoforms are expressed in human neutrophils, with PLD1 expressed at a higher level. Endogenous PLD1 was detected by immunoprecipitation and Western blotting, and was predominantly membrane-associated under control and primed/stimulated conditions. Immunofluorescence showed that PLD had a punctate distribution throughout the cell, which was not altered after stimulation by soluble agonists. In contrast, PLD localized to the phagolysosome membrane after ingestion of nonopsonized zymosan particles. We also demonstrate that tumour necrosis factor alpha greatly potentiates agonist-stimulated PLD activation, myeloperoxidase release, and superoxide anion generation, and that PLD activation occurs via a phosphatidylinositol 3-kinase-sensitive and brefeldin-sensitive ADP-ribosylation factor GTPase-regulated mechanism. Moreover, propranolol, which causes an increase in PLD-derived phosphatidic acid accumulation, caused a selective increase in agonist-stimulated myeloperoxidase release. Our results indicate that priming is a critical regulator of PLD activation, that the PLD-generated lipid products exert divergent effects on neutrophil functional responses, that PLD1 is the major PLD isoform present in human neutrophils, and that PLD1 actively translocates to the phagosomal wall after particle ingestion.