Biomedical subjects

William MacNee

Publications and source records attributed to William MacNee.

32 records · Page 2Linked to original sources

PubMed2004

Haplotypes of the alpha-1 antitrypsin gene in healthy controls and Z deficiency patients.

Alpha-1 antitrypsin (AAT; HUGO symbol, SERPINA1) is one of the major serine protease inhibitors (serpins) in human plasma. Deficiency of AAT is a recognised risk factor for chronic obstructive pulmonary disease (COPD), attributed to uninhibited neutrophil elastase released into the lung tissue during inflammatory states. In this study we used sequencing to screen the exonic regions, 5' and 3' flanking sequence of the AAT gene in order to generate a high density map of single nucleotide polymorphisms (SNPs). 16 SNPs were identified throughout AAT. Haplotypes based on SNPs with a minor allele frequency of > or =5% were estimated using genotypic information from 225 healthy control individuals and 41 AAT deficient Pi-ZZ individuals. AAT shows a large amount of variation in the control population, with 17 haplotypes accounting for 88% of the observed variation. The haplotype distribution of the common deficiency Pi-Z variant of AAT was significantly different when compared to the normal variants. In addition to the haplotype information, we present evidence for a functional effect of a SNP in intron 1.

Cell Line↗

PubMed2004

Regulation of LPS-mediated inflammation in vivo and in vitro by the thiol antioxidant Nacystelyn.

Increased levels of proinflammatory cytokines are present in bronchoalveolar lavage fluid in various lung diseases. Redox-sensitive transcription factors such as NF-kappaB regulate gene transcription for these cytokines. We therefore studied the effect of a new thiol antioxidant compound, Nacystelyn (NAL), on IL-8 regulation in a human macrophage-derived cell line (THP-1). LPS (10 microg/ml) increased IL-8 release compared with control levels. This LPS activation was inhibited by coincubation with NAL (1 and 5 mM). Pretreatment with cycloheximide or okadaic acid, protein synthesis, and serine/threonine phosphatase inhibitors, respectively, did not modify inhibition of IL-8 release caused by NAL. NF-kappaB and C/EBP DNA binding were increased after LPS treatment compared with control, an effect inhibited by cotreatment with NAL. Activator protein (AP)-1 DNA binding was unaffected. The enhanced neutrophil chemotaxis produced by conditioned media from LPS-treated cells was inhibited when cells were cotreated with NAL. The selectivity of NAL inhibition upon IL-8 expression was studied. LPS-treated THP-1 cells also had higher levels of TNF-alpha, transforming growth factor (TGF)-beta1 and -3, MIP-1alpha and -beta, and RANTES gene expression. However, only LPS-induced IL-8 and TGF-beta1 expressions were inhibited by NAL. An anti-inflammatory effect of NAL was confirmed in vivo as shown by a reduction in LPS-induced neutrophil recruitment to the lungs following instillation of NAL into the lungs. Our studies demonstrate that NAL has anti-inflammatory properties in vitro and in vivo, may therefore have a therapeutic role in lung inflammation, and has the advantage over other antioxidant agents in that it may be administrated by inhalation.

Acetylcysteine↗

PubMed2004

Expression of adhesion molecules during apoptosis of circulating neutrophils in COPD.

STUDY OBJECTIVES: Neutrophil accumulation occurs in the lungs of patients with COPD. This can be due to increased recruitment and/or delayed tissue clearance. Previous studies have described alterations in circulating neutrophils in these patients that can facilitate the former. Dysregulation of neutrophil apoptosis may contribute to the latter. This study investigated the potential abnormalities of the apoptotic process in COPD patients. DESIGN: Prospective study. SETTINGS: Outpatient clinic in a urban, tertiary hospital. PATIENTS: Fourteen stable patients with COPD, 8 smokers with normal lung function, and 8 healthy nonsmoking subjects. MEASUREMENTS AND RESULTS: We cultured circulating neutrophils that had been harvested from the study subjects at 2, 6, and 24 h. Apoptosis was assessed using flow cytometry by annexin binding and CD16 expression. The surface expression of the adhesion molecules Mac-1 (CD11b) and L-selectin (CD62L) also was determined by flow cytometry. The percentage of apoptotic neutrophils increased with time similarly in all groups. However, the surface expression of Mac-1 (CD11b) was higher, and that of L-selectin (CD62L) was lower, during apoptosis in the neutrophils of patients with COPD. CONCLUSIONS: These results show that, quantitatively, in vitro neutrophil apoptosis in COPD patients occurred at a rate similar to that found in healthy individuals and smokers with normal lung function. Qualitatively, however, the increased surface expression of Mac-1 (CD11b) and the decreased surface expression of L-selectin (CD62L) observed in the apoptotic neutrophils of COPD patients indicate increased activation during the apoptotic process. This may be relevant for the pathogenesis of COPD.

Apoptosis↗

PubMed2003

Oxidative stress and calcium signaling in the adverse effects of environmental particles (PM10).

This review focuses on the potential role that oxidative stress plays in the adverse effects of PM(10). The central hypothesis is that the ability of PM(10) to cause oxidative stress underlies the association between increased exposure to PM(10) and both exacerbations of lung disease and lung cancer. Pulmonary inflammation may also underlie the cardiovascular effects seen following increased PM(10), although the mechanisms of the cardiovascular effects of PM(10) are not well understood. PM(10) is a complex mix of various particle types and several of the components of PM(10) are likely to be involved in the induction of oxidative stress. The most likely of these are transition metals, ultrafine particle surfaces, and organic compounds. In support of this hypothesis, oxidative stress arising from PM(10) has been shown to activate a number of redox-responsive signaling pathways in lung target cells. These pathways are involved in expression of genes that play a role in responses relevant to inflammation and pathological change, including MAPKs, NF-kappaB, AP-1, and histone acetylation. Oxidative stress from particles is also likely to play an important role in the carcinogenic effects associated with PM(10) and hydroxyl radicals from PM(10) cause DNA damage in vitro.

Animals↗

PubMed2003

Cigarette smoke prevents apoptosis through inhibition of caspase activation and induces necrosis.

Emphysema is characterized by enlargement of the distal airspaces in the lungs due to destruction of alveolar walls. Alveolar endothelial and epithelial cell apoptosis induced by cigarette smoke is thought to be a possible mechanism for this cell loss. In contrast, our studies show that cigarette smoke condensate (CSC) induces necrosis in alveolar epithelial cells and human umbilical vein endothelial cells. Furthermore, study of the cell death pathway in a model system using Jurkat cells revealed that in addition to inducing necrosis, CSC inhibited apoptosis induced by staurosporine or Fas ligation, with both effects prevented by the antioxidants glutathione and dithiothreitol. Time course experiments revealed that CSC inhibited an early step in the caspase cascade, whereby caspase-3 was not activated. Moreover, cell-free reconstitution of the apoptosome in cytoplasmic extracts from CSC-treated cells, by addition of cytochrome-c and dATP, did not result in activation of caspases-3 or -9. Thus, smoke treatment may alter the levels of pro- and antiapoptogenic factors downstream of the mitochondria to inhibit active apoptosome formation. Therefore, unlike previous studies, cell death in response to cigarette smoke by necrosis and not apoptosis may be responsible for the loss of alveolar walls and inflammation observed in emphysema.

Apoptosis↗

PubMed2003

Ultrafine carbon black particles inhibit human lung fibroblast-mediated collagen gel contraction.

Both acute and chronic exposure to particulates have been associated with increased mortality and morbidity from a number of causes, including chronic obstructive pulmonary disease and other chronic lung diseases. The current study evaluated the hypothesis that ultrafine carbon particles, a component of ambient particulates, could affect tissue repair. To assess this, the three-dimensional collagen gel contraction model was used. Ultrafine carbon black particles, but not fine carbon black, inhibited fibroblast-mediated collagen gel contraction. Although previous research has indicated that inflammatory effects of ultrafine carbon black particles are mediated by oxidant mechanisms, the current study suggests that ultrafine carbon black's inhibition of fibroblast gel contraction is mediated by the binding of both fibronectin and transforming growth factor (TGF)-beta to the ultrafine particles. Binding of TGF-beta was associated with a reduction in nuclear localization of Smads, indicative of inhibition of TGF-beta signal transduction. There was also a decrease in fibronectin mRNA, consistent with a decrease in TGF-beta-mediated response. Taken together, these results demonstrate the ability of ultrafine particles to contribute to altered tissue repair and extend the known mechanisms by which these biologically active particles exert their effects.

Base Sequence↗

PubMed2002

Histone acetylation regulates epithelial IL-8 release mediated by oxidative stress from environmental particles.

Increases in the levels of environmental particulate matter with a diameter of <10 microm diameter (PM(10)) in the air are associated with a variety of adverse health effects, particularly chronic lung and cardiovascular diseases. The expression of many inflammatory genes involves the remodeling of the chromatin structure provided by histone proteins. Histone acetylation causes the unwinding of chromatin structure, therefore allowing transcription factor access to promoter sites. Acetylation is reversible and is regulated by histone acetyltransferases (HATs), which promote acetylation, and deacetylases, which promote deacetylation. PM(10) and H(2)O(2) increased IL-8 protein release from A549 cells after 24-h treatment, and this was enhanced by histone deacetylase inhibition by trichostatin A (cotreatment). PM(10) and H(2)O(2) treatment also increased HAT activity as well as the level of acetylated histone 4 (H4). PM(10) enhanced H4 acetylation that was mediated by oxidative stress as shown by thiol antioxidant inhibition. Acetylation of H4 mediated by PM(10) was associated with the promoter region of the IL-8 gene. These data suggest that remodeling of chromatin by histone acetylation plays a role in PM(10)-mediated responses in the lungs.

Acetylation↗

PubMed2002

4-Hydroxy-2-nonenal, a specific lipid peroxidation product, is elevated in lungs of patients with chronic obstructive pulmonary disease.

Cigarette smoking results in oxidative stress and inflammation in the lungs, which are involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). 4-Hydroxy-2-nonenal (4-HNE), a highly reactive diffusible product of lipid peroxidation, is a key mediator of oxidant-induced cell signaling and apoptosis. 4-HNE has a high affinity toward cysteine, histidine, and lysine groups and forms direct protein adducts. We investigated the presence of 4-HNE-modified proteins in lung tissue obtained from subjects with and without COPD. We studied 23 current or ex-smokers with similar smoking histories with COPD (n = 11; FEV(1) < 70% predicted) or without COPD (n = 12; FEV(1) > 84% predicted) who had undergone lung resection. As 4-HNE and transforming growth factor-beta(1) (TGF-beta(1)) can modulate gamma-glutamylcysteine synthetase (gamma-GCS) mRNA levels in lung cells, we assessed the relations between 4-HNE-modified protein levels, FEV(1), gamma-GCS, and TGF-beta(1). 4-HNE-modified protein levels were elevated in airway and alveolar epithelial cells, endothelial cells, and neutrophils in subjects with COPD, compared with the levels in subjects without COPD (p < 0.01). We also observed a significant inverse correlation between the levels of 4-HNE adducts in alveolar epithelium, airway endothelium, and neutrophils and FEV(1) (p < 0.05) and a positive correlation between 4-HNE adducts and TGF-beta(1) protein and mRNA as well as gamma-GCS mRNA levels in airway and alveolar epithelium (p < 0.01). The elevated levels of 4-HNE may play a role in the signaling events in lung inflammation leading to the imbalance of the expression of both proinflammatory mediators and protective antioxidant genes in COPD.

Aldehydes↗

PubMed2002

Molecular mechanism of transforming growth factor (TGF)-beta1-induced glutathione depletion in alveolar epithelial cells. Involvement of AP-1/ARE and Fra-1.

Glutathione (GSH) is a ubiquitous antioxidant in lung epithelial cells and lung lining fluid. Transforming growth factor beta1 (TGF-beta1) is a pleiotropic cytokine involved in cellular proliferation and differentiation. The level of TGF-beta1 is elevated in many chronic inflammatory lung disorders associated with oxidant/antioxidant imbalance. In this study, we show that TGF-beta1 depletes GSH by down-regulating expression of the enzyme responsible for its formation, gamma-glutamylcysteine synthetase (gamma-GCS) and induces reactive oxygen species production in type II alveolar epithelial cells (A549). To investigate the molecular mechanisms of inhibition of glutathione synthesis, we employed reporters containing fragments from the promoter region of the gamma-GCS heavy subunit (h), the gene that encodes the catalytic subunit of gamma-GCS. We found that TGF-beta1 reduced the expression of the long gamma-GCSh construct (-3802/GCSh-5'-Luc), suggesting that an antioxidant response element (ARE) may be responsible for mediating the TGF-beta1 effect. Interestingly, the electrophoretic mobility shift assay revealed that the DNA binding activity of both activator protein-1 (AP-1) and ARE was increased in TGF-beta1-treated epithelial cells. The gamma-GCSh ARE contains a perfect AP-1 site embedded within it, and mutation of this internal AP-1 sequence, but not the surrounding ARE, prevented DNA binding. Further studies revealed that c-Jun and Fra-1 dimers, members of the AP-1 family previously shown to exert a negative effect on phase II gene expression, bound to the ARE sequence. We propose a novel mechanism of gamma-GCSh down-regulation by TGF-beta1 that involves the binding of c-Jun and Fra-1 dimers to the distal promoter. The findings of this study provide important information, which may be used for the modulation of glutathione biosynthesis in inflammation.

Antioxidants↗

PubMed2002

Nacystelyn inhibits oxidant-mediated interleukin-8 expression and NF-kappaB nuclear binding in alveolar epithelial cells.

Nacystelyn (NAL), a recently developed lysine salt of N-acetyl-L-cytokine (NAC) has mucolytic and antioxidant properties. In this study, we investigated the effect of NAL upon oxidant-mediated interleukin (IL)-8 release and the activation of the redox-sensitive transcription factors AP-1, NF-kappaB, and C/EBP in a human alveolar epithelial cell line (A549). NAL (5 mM) enhanced intracellular glutathione (GSH) after 4 h and abolished H(2)O(2)-induced IL-8 release from A549 cells. This was associated with inhibition of NF-kappaB and C/EBP DNA-binding, measured by the Electrophoretic Mobility Shift Assay (EMSA). NAL also abolished the transcriptional activation of IL-8 in an IL-8-chloramphenicol acetyl transferase (CAT) reporter system, transfected into A549 cells. Supernatants obtained from H(2)O(2)-treated A549 cells induced chemotaxis of polymorphonuclear neutrophils, which could be inhibited by co-incubation with NAL. These data indicate that NAL may be used to modulate pro-inflammatory process by inhibiting cytokine release in the lungs and thus has therapeutic potential in inflammatory lung diseases.

Acetylcysteine↗

PubMed2002

Potential role of IL-8, platelet-activating factor and TNF-alpha in the sequestration of neutrophils in the lung: effects on neutrophil deformability, adhesion receptor expression, and chemotaxis.

The microvasculature of the normal lung contains a pool of sequestered neutrophils, which is markedly enhanced in acute lung inflammation. Lung neutrophil sequestration is determined by the cells' deformability and adhesivity to capillary endothelium, and is a pre-requisite for emigration into the airspaces. We assessed the effect of several pro-inflammatory mediators associated with acute lung inflammation on these factors. Platelet-activating factor, IL-8 and formyl-Met-Leu-Phe (fMLP) induced a marked, but transient reduction in neutrophil deformability. Also, increased surface expression of the beta(2)-integrin and CD11b, and shedding of L-selectin (CD62L) was observed for these stimuli. TNF-alpha in contrast caused a small decrease in cell deformability only after 30 min, and shedding of L-selectin, but no change in CD11b levels. However, TNF-alpha-pretreatment markedly enhanced the fMLP response for cell deformability, CD11b expression and CD62L loss. Moreover, all pre-treatments were found to induce chemokinesis, and all except fMLP, enhanced fMLP-directed chemotaxis. We were able to demonstrate, using specific TNF-alpha receptor antagonists, that the TNF-alpha-induced changes in chemotaxis were mediated through the 55-kDa receptor. Also, inhibitors of the mitogen activated protein (MAP) kinase signaling pathway showed that the p38 MAP kinase pathway was involved for fMLP-directed chemotaxis of TNF-pretreated neutrophils, although activation of the extracellular signal-regulated kinase (ERK) pathway was also seen. These data demonstrate the differential role of pro-inflammatory mediators in controlling neutrophil sequestration and migration, which may orchestrate the severity of the inflammatory response in such respiratory diseases as chronic obstructive pulmonary disease and asthma.

Cell Adhesion↗

PubMed2002

The pulmonary toxicology of ultrafine particles.

Ultrafine particles are a component of air pollution, derived from primary combustion sources, and so we have undertaken a programme of study on the mechanisms of lung injury caused by ultrafine particles. Ultrafine particles made of low-solubility, low-toxicity materials are more inflammogenic in the rat lung than fine respirable, particles made from the same material. Ultrafine particles can cause inflammation via processes independent of the release of transition metals, as shown by the fact that soluble products from ultrafine carbon black have no ability to cause inflammation. The property that drives the greater inflammogenicity of ultrafines is unknown but very likely relates to particle surface area and involves oxidative stress. Increases in intracellular Ca(++) may underlie the cellular effects of ultrafines, although the mechanism whereby ultrafines have this effect is not understood. However, increased influx of Ca(++) into macrophages occurs via the membrane Ca(++) channels following contact with ultrafine particles, and involves oxidative stress. Increased Ca(++) in macrophages exposed to ultrafines can lead to the transcription of key pro-inflammatory genes such as TNFalpha. Ultrafine particles can also impair the ability of macrophages to phagocytose and clear other particles, and this may be pro-inflammogenic.

Aerosols↗

PubMed2002

Glutathione prevents inhibition of fibroblast-mediated collagen gel contraction by cigarette smoke.

Cigarette smoke, the major risk factor for the development of emphysema, contains over 4,700 chemical compounds, including free radicals and other oxidants (10(14)/puff). An imbalance between oxidants and antioxidants has been proposed in the pathogenesis of chronic obstructive pulmonary disease. Inhibition of repair processes has been suggested to be one pathway contributing to the development of emphysema. We hypothesized that cigarette smoke inhibition of repair might result from a shift of the oxidant/antioxidant balance in favor of oxidants. To evaluate this hypothesis, N-acetyl-L-cysteine (NAC), which serves as a substrate for glutathione (GSH) production, and buthionine sulfoximine (BSO), which inhibits GSH production, were incubated in the presence and absence of cigarette smoke extract (CSE) with fibroblasts in three-dimensional collagen gels. Neither agent alone altered gel contraction. CSE inhibition of gel contraction, however, was mitigated by NAC and potentiated by BSO. Parallel effects were observed on cigarette smoke inhibition of fibronectin production and mRNA expression as well as by changes in intracellular GSH content. Pretreatment of fibroblasts with NAC or BSO resulted in similar effects, suggesting that neither agent was acting directly on smoke but, rather, was altering cellular response to smoke. In conclusion, smoke inhibition of fibroblast repair, as reflected by collagen gel contraction and fibronectin production, may be modulated by intracellular GSH levels.

Acetylcysteine↗

PubMedDate not supplied

Oxidative stress and TNF-alpha induce histone acetylation and NF-kappaB/AP-1 activation in alveolar epithelial cells: potential mechanism in gene transcription in lung inflammation.

Oxidants and inflammatory mediators such as tumour necrosis factor-alpha (TNF-alpha) activate nuclear factor kappa B (NF-kappaB) and activator protein-1 (AP-1) transcription factors, and enhance the expression of both pro-inflammatory and protective antioxidant genes. Remodelling of chromatin within the nucleus, controlled by the degree of acetylation/deacetylation of histone residues on the histone core around which DNA is coiled, is important in allowing access for transcription factor DNA binding and hence gene transcription. Unwinding of DNA is important in allowing access for transcription factor DNA binding and hence gene transcription. Nuclear histone acetylation is a reversible process, and is regulated by a group of acetyltransferases (HATs) which promote acetylation, and deacetylases (HDACs) which promote deacetylation. The aim of this study was to determine whether oxidative stress and the pro-inflammatory mediator, TNF-alpha, altered histone acetylation/deacetylation and the activation of NF-kappaB and AP-1, leading to the release ofthe pro-inflammatory cytokine IL-8 in human alveolar epithelial cells (A549). Hydrogen peroxide (H2O2) (100 microM) and TNF-alpha (10 ng/ml) imposed oxidative stress in A549 cells as shown by depletion of the antioxidant reduced glutathione (GSH) concomitant with increased levels of oxidised glutathione (GSSG). Treatment of A549 cells with H2O2, TNF-alpha and the HDAC inhibitor, trichostatin A, TSA (100 ng/ml) significantly increased acetylation of histone proteins shown by immunostaining of cells and increased HAT activity, compared to the untreated cells. H2O2, and TNF-a, and TSA all increased NF-kappaB and AP-1 DNA binding to their consensus sites assessed by the electrophoretic mobility shift assay. TSA treatment potentiated the increased AP-1 and NF-KB binding, produced by H2O2 or TNF-alpha treatments in A549 cells. Both H2O2 and TNF-alpha significantly increased IL-8 release, which was further enhanced by pre-treatment of A549 cells with TSA compared to the individual treatments. This study shows that the oxidant H2O2 and the pro-inflammatory mediator, TNF-a induce histone acetylation which is associated with decreased GSH levels and increased AP-1 and NF-kappaB activation leading to enhanced proinflammatory IL-8 release in alveolar epithelial cells. This indicates a mechanism for the pro-inflammatory effects of oxidative stress.

Acetylation↗