Pulmonari Oxdative Stress

Pulmonary and Systemic Oxidant/AntioxidantImbalance in Chronic Obstructive Pulmonary DiseaseWilliam MacNee

ELEGI, Colt Research Laboratories, MRC Centre for Inflammation Research, Medical School, University of Edinburgh, Edinburgh, Scotland,United Kingdom

An imbalance between oxidants and antioxidants is considered toplay a role in the pathogenesis of chronic obstructive pulmonarydisease (COPD). There is considerable evidence that an increasedoxidative burden occurs in the lungs of patients with this disorder,and this may be involved in many of the pathogenic processes,such asdirect injury to lung cells,mucushypersecretion, inactivationof antiproteases, and enhancing lung inflammation through activa-tion of redox-sensitive transcription factors. COPD is now recog-nized to have multiple systemic consequences, such as weight lossand skeletal muscle dysfunction. Moreover, it is appreciated thatoxidative stress extends beyond the lung and may, through similaroxidative stress mechanisms as those in the lung, contribute toseveral of the systemic manifestations in COPD.

Keywords: chronic obstructive pulmonary disease; oxidant/antioxidantimbalance; oxidative stress; smoking; systemic inflammation

The lungs are exposed continuously to oxidants generated eitherendogenously from phagocytes and other cell types or exoge-nously from air pollutants or cigarette smoke. In addition, intra-cellular oxidants, such as those derived from mitochondrial elec-tron transport, are involved in many cellular signaling pathways.Lung cells are protected against this oxidative challenge by well-developed enzymatic and nonenzymatic antioxidant systems (1).When the balance between oxidants and antioxidants shifts infavor of the former, from either an excess of oxidants and/ordepletion of antioxidants, oxidative stress occurs. Oxidative stressproduces not only direct injurious effects in the lungs but alsoactivates molecular mechanisms that initiate lung inflammation (2).

Smoking is the main etiologic factor in chronic obstructivepulmonary disease (COPD). Cigarette smoke contains around1017 oxidant molecules per puff, and this, together with a largebody of evidence demonstrating increased oxidative stress insmokers with and without COPD, has led to the proposed roleof oxidant/antioxidant imbalance in the pathogenesis of this con-dition (3). Increasingly, COPD is recognized to affect not onlythe lungs but also to have significant systemic consequences,such as muscle dysfunction and weight loss (4). Oxidative stressis also believed to play an important role in the systemic manifes-tations of COPD (5).

HOW IS OXIDATIVE STRESS MEASURED?

Oxidative stress can bemeasured in several different ways, eitherby direct measurements of the oxidative burden, as the responsesto oxidative stress, or by the effects of oxidative stress on targetmolecules (Table 1). Direct measurements of the oxidative bur-den in airspaces can be derived from measurement of hydrogen

(Received in original formNovember 11, 2004; accepted in final formDecember 21,2004)

Correspondence and requests for reprints should be addressed toW.MacNee, M.B.Ch.B., M.D., ELEGI, Colt Research Laboratories, Wilkie Building, Medical School,Teviot Place, Edinburgh EH8 9AG, Scotland, UK. E-mail: [email protected]

Proc Am Thorac Soc Vol 2. pp 5060, 2005DOI: 10.1513/pats.200411-056SFInternet address: www.atsjournals.org

peroxide (H2O2) in bronchoalveolar lavage (BAL) fluid or inexhaled breath condensate. Airspace leukocytes derived fromBAL can also be assessed ex vivo for their ability to producereactive oxygen species (ROS). Nitric oxide (NO) is producedin the lungs by the catalytic activity of NO synthase (NOS),which exists in both constitutive isoforms (cNOS) and an induc-ible isoform (iNOS) (6). The latter is induced by inflammatorystimuli, and NO in exhaled breath is considered a marker ofinflammation and indirectly as a marker of oxidative stress.

Measurements of the responses to oxidative stress can beobtained by assessing changes in antioxidants in BAL fluid orin induced sputum.The depletion of antioxidants or upregulationof antioxidant enzymes can be assessed in lung tissue (7). Perhapsmore important than the presence of oxidative stress are mea-surements of the effects of oxidative stress on target molecules.Oxidative stress renders proteins more susceptible to proteolyticdegradation by modifying amino acid chains to form proteinaggregates and cleaving peptide bonds (8). As part of this pro-cess, some amino acid residues are converted to carbonyl resi-dues. Exposure of human plasma to cigarette smoke in vitroresults in depletion of plasma protein sulfhydryl groups andelevation of protein carbonyl levels (9). Plasma proteins can alsobe degraded through nitration and oxidation by reactive nitrogenspecies, the formation of which is stimulated by cigarette smok-ing (10).

Oxidized proteins can be measured as protein carbonyls, ni-trotyrosine, or oxidative damage to DNA producing 8-hydroxy-deoxyguanine. The reaction of NO and superoxide anion pro-duces peroxynitrite (Figure 1), which can then cause the nitrationof tyrosine to produce nitrotyrosine, a marker of peroxynitritethat can be measured in blood, breath condensate, BAL, andlung tissue as an indicator of free-radical protein damage (11).NO may form stable nitrosothiols with low-molecular-weightthiols, such as glutathione, to enhance its bioactivity (12). Nitriteis a further end-product of NO.

Lipid peroxidation is a process of abstraction of a protonfrom a side chain of a fatty acid to produce a carbon-centeredradical, which itself can then react with the fatty acid to produce alipid peroxidation product and a further carbon-centered radical,which can react again, resulting in a chain reaction (13). Lipidperoxidation products, such as F2-isoprostanes, 4-hydroxyno-nenal (4-HNE), and hydrocarbons, can be measured in breath,breath condensate, BAL, and lung tissue.

EVIDENCE OF LOCAL OXIDATIVE STRESSIN THE LUNGS

Numerous studies have shown that oxidative stress is increasedin the lungs of patients with COPD compared with healthysubjects, but also compared with smokers with similar smokinghistory but who have not developed airways obstruction (3).Smokers and patients with COPD have higher levels of H2O2 inexhaled breath condensate, a direct measurement of airspaceoxidative burden, than ex-smokers with COPD or nonsmokers(14, 15). H2O2 levels are even higher during exacerbations ofCOPD (Figure 2) (14). The elevated level of H2O2 in the exhaled

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MacNee: Systemic Oxidative Stress in COPD 51

TABLE 1. MEASUREMENTS OF OXIDATIVE STRESS

Direct measurements of oxidative burden Hydrogen peroxide in breath condensate or BAL fluidBAL/peripheral blood leukocyte reactive oxygen species productionNitric oxide in exhaled breath

Responses to oxidative stressCarbon monoxide in breath (reflecting hemoxygenase activity)Antioxidants, antioxidant enzymes in blood, sputum, BAL, and lung tissue

Effects of oxidative stress on target moleculesOxidized proteins (e.g., carbonyl residues, oxidase, and nitrated proteins)Lipid peroxidation products (e.g., F2-isoprostains, 4-hydroxy-2-nonenal,hydrocarbons) in breath condensate, sputum,BAL, blood, urine, lung tissue

Definition of abbreviation: BAL bronchoalveolar lavage.

breathof smokers is believed toderive partly from increased releaseof superoxide anion (O2) by alveolar macrophages (16, 17).

Because the iron content of smokers alveolar macrophagesis also increased compared with that of nonsmokers (18), theresultant increased free iron in the airspaces of smokers wouldstimulate the generation of ROS through the Fenton reaction(10). A further source of both O2 and H2O2 is the xanthine/xanthine oxidase reaction. Xanthine oxidase activity is increasedin cell-free BAL fluid and in the plasma of smokers and inpatients with COPD compared with healthy subjects and non-smoking subjects, respectively (19, 20).

NO has been used as a marker of airway inflammation andindirectly as a measure of oxidative stress. Increased NO levelsin exhaled breath occurs in some studies of COPD but are not ashigh as the NO levels reported in asthma (2123). Other studieshave found either normal or even lower exhaled NO in patientswith stable COPD, compared with normal subjects (24, 25).

The rapid reaction of NO with O2, as described previously,or with thiols may alter breath NO levels. Nitrosothiol levelshave been shown to be higher in breath condensate in smokersand patients with COPD compared with subjects who do notsmoke (Figure 3) (6). Smoking increases directly exhaled NOlevels, which limits the usefulness of this marker in COPD.Peroxynitrite, formed by the reaction of NO with superoxideanion, can cause nitrosylation of tyrosine to produce nitrotyro-sine (11). Nitrotyrosine levels are elevated in sputum leukocytesof patients with COPD (Figure 3) and are correlated negativelywith the FEV1 (26).

Exhaled carbon monoxide (CO) as a measure of the responseof heme oxygenase to oxidative stress (Figure 4) has been shownto be elevated in exhaled breath in patients with COPD com-

Figure 1. Nitric oxide (NO) and NO-related products. FAD flavinadenine dinucleotide; FMN flavin mononucleotide; NADP nicotin-amide adenine dinucleotide phosphate; NADPH reduced form ofNADP; S-GSNO S-nitrosoglutathione. Adapted from Reference 6.

Figure 2. Individual data for hydrogen peroxide (H2O2) concentrationsin breath condensate in control subjects and in patients with stable andunstable chronic obstructive pulmonary disease (COPD). Modified fromReference 14 by permission.

pared with normal subjects (Figure 5) (27). Again, CO is presentin cigarette smoke, which limits the usefulness of this markerof oxidative stress in patients who smoke.

Lipid peroxidation products, such as thiobarbituric acid-reacting substances, are elevated in sputum in patients withCOPD,and correlate negatively with the FEV1 (28, 29). Isoprostanes,produced by ROS-mediated peroxidation of arachidonic acid,circulate in plasma and can be excreted in the urine (30). Thelevels of 8-isoprostane in breath condensate have been shownto be elevated in patients with COPD compared with normalsubjects and smokers who have not developed the disease, andcorrelate with the degree of obstruction (31). The levels of8-isoprostane in breath condensate in patients with COPD alsoshow a positive correlation with the percentage of neutrophilsin induced sputum, suggesting a role for oxidative stress in theairway inflammation (Figure 6) (32). Isoprostanes may also re-flect systemic effects caused by ROS. In plasma, the levels offree F2-isoprostanes are increased in smokers and decreasedafter smoking cessation (33). In urine, the levels of F2-isopros-tanes are elevated in patients with COPD in comparison tohealthy control subjects, with the highest levels observed duringexacerbations (34).

Lipid peroxides can interact with enzymatic or nonenzymaticantioxidants or decompose by reacting with metal ions or iron-

Figure 3. Left: nitrosothiols, breath condensate in smokers and patientswith COPD. Right: increased inducible NO (iNOS) and nitrotyrosineimmunoreactivity in sputum leukocytes in COPD. *p 0.05. Modifiedby permission from Reference 26.



52 PROCEEDINGS OF THE AMERICAN THORACIC SOCIETY VOL 2 2005

Figure 4. Hemeoxygenase system-synthesis of carbon monoxide (CO).

containing proteins, thereby forming hydrocarbon gases and un-saturated aldehydes (13). Hydrocarbons are by-products of fattyacid peroxidation (27). Patients with COPD show an increasedlevel of exhaled ethane in breath compared with control subjects,the levels being correlated negatively with lung function (28, 35).

There is increasing evidence that these markers of oxidativestress are also reflected in lung tissue in patients with COPD.Increased nitrotyrosine immunoreactivity has been shown insputum leukocytes and lung tissue in patients with COPD com-pared with healthy subjects (26). The lipid peroxidation product4-HNE reacts quickly with cellular proteins to form adducts.These adducts have been shown to be present in greater quanti-ties in airway epithelial and endothelial cells in the lungs ofpatients with COPD, compared with smokers with a similarsmoking history who have not developed the disease (Figure 7)(36). Lipid peroxidation products, such as 8-isoprostane, canact as signaling molecules and cause release of inflammatorymediators, such as interleukin 8 (IL-8) from lung cells (37).4-HNE can cause the upregulation of transforming growth factor (TGF-) (38) and oxidant enzyme gene expression (39).

Thus, many studies show increased markers of airway oxida-tive stress in exhaled breath or breath condensate and in lungtissue in patients with COPD compared with normal subjectsand smokers who have not developed the disease. In addition,many of thesemarkers correlatewith the degree of airflow limita-tion in COPD, suggesting a role for oxidative stress in the declinein lung function in COPD. Furthermore, products of oxidativestress, such as lipid peroxides, can act as signaling molecules toenhance inflammation in COPD.

ANTIOXIDANTS AND COPD

Several studies have investigated a relationship between anti-oxidants, pulmonary function, and the development of COPD(40, 41). The National Health and Nutrition Examination Survey(NHANES) and the Dutch Monitoring Project for the RiskFactors for Chronic Diseases (MORGEN) suggested relation-ships between dietary intake and airflow limitation. In NHANESI, lower dietary vitamin C related directly to lower FEV1 levelsin a population survey study and, in addition, the protectiveeffect of vitamin C was even greater in subjects with bronchitis(40). Data from NHANES II showed an inverse relationshipbetween both dietary and serum vitamin C and chronic respira-tory symptoms (41). Moreover, in NHANES III, the levels ofdietary vitamin C, vitamin E, selenium, and beta-carotene werepositively associated with lung function (42). Analysis of thedata obtained in the MORGEN study also shows higher intakeof vitamin C and beta-carotene is associated with a higher levelFEV1, comparedwith a low intake of these antioxidants (43). Theassociation between dietary vitaminE intake and lung function isless consistent (4345). Several studies, including theMORGEN

Figure 5. Exhaled CO in patients with COPD with and without cortico-steroid therapy. ns not significant. Modified by permission fromReference 27.

study, have shown associations between fruit intake and higherFEV1 and lower symptoms in patients with COPD (4649). Datafrom the MORGEN study have also shown the beneficial effectsof flavonoids on FEV1 (50).

IS OXIDATIVE STRESS IMPORTANT IN THEPATHOGENESIS OF COPD?

Biomarkers of oxidative stress have been related to the develop-ment of airflow limitation, and many studies have shown higheroxidant levels in patients with COPD, compared with healthysmokers. Furthermore, several studies show relationships be-tween oxidative stress markers and the degree of airflow limita-tion in COPD (3, 5, 51). However, the presence of oxidativestress and its relationship to airflow limitation may be epipheno-mena, because it occurs in inflammation, a characteristic featureof COPD (10). There are as yet no longitudinal studies showingthat the presence of enhanced oxidative stress relates to thedecline in FEV1 or to the progression of the disease.

OXIDATIVE STRESS ANDPROTEASE/ANTIPROTEASE IMBALANCE

An increased protease burden in the lungs occurs as a result ofthe influx and activation of inflammatory leukocytes. It has beenproposed that a relative deficiency of antiproteases, such as1-antitrypsin, because of their inactivation by oxidants, createsa protease/antiprotease imbalance in the lungs. This forms the

Figure 6. Isoprostane levels in exhaled breath condensate (EBC) insmokers with COPD (left). Significant correlation between 8-isoprostanelevels in EBC and percentage of neutrophils in induced sputum (right).Adapted from Reference 32.




Figure 7. Immunostaining for lipidperoxidation product 4-hydroxy-2-nonenal (4-HNE) adduct in thelungs of smokers with and withoutCOPD. (A ) Immunohistochemistryshowing increased staining in bothbronchial and alveolar epitheliumin patients with COPD who havethe same smoking history as agroup of smokers without COPD.(B ) Staining score showing in-creased staining in COPD. (C ) Cor-relation between 4-HNE stainingscore in the lungs and percent pre-dicted FEV1. Modified by permis-sion from Reference 36.

basis of the protease/antiprotease theory of the pathogenesis ofemphysema (52, 53). Inactivation of 1-antitrypsin occurs byoxidation of a critical methionine residue at its active site byoxidants from cigarette smoke or released from inflammatoryleukocytes, resulting in a dramatic reduction in its inhibitorycapacity in vitro (54, 55). The acute effects of cigarette smokeon functional activity of 1-antitrypsin have been studied in vivo,and show a transient, but nonsignificant, fall in the antiproteaseactivity of BAL fluid 1 hour after smoking (56). However, aprotease/antiprotease imbalance involving 1-antitrypsin andneutrophil elastase is likely an oversimplification, because otherproteases and other antiproteases are likely to have a role inthe pathogenesis of COPD.

OXIDANTS AND MUCUS HYPERSECRETION

Oxidant-generating systems, such as xanthine/xanthine oxidase,can cause airway epithelial mucus secretion (57). Oxidants arealso involved in the signaling pathways for epidermal growthfactor, which has an important role in mucus production (58).H2O2 or HOCl, in relatively low concentrations (100 M), causessignificant impairment of ciliary beating and even complete stasis(59). Oxidant-mediated hypersecretion and impaired mucocili-ary clearance may result in airway mucus accumulation contrib-uting to airflow limitation (60).

OXIDATIVE STRESS AND AIRSPACE EPITHELIAL INJURY

Because of their direct contact with the environment, the lungepithelial surfaces are especially vulnerable to the effects ofoxidative stress. Injury to the airway epithelium is an importantearly event after exposure to cigarette smoke as shown by anincrease in airspace epithelial permeability. This increased per-meability can be shown to result from cigarette smoke exposure

both in vitro and in vivo (6163) and is partially reversible byantioxidants. Extra- and intracellular glutathione appears to becritical to the maintenance of epithelial integrity after exposureto smoke. This has been shown in studies of the permeabilityof epithelial cell monolayers in vitro and in animal models in vivoafter exposure to cigarette smoke condensate, which is associatedwith profound changes in the homeostasis of the antioxidant gluta-thione (6366). Depletion of lung glutathione alone can induceincreased airspace epithelial permeability (63, 66). Interindividualvariability in antioxidant defenses may be one factor in determin-ing whether COPD develops after cigarette smoking.

OXIDATIVE STRESS AND NEUTROPHIL INFLUXIN THE LUNGS

The lungs contain a large pool of noncirculating (marginated)neutrophils, which either are retained ormove only slowly withinthe pulmonary microcirculation (67). This is because of thesmaller average diameter of the neutrophil ( 7 m) comparedwith the pulmonary capillary diameter ( 5 m), and results ina proportion of the circulating neutrophils having to deform andthus move slowly to negotiate the small capillaries. In normalsubjects, there is a correlation between neutrophil deformability,measured in vitro, and their subsequent sequestration in thepulmonary microcirculation after reinjectionthe less deform-able the cells, the greater their sequestration (68). This mecha-nism of neutrophil sequestration in the pulmonary bed allowstime to interact and adhere to capillary endothelium before theirsubsequent migration across the alveolarcapillary membraneto the interstitial airspaces. During smoking, there is a transientincrease in neutrophil sequestration in the lungs (69) becauseof a decrease in circulating neutrophil deformability (67, 70).Studies in vitro show that the decreased neutrophil deformabilityinduced by cigarette smoke is abolished by antioxidants (71).




Figure 8. Oxidative stress in exacerbations of COPD. Super-oxide anion release from peripheral blood neutrophils (PMN;spontaneous andphorbolmyristate acetate [PMA] activated)in normal subjects and in patients with acute and stableCOPD (left). Plasma antioxidant capacity (Trolox equivalentantioxidant capacity [TEAC]) in normal subjects, and in thosewith stable and exacerbated COPD (right). ROS reactiveoxygen species. *p 0.05; **p 0.01. Adapted from Refer-ence 108.

Oxidative stress occurs systemically during smoking as shownby a profound decrease in the antioxidant capacity of plasma(Figure 8), which may reduce neutrophil deformability becauseof actin polymerization (71). These may be the initial eventsthat cause the influx of neutrophils into the lungs of cigarettesmokers, and variability in this effect (69, 70) may be one reasonwhy not all smokers develop enhanced lung inflammation andtherefore COPD. It is possible that activation of neutrophilssequestered in the pulmonary microvasculature could also in-duce the release of ROS and proteases within a microenviron-ment, which limits access for free-radical scavengers and anti-proteases. Thus, destruction of the alveolar wall, as occurs inemphysema, could result from a proteolytic/oxidant insult de-rived from the intravascular space without the need for theneutrophils to migrate into the airspaces.

OXIDATIVE STRESS AND LUNG INFLAMMATION

As mentioned previously, oxidative stress is present whereverthere is inflammation. Regardless, oxidative stress may also bea mechanism for enhancing the airspace inflammation that is acharacteristic feature of COPD (10). Oxidative stress can causethe release of chemotactic factors, such as IL-8, from airwayepithelial cells (72), and epithelial cells from patients with COPDrelease more IL-8 than those of smokers or healthy individuals(73). Numerousmarkers of inflammation, such as IL-8 and tumornecrosis factor-, have been shown to be elevated in the sputumof patients with COPD (74), and there is overwhelming evidencethat COPD is associated with enhanced airway inflammation asconfirmed by biopsy studies (75). Recent studies have also showna relationship between markers of oxidative stress in breath andthe number of neutrophils in induced sputum (32). Epidemio-logic studies have shown a relationship between circulating neu-trophil numbers and FEV1 (76) and between the changes inperipheral blood neutrophil numbers and airflow limitation overtime (77). There is also a relationship between peripheral bloodneutrophil oxidant release and measures of airflow limitation inyoung smokers (78). In addition, lipid peroxidation products inplasma have also been shown to correlate inversely with thepercent predicted FEV1 in a population study (79).

Oxidative stress may have a fundamental role enhancing in-flammation through the upregulation of redox-sensitive tran-scription factors, such as nuclear factor B (NF-B) and activat-ing protein 1 (AP-1), and also the extracellular signal-regulatedkinase (ERK), c-Jun N-terminal kinase (JNK), and p38mitogen-activated protein kinase pathways. Cigarette smoke has beenshown to activate all of these signaling mechanisms (2, 8082).Genes for many inflammatory mediators are regulated by NF-B,

which is present in the cytosol in an inactive form linked to itsinhibitory protein IB. Many stimuli, including cytokines andoxidants, result in activation of IBkinase, resulting in phosphor-ylation of IB, cleaving of IB from NF-B, and the destructionof IB in the proteasome. This critical event in the inflammatoryresponse is redox-sensitive (83, 84). Studies in macrophage celllines and alveolar and bronchial epithelial cells show that oxi-dants cause the release of inflammatory mediators, such as IL-8,IL-1, andNO, and that these events are associatedwith increasedexpression of the genes for these inflammatory mediators andincreased nuclear binding or activation of NF-B (85, 86). Thelinking of NF-B to its consensus site in the nucleus leads toenhanced transcription of proinflammatory genes and thereforeinflammation, which itself will produce more oxidative stress,creating a vicious circle of enhanced inflammation resulting fromthe increased oxidative stress (Figure 9). Animal models ofsmoke exposure show that neutrophil influx in the lungs is associ-ated with increased IL-8 gene expression and protein releaseandwithNF-Bactivation (87).All of these events are associatedwith oxidative stress because they can be abrogated by antioxi-dant therapy (Figure 10). NF-B can be activated and translo-cated to the nucleus in lung tissue in smokers and in patients withCOPD compared with healthy subjects (88). NF-B activation inlung tissue has been shown to correlate with the FEV1 (89).

A further event induced by oxidative stress, which may en-hance lung inflammation, is chromatin remodeling. Under nor-mal circumstances, DNA is wound tightly around a core of his-tones, and in this configuration prevents excessive transcriptionby factors such as NF-B, as well as reduced access of RNApolymerase to DNA, thereby resulting in transcriptional repres-sion and gene silencing (Figure 11). Under the influence of his-tone acetyltransferases, histone residues are acetylated, causinga change in their charge and the unwinding of DNA. This thenallows access for transcription factors such as NF-B and/or RNApolymerase to the transcriptional machinery, therefore enhanc-ing gene expression. This process is reversed by histone deacety-lases (HDAC), enzymes that deacetylate histone residues, re-sulting in the rewinding of DNA and gene silencing. Theseprocesses are known to be redox-sensitive. Histone acetylationcan be shown to occur after cigarette smoke exposure of epithe-lial cells and is prevented by the antioxidant N-acetylcysteine,indicating that the process is redox-sensitive (90). Furthermore,animal models of cigarette smoke exposure have been shownto result in increased acetylated histone in lung and decreasedHDAC activity, and both of these events would enhance geneexpression (91). HDAC activity in alveolar macrophages, specifi-cally HDAC2 activity, has also been shown to be downregulated




Figure 9. Activation of signal pathways by oxidative stress. AP-1 acti-vating protein 1; ERK extracellular signal-related kinase; ikK inhibitorkB kinase; JNK c-Jun N-terminal kinase; NF-B nuclear factor B;P phosphate.

in alveolar macrophages obtained from cigarette smokers, a pro-cess that would enhance gene expression (Figure 12) (92). Recentstudies suggest that acetylatedhistone residues, specifically histone4 (H4), are present to a greater extent in smokers and in patientswith COPD who smoke, with an associated decrease in HDAC2,specifically in patients with COPD who smoke and in patientswith severe COPD (88). Acetylated histone 3 appears to beelevated in lung tissue in ex-smoking patients with COPD andmay be a mechanism for persistent inflammation in COPD aftersmoking cessation (88). A correlation has also been shown be-tween decreased HDAC activity in lung tissue and FEV1 inpatients with COPD (88, 93).

Many of the markers of oxidative stress do not respond totherapy with corticosteroids, and it is believed that oxidativestress may be involved in the relative resistance to these drugsin COPD. HDAC recruitment is believed to be required forthe antiinflammatory action of corticosteroids in smokers and inpatients with COPD. Amechanism involving nitration of HDACs,and therefore downregulation of HDACs in lung tissue, mayprevent the action of corticosteroids (94).

Recent evidence suggests that latent adenoviral infectionmaybe associated with the pathogenesis of COPD by enhancing lunginflammation (95). E1A protein derived from latent adenoviral

Figure 10. Effect of recombinant superoxide dismutase (rhSOD) on cig-arette smoke (CS-)induced neutrophil influx, interleukin (IL-)-8 geneexpression, and NF-B nuclear binding in guinea pig lungs. *p 0.05;**p 0.01. Modified by permission from Reference 86.

Figure 11. Model of histone acetylation/deacetylation. HAT histoneacetyltransferase.

infection interacts with transcription factor cofactors and en-hances nuclear binding of transcription factors (95). E1A hasbeen noted to be more prevalent in the lungs of smokers whodevelop COPD than in smokers who do not develop the disease(96). Cells transfected with the E1A protein have also beenshown to have increased IL-8 release in response to oxidants,such as H2O2, and enhanced NF-B activation (97). Latent ade-noviral infection may, therefore, render lung cells more suscepti-ble to oxidative stress, thus enhancing the release of proinflam-matory mediators. The presence of E1A also enhances smoke-induced inflammation and emphysema in animal models (98).

The pathogenesis of emphysema has been proposed recentlyto result from loss of alveolar endothelial cells via apoptosis,and this may be as an initial event in the development of emphy-sema (99). Apoptosis occurs to a greater extent in emphysema-tous lungs than in lungs of nonsmokers (100). The process ofendothelial apoptosis is believed to be under the influence ofvascular endothelial growth factor (VEGF)R2 receptors. Down-regulation of VEGF-R2 has been shown in animals to produceemphysema (100), and reduced expression of VEGF-R2 is evi-dent in emphysematous human lungs (100). Studies have alsoshown that the apoptosis/emphysema produced by VEGF in-hibition in animal models is associated with increased markersof oxidative stress and prevented by antioxidants (101).

Ablation in mice of nuclear factor, erythroid-derived 2, like2 (Nrf2), a redox-sensitive transcription factor that is involvedin the regulation of many antioxidant genes results in moreextensive cigarette smokeinduced emphysema than occurs inwild-type mice (102), and this was associated with increasedmarkers of oxidative stress and increased apoptosis in the lungs,suggesting a role for theNrf2 pathway in determining the suscep-tibility to tobacco smokeinduced emphysema through a mecha-nism involving upregulation of antioxidant defenses.

Other events involving oxidative stress and epithelial cellsmay be relevant to the development of COPD. For example,TGF-1 expression is increased in small airway epithelial cellsof smokers compared with nonsmokers and increased evenmorein patients with COPD (101, 102). TGF- mRNA levels corre-lated positively with smoking history and the degree of airwayobstruction (103, 104).

Furthermore, TGF-1 itself may increase oxidative stress be-cause this substance produces profound changes in endothelialand epithelial cell glutathione (105, 106) by a mechanism involv-ing the downregulation of -glutamylcysteine synthetase (GCS)RNA in alveolar epithelial cells (39, 106). In addition, oxidativestress has been shown to activate TGF-.

Studies in vitro show that the lipid peroxidation product




Figure 12. Histone deacetylase(HDAC) activity (left) and im-munoreactivity (right) in alveolarmacrophages from smokers andnonsmokers. Modified by permis-sion from Reference 90.

4-HNE is present in increased amounts in lung tissue in patientswith COPD (36). 4-HNE increases TGF- expression by amech-anism dependent on the activation of macrophage AP-1 (38).Furthermore, 4-HNE induces GCS in alveolar epithelial cells(39), and increased GCS expression has been demonstrated inthe lungs of patients with COPD (107). Thus, it has been sug-gested that 4-HNE can act as a second messenger in the regula-tion of protective antioxidant genes, such as GCS, and a varietyof other genes, such as TGF- (107).

Thus, as a response to oxidative stress induced by cigarettesmoke, there appears to be upregulation of protective antioxi-dant genes. Glutathione is concentrated in epithelial lining fluidcompared with plasma (108). Human studies have shown thatglutathione is elevated in the epithelial lining fluid in chroniccigarette smokers compared with nonsmokers, an increase thatdoes not occur during acute cigarette smoking (61). The effectsof chronic cigarette smoking can be mimicked by exposure ofairspace epithelial cells to cigarette smoke condensate in vitro.This produces an initial decrease in intracellular glutathione(GSH) with a rebound increase after 24 hours (64, 109). Thiseffect is mimicked by a similar change in glutathione in rat lungsin vivo after exposure to cigarette smoke (109), associated withan increase in the oxidized form of glutathione (GSSG). Theincrease in glutathione after cigarette smoke exposure is causedby transcriptional upregulation of GCSmRNA, the rate-limitingenzyme in GSH synthesis (109). Themechanism of the upregula-tion of GCS mRNA is by the activation by cigarette smoke ofthe redox-sensitive transcription factor AP-1 (108, 110). Theseevents likely account for the increased glutathione levels seenin epithelial lining fluid in chronic cigarette smokers, a protectivemechanism. The injurious effects of cigarette smoke may occurrepeatedly during and immediately after cigarette smoking whenthe lung is depleted of antioxidants, including glutathione.TNF-, which is believed to have a role in the lung inflammationof COPD, also decreases intracellular glutathione levels initiallyin epithelial cells by a mechanism involving intracellular oxida-tive stress, which is followed 24 hours thereafter by a reboundincrease in intracellular glutathione as a result of AP-1 activationand increased GCS expression (108). Animals exposed to wholecigarette smoke for up to 14 days also show increased expressionof a number of antioxidant genes, including manganese super-oxidedismutase,metallothionine, andglutathioneperoxidase(111).

Sensitivity to oxidative stress may also be a cofactor in thedevelopment of a protease/antiprotease imbalance and conse-quent emphysema. Strains of mice that decreased their lung anti-oxidant defenses developed emphysema when exposed chroni-cally to cigarette smoke, whereas emphysema did not developin strains of mice that showed upregulation of antioxidants inresponse to smoke (112).

SYSTEMIC OXIDATIVE STRESS

An increased systemic oxidative burden has been shown to occurin smokers. In COPD, peripheral blood neutrophils have beenshown to release more ROS than in normal subjects and this isenhanced still further in exacerbation, being associated withmarked depletion of the plasma antioxidant capacity, indicatingincreased systemic oxidative stress (Figure 8) (113). Products oflipid peroxidation are also increased in plasma in smokers withCOPD, particularly during exacerbations (113). Increased levelsof nitrotyrosine have also been shown to occur in the plasma ofpatients with COPD (25).

Exacerbations of COPD result from increased levels of airpollutants, specifically particulate air pollution (see article in thisissue by van Eeden and coworkers, pp. 6167) (114). Particulateair pollution causes oxidative stress in the airways (115), andenhanced inflammation by mechanisms similar to those de-scribed previously, through oxidative stressinducedNF-B acti-vation, decreased histone deacetylation, and increased histoneacetylation (72, 86). Air pollution has also been associated withincreased cardiovascular deaths. The mechanism of this effectmay involve oxidative stressinduced changes in fibrinolytic bal-ance, resulting in enhanced plasminogen activator inhibitor type1 and decreased tissue-type plasminogen activator, both of whichwould reduce fibrinolysis of clots forming on ruptured athero-sclerotic plaques and therefore lead to acute cardiac events, suchas myocardial infarction and death (discussed in detail in thearticles in this issue by MacCallum, pp. 3443, and Tapson, pp.7177) (116). Furthermore, COPD is an independent risk factorfor the development of ischemic heart disease and death frommyocardial infarction, as reviewed by Sin and Man in this issue,pp. 811 (117).

It is now recognized that COPD is not only a disease thataffects the lungs but has important systemic consequences, such




Figure 13. Altered glutamatemetabolism associatedwith reducedmus-cle glutathione levels in patients with emphysema. **p 0.01; ***p 0.001. Adapted from Reference 125.

as cachexia of skeletalmuscle function (118). Increasing evidencesuggests that similar mechanisms involving oxidative stress andinflammation in the lungs may also be responsible for many ofthe systemic effects of COPD (5, 118).

Patients with COPD often display weight loss, which is seenas an independent predictor of outcome (119, 120). Loss of fat-free mass also results in peripheral muscle dysfunction, de-creased exercise capacity, and reduced health status (121123).There are several factors that influence the loss of weight andfat-free mass in patients with COPD, including malnutrition,imbalance in overall protein turnover and hormones involvedin this process, tissue hypoxia, and pulmonary inflammation (118,122, 124, 125). The roles of body weight and fat-free mass inthe pathogenesis andmorbidity of COPD are reviewed byWout-ers in this issue (pp. 2633).

The cachexia and loss of fat-free mass that occur in COPDmay involve oxidative stress. Skeletal muscle is exposed continu-ously to changes in the redox environment as occurs duringexercise. There is increased ROS production by the mitochon-drial respiratory chain after exercise in patients with COPD. Ithas been shown that lipid peroxidation products increase inserum during exercise accompanied by an increase in GSSG/GSH ratio (125128). The increased oxidative stress was muchgreater than in healthy individuals. Thus, although redox homeo-stasis seems to be conserved in COPD, it requires only a littlestress to disturb this balance. Skeletal muscle cells adapt tooxidative stress by upregulating antioxidant enzymes, such asmanganese, copper, and zinc superoxide dismutase (SOD), cata-lase, and glutathione peroxidase (129). In patients with COPDsubmitted to a training protocol, GSSG/GSH ratios increased,whereas no such increase was seen in healthy individuals (130).This finding suggests that the adaptor response of skeletal muscleto oxidative stress may be impaired in COPD. There is alsoevidence of disturbed redox homeostasis in the phenotype ofCOPD associated with emphysema, in which glutathione levelsin the vastus lateralis muscle were decreased. This was associatedwith reduced concentrations of glutamate, an important sub-strate in the synthesis of glutamine and glutathione (Figure 13)(131). This suggests that glutathione metabolism may be im-paired in COPD, which is supported further by studies thatdemonstrate a decrease in glutathione peroxidase activity, ele-vated glutathione reductase activity, and increased lipid peroxi-dation indicative of oxidative damage in skeletalmuscle of exper-imental emphysema in hamsters (132).

The mitochondrial electron transport chain may be responsi-ble for the increased ROS production in skeletal muscle duringexercise and the mitochondrial electron transport chain may bestimulated by TNF (133), which is elevated in the circulation in

patients with COPD who lose weight (134). Leukocytes thatinfiltrate muscles in patients with COPD may be another sourceof ROS (135). Moreover, exercise also increases xanthine/xan-thine oxidase activity, another source of ROS (136). Reactivenitrogen species may also contribute to oxidative stress. Induc-ible NO expression in skeletal muscle in response to inflamma-tory cytokines has been reported and depends on NF-B activa-tion (135). Muscle function may be directly compromised byoxidative stress, which has been shown in vitro to decrease con-tractility and ex vivo to increase fragility of muscle to oxidants(136, 137). Proteins in the contractile apparatus of musclemay beoxidized by ROS, critically sulfhydryl residues in the contractileproteins, which may impair force development (138). In additionto impairing muscle contractility and muscle fatigue, oxidativestress may also compromise muscle function directly and inducemuscle atrophy. Muscle mass can be reduced as a result of animbalance in muscle protein metabolism, which has been de-scribed in studies of oxidative stressinduced inhibition of mus-cle-specific protein expression (139, 140). In addition, oxidativestress may result in apoptosis, which has also been described forskeletal monocytes and may contribute to oxidative stressdependent muscle atrophy (141, 142).

In conclusion, there is now considerable evidence of bothlocal and systemic oxidative stress in patients with COPD. Thereis also increasing evidence that oxidative stress is involved inthe pathogenesis of local lung inflammation as well as in systemicphenomena, such as skeletal muscle dysfunction, and perhapseven the increased cardiovascular risk of mortality that resultsfrom COPD.

Conflict of Interest Statement : W.M. has been reimbursed for travel by Glaxo-SmithKline, Zambon, AstraZeneca, Boehringer Ingelheim, Pfizer, and Micromet forattending conferences and has received honoraria from GlaxoSmtihKline, Astra-Zeneca, Zambon, and Pfizer for participating as a speaker in scientific meetings,and serves on advisory boards for GlaxoSmithKline, Pfizer, Almirall, Amgen, Bayer,and Micromet, and serves as a consultant for Pfizer and SMB Pharmaceuticals, andresearch grants to support work carried out in his laboratory come from SMB, Pfizer,Ceremedix, GlaxoSmithKline, Chugi, and Novartis.

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