Proc Am Thorac Soc 2005.2:50-60.

Pulmonary and Systemic Oxidant/Antioxidant

Imbalance in Chronic Obstructive Pulmonary Disease
William 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 to

play a role in the pathogenesis of chronic obstructive pulmonary
disease (COPD). There is considerable evidence that an increased
oxidative burden occurs in the lungs of patients with this disorder,
and this may be involved in many of the pathogenic processes,
such as direct injury to lung cells, mucus hypersecretion, inactivation
of antiproteases, and enhancing lung inflammation through activation of redox-sensitive transcription factors. COPD is now recognized to have multiple systemic consequences, such as weight loss
and skeletal muscle dysfunction. Moreover, it is appreciated that
oxidative stress extends beyond the lung and may, through similar
oxidative stress mechanisms as those in the lung, contribute to
several of the systemic manifestations in COPD.
Keywords: chronic obstructive pulmonary disease; oxidant/antioxidant
imbalance; oxidative stress; smoking; systemic inflammation

The lungs are exposed continuously to oxidants generated either

endogenously from phagocytes and other cell types or exogenously from air pollutants or cigarette smoke. In addition, intracellular oxidants, such as those derived from mitochondrial electron transport, are involved in many cellular signaling pathways.
Lung cells are protected against this oxidative challenge by welldeveloped enzymatic and nonenzymatic antioxidant systems (1).
When the balance between oxidants and antioxidants shifts in
favor of the former, from either an excess of oxidants and/or
depletion of antioxidants, oxidative stress occurs. Oxidative stress
produces not only direct injurious effects in the lungs but also
activates molecular mechanisms that initiate lung inflammation (2).
Smoking is the main etiologic factor in chronic obstructive
pulmonary disease (COPD). Cigarette smoke contains around
1017 oxidant molecules per puff, and this, together with a large
body of evidence demonstrating increased oxidative stress in
smokers with and without COPD, has led to the proposed role
of oxidant/antioxidant imbalance in the pathogenesis of this condition (3). Increasingly, COPD is recognized to affect not only
the lungs but also to have significant systemic consequences,
such as muscle dysfunction and weight loss (4). Oxidative stress
is also believed to play an important role in the systemic manifestations of COPD (5).

HOW IS OXIDATIVE STRESS MEASURED?

Oxidative stress can be measured in several different ways, either
by direct measurements of the oxidative burden, as the responses
to oxidative stress, or by the effects of oxidative stress on target
molecules (Table 1). Direct measurements of the oxidative burden in airspaces can be derived from measurement of hydrogen

(Received in original form November 11, 2004; accepted in final form December 21, 2004)
Correspondence and requests for reprints should be addressed to W. MacNee, M.B.
Ch.B., M.D., ELEGI, Colt Research Laboratories, Wilkie Building, Medical School,
Teviot Place, Edinburgh EH8 9AG, Scotland, UK. E-mail: w.macnee@ed.ac.uk
Proc Am Thorac Soc Vol 2. pp 5060, 2005
DOI: 10.1513/pats.200411-056SF
Internet address: www.atsjournals.org

peroxide (H2O2) in bronchoalveolar lavage (BAL) fluid or in

exhaled breath condensate. Airspace leukocytes derived from
BAL can also be assessed ex vivo for their ability to produce
reactive oxygen species (ROS). Nitric oxide (NO) is produced
in the lungs by the catalytic activity of NO synthase (NOS),
which exists in both constitutive isoforms (cNOS) and an inducible isoform (iNOS) (6). The latter is induced by inflammatory
stimuli, and NO in exhaled breath is considered a marker of
inflammation and indirectly as a marker of oxidative stress.
Measurements of the responses to oxidative stress can be
obtained by assessing changes in antioxidants in BAL fluid or
in induced sputum. The depletion of antioxidants or upregulation
of antioxidant enzymes can be assessed in lung tissue (7). Perhaps
more important than the presence of oxidative stress are measurements of the effects of oxidative stress on target molecules.
Oxidative stress renders proteins more susceptible to proteolytic
degradation by modifying amino acid chains to form protein
aggregates and cleaving peptide bonds (8). As part of this process, some amino acid residues are converted to carbonyl residues. Exposure of human plasma to cigarette smoke in vitro
results in depletion of plasma protein sulfhydryl groups and
elevation of protein carbonyl levels (9). Plasma proteins can also
be degraded through nitration and oxidation by reactive nitrogen
species, the formation of which is stimulated by cigarette smoking (10).
Oxidized proteins can be measured as protein carbonyls, nitrotyrosine, or oxidative damage to DNA producing 8-hydroxydeoxyguanine. The reaction of NO and superoxide anion produces peroxynitrite (Figure 1), which can then cause the nitration
of tyrosine to produce nitrotyrosine, a marker of peroxynitrite
that can be measured in blood, breath condensate, BAL, and
lung tissue as an indicator of free-radical protein damage (11).
NO may form stable nitrosothiols with low-molecular-weight
thiols, such as glutathione, to enhance its bioactivity (12). Nitrite
is a further end-product of NO.
Lipid peroxidation is a process of abstraction of a proton
from a side chain of a fatty acid to produce a carbon-centered
radical, which itself can then react with the fatty acid to produce a
lipid peroxidation product and a further carbon-centered radical,
which can react again, resulting in a chain reaction (13). Lipid
peroxidation products, such as F2-isoprostanes, 4-hydroxynonenal (4-HNE), and hydrocarbons, can be measured in breath,
breath condensate, BAL, and lung tissue.

EVIDENCE OF LOCAL OXIDATIVE STRESS

IN THE LUNGS
Numerous studies have shown that oxidative stress is increased
in the lungs of patients with COPD compared with healthy
subjects, but also compared with smokers with similar smoking
history but who have not developed airways obstruction (3).
Smokers and patients with COPD have higher levels of H2O2 in
exhaled breath condensate, a direct measurement of airspace
oxidative burden, than ex-smokers with COPD or nonsmokers
(14, 15). H2O2 levels are even higher during exacerbations of
COPD (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 fluid
BAL/peripheral blood leukocyte reactive oxygen species production
Nitric oxide in exhaled breath
Responses to oxidative stress
Carbon monoxide in breath (reflecting hemoxygenase activity)
Antioxidants, antioxidant enzymes in blood, sputum, BAL, and lung tissue
Effects of oxidative stress on target molecules
Oxidized 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.

breath of smokers is believed to derive partly from increased release

of superoxide anion (O2) by alveolar macrophages (16, 17).
Because the iron content of smokers alveolar macrophages
is also increased compared with that of nonsmokers (18), the
resultant increased free iron in the airspaces of smokers would
stimulate 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 increased
in cell-free BAL fluid and in the plasma of smokers and in
patients with COPD compared with healthy subjects and nonsmoking subjects, respectively (19, 20).
NO has been used as a marker of airway inflammation and
indirectly as a measure of oxidative stress. Increased NO levels
in exhaled breath occurs in some studies of COPD but are not as
high as the NO levels reported in asthma (2123). Other studies
have found either normal or even lower exhaled NO in patients
with 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 levels
have been shown to be higher in breath condensate in smokers
and patients with COPD compared with subjects who do not
smoke (Figure 3) (6). Smoking increases directly exhaled NO
levels, which limits the usefulness of this marker in COPD.
Peroxynitrite, formed by the reaction of NO with superoxide
anion, can cause nitrosylation of tyrosine to produce nitrotyrosine (11). Nitrotyrosine levels are elevated in sputum leukocytes
of patients with COPD (Figure 3) and are correlated negatively
with the FEV1 (26).
Exhaled carbon monoxide (CO) as a measure of the response
of heme oxygenase to oxidative stress (Figure 4) has been shown
to be elevated in exhaled breath in patients with COPD com-

Figure 1. Nitric oxide (NO) and NO-related products. FAD flavin

adenine dinucleotide; FMN flavin mononucleotide; NADP nicotinamide adenine dinucleotide phosphate; NADPH reduced form of
NADP; S-GSNO S-nitrosoglutathione. Adapted from Reference 6.

Figure 2. Individual data for hydrogen peroxide (H2O2) concentrations

in breath condensate in control subjects and in patients with stable and
unstable chronic obstructive pulmonary disease (COPD). Modified from
Reference 14 by permission.

pared with normal subjects (Figure 5) (27). Again, CO is present

in cigarette smoke, which limits the usefulness of this marker
of oxidative stress in patients who smoke.
Lipid peroxidation products, such as thiobarbituric acidreacting substances, are elevated in sputum in patients with COPD,
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). The
levels of 8-isoprostane in breath condensate have been shown
to be elevated in patients with COPD compared with normal
subjects and smokers who have not developed the disease, and
correlate with the degree of obstruction (31). The levels of
8-isoprostane in breath condensate in patients with COPD also
show a positive correlation with the percentage of neutrophils
in induced sputum, suggesting a role for oxidative stress in the
airway inflammation (Figure 6) (32). Isoprostanes may also reflect systemic effects caused by ROS. In plasma, the levels of
free F2-isoprostanes are increased in smokers and decreased
after smoking cessation (33). In urine, the levels of F2-isoprostanes are elevated in patients with COPD in comparison to
healthy control subjects, with the highest levels observed during
exacerbations (34).
Lipid peroxides can interact with enzymatic or nonenzymatic
antioxidants or decompose by reacting with metal ions or iron-

Figure 3. Left: nitrosothiols, breath condensate in smokers and patients

with COPD. Right: increased inducible NO (iNOS) and nitrotyrosine
immunoreactivity in sputum leukocytes in COPD. *p 0.05. Modified
by permission from Reference 26.

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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 unsaturated aldehydes (13). Hydrocarbons are by-products of fatty
acid peroxidation (27). Patients with COPD show an increased
level 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 oxidative
stress are also reflected in lung tissue in patients with COPD.
Increased nitrotyrosine immunoreactivity has been shown in
sputum leukocytes and lung tissue in patients with COPD compared with healthy subjects (26). The lipid peroxidation product
4-HNE reacts quickly with cellular proteins to form adducts.
These adducts have been shown to be present in greater quantities in airway epithelial and endothelial cells in the lungs of
patients with COPD, compared with smokers with a similar
smoking history who have not developed the disease (Figure 7)
(36). Lipid peroxidation products, such as 8-isoprostane, can
act as signaling molecules and cause release of inflammatory
mediators, 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 oxidative stress in exhaled breath or breath condensate and in lung
tissue in patients with COPD compared with normal subjects
and smokers who have not developed the disease. In addition,
many of these markers correlate with the degree of airflow limitation in COPD, suggesting a role for oxidative stress in the decline
in lung function in COPD. Furthermore, products of oxidative
stress, such as lipid peroxides, can act as signaling molecules to
enhance inflammation in COPD.

ANTIOXIDANTS AND COPD

Several studies have investigated a relationship between antioxidants, pulmonary function, and the development of COPD
(40, 41). The National Health and Nutrition Examination Survey
(NHANES) and the Dutch Monitoring Project for the Risk
Factors for Chronic Diseases (MORGEN) suggested relationships between dietary intake and airflow limitation. In NHANES
I, lower dietary vitamin C related directly to lower FEV1 levels
in a population survey study and, in addition, the protective
effect of vitamin C was even greater in subjects with bronchitis
(40). Data from NHANES II showed an inverse relationship
between both dietary and serum vitamin C and chronic respiratory symptoms (41). Moreover, in NHANES III, the levels of
dietary vitamin C, vitamin E, selenium, and beta-carotene were
positively associated with lung function (42). Analysis of the
data obtained in the MORGEN study also shows higher intake
of vitamin C and beta-carotene is associated with a higher level
FEV1, compared with a low intake of these antioxidants (43). The
association between dietary vitamin E intake and lung function is
less consistent (4345). Several studies, including the MORGEN

Figure 5. Exhaled CO in patients with COPD with and without corticosteroid therapy. ns not significant. Modified by permission from
Reference 27.

study, have shown associations between fruit intake and higher

FEV1 and lower symptoms in patients with COPD (4649). Data
from the MORGEN study have also shown the beneficial effects
of flavonoids on FEV1 (50).

IS OXIDATIVE STRESS IMPORTANT IN THE

PATHOGENESIS OF COPD?
Biomarkers of oxidative stress have been related to the development of airflow limitation, and many studies have shown higher
oxidant levels in patients with COPD, compared with healthy
smokers. Furthermore, several studies show relationships between oxidative stress markers and the degree of airflow limitation in COPD (3, 5, 51). However, the presence of oxidative
stress and its relationship to airflow limitation may be epiphenomena, because it occurs in inflammation, a characteristic feature
of COPD (10). There are as yet no longitudinal studies showing
that the presence of enhanced oxidative stress relates to the
decline in FEV1 or to the progression of the disease.

OXIDATIVE STRESS AND

PROTEASE/ANTIPROTEASE IMBALANCE
An increased protease burden in the lungs occurs as a result of
the influx and activation of inflammatory leukocytes. It has been
proposed that a relative deficiency of antiproteases, such as
1-antitrypsin, because of their inactivation by oxidants, creates
a protease/antiprotease imbalance in the lungs. This forms the

Figure 6. Isoprostane levels in exhaled breath condensate (EBC) in

smokers with COPD (left). Significant correlation between 8-isoprostane
levels in EBC and percentage of neutrophils in induced sputum (right).
Adapted from Reference 32.

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Figure 7. Immunostaining for lipid

peroxidation product 4-hydroxy2-nonenal (4-HNE) adduct in the
lungs of smokers with and without
COPD. (A ) Immunohistochemistry
showing increased staining in both
bronchial and alveolar epithelium
in patients with COPD who have
the same smoking history as a
group of smokers without COPD.
(B ) Staining score showing increased staining in COPD. (C ) Correlation between 4-HNE staining
score in the lungs and percent predicted FEV1. Modified by permission from Reference 36.

basis of the protease/antiprotease theory of the pathogenesis of

emphysema (52, 53). Inactivation of 1-antitrypsin occurs by
oxidation of a critical methionine residue at its active site by
oxidants from cigarette smoke or released from inflammatory
leukocytes, resulting in a dramatic reduction in its inhibitory
capacity in vitro (54, 55). The acute effects of cigarette smoke
on functional activity of 1-antitrypsin have been studied in vivo,
and show a transient, but nonsignificant, fall in the antiprotease
activity of BAL fluid 1 hour after smoking (56). However, a
protease/antiprotease imbalance involving 1-antitrypsin and
neutrophil elastase is likely an oversimplification, because other
proteases and other antiproteases are likely to have a role in
the pathogenesis of COPD.

OXIDANTS AND MUCUS HYPERSECRETION

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

OXIDATIVE STRESS AND AIRSPACE EPITHELIAL INJURY

Because of their direct contact with the environment, the lung
epithelial surfaces are especially vulnerable to the effects of
oxidative stress. Injury to the airway epithelium is an important
early event after exposure to cigarette smoke as shown by an
increase in airspace epithelial permeability. This increased permeability can be shown to result from cigarette smoke exposure

both in vitro and in vivo (6163) and is partially reversible by

antioxidants. Extra- and intracellular glutathione appears to be
critical to the maintenance of epithelial integrity after exposure
to smoke. This has been shown in studies of the permeability
of epithelial cell monolayers in vitro and in animal models in vivo
after exposure to cigarette smoke condensate, which is associated
with profound changes in the homeostasis of the antioxidant glutathione (6366). Depletion of lung glutathione alone can induce
increased airspace epithelial permeability (63, 66). Interindividual
variability in antioxidant defenses may be one factor in determining whether COPD develops after cigarette smoking.

OXIDATIVE STRESS AND NEUTROPHIL INFLUX

IN THE LUNGS
The lungs contain a large pool of noncirculating (marginated)
neutrophils, which either are retained or move only slowly within
the pulmonary microcirculation (67). This is because of the
smaller average diameter of the neutrophil ( 7 m) compared
with the pulmonary capillary diameter ( 5 m), and results in
a proportion of the circulating neutrophils having to deform and
thus move slowly to negotiate the small capillaries. In normal
subjects, there is a correlation between neutrophil deformability,
measured in vitro, and their subsequent sequestration in the
pulmonary microcirculation after reinjectionthe less deformable the cells, the greater their sequestration (68). This mechanism of neutrophil sequestration in the pulmonary bed allows
time to interact and adhere to capillary endothelium before their
subsequent migration across the alveolarcapillary membrane
to the interstitial airspaces. During smoking, there is a transient
increase in neutrophil sequestration in the lungs (69) because
of a decrease in circulating neutrophil deformability (67, 70).
Studies in vitro show that the decreased neutrophil deformability
induced by cigarette smoke is abolished by antioxidants (71).

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Figure 8. Oxidative stress in exacerbations of COPD. Superoxide anion release from peripheral blood neutrophils (PMN;
spontaneous and phorbol myristate acetate [PMA] activated)
in normal subjects and in patients with acute and stable
COPD (left). Plasma antioxidant capacity (Trolox equivalent
antioxidant capacity [TEAC]) in normal subjects, and in those
with stable and exacerbated COPD (right). ROS reactive
oxygen species. *p 0.05; **p 0.01. Adapted from Reference 108.

Oxidative stress occurs systemically during smoking as shown

by a profound decrease in the antioxidant capacity of plasma
(Figure 8), which may reduce neutrophil deformability because
of actin polymerization (71). These may be the initial events
that cause the influx of neutrophils into the lungs of cigarette
smokers, and variability in this effect (69, 70) may be one reason
why not all smokers develop enhanced lung inflammation and
therefore COPD. It is possible that activation of neutrophils
sequestered in the pulmonary microvasculature could also induce the release of ROS and proteases within a microenvironment, which limits access for free-radical scavengers and antiproteases. Thus, destruction of the alveolar wall, as occurs in
emphysema, could result from a proteolytic/oxidant insult derived from the intravascular space without the need for the
neutrophils to migrate into the airspaces.

OXIDATIVE STRESS AND LUNG INFLAMMATION

As mentioned previously, oxidative stress is present wherever
there is inflammation. Regardless, oxidative stress may also be
a mechanism for enhancing the airspace inflammation that is a
characteristic feature of COPD (10). Oxidative stress can cause
the release of chemotactic factors, such as IL-8, from airway
epithelial cells (72), and epithelial cells from patients with COPD
release more IL-8 than those of smokers or healthy individuals
(73). Numerous markers of inflammation, such as IL-8 and tumor
necrosis factor-, have been shown to be elevated in the sputum
of patients with COPD (74), and there is overwhelming evidence
that COPD is associated with enhanced airway inflammation as
confirmed by biopsy studies (75). Recent studies have also shown
a relationship between markers of oxidative stress in breath and
the number of neutrophils in induced sputum (32). Epidemiologic studies have shown a relationship between circulating neutrophil numbers and FEV1 (76) and between the changes in
peripheral blood neutrophil numbers and airflow limitation over
time (77). There is also a relationship between peripheral blood
neutrophil oxidant release and measures of airflow limitation in
young smokers (78). In addition, lipid peroxidation products in
plasma have also been shown to correlate inversely with the
percent predicted FEV1 in a population study (79).
Oxidative stress may have a fundamental role enhancing inflammation through the upregulation of redox-sensitive transcription factors, such as nuclear factor B (NF-B) and activating protein 1 (AP-1), and also the extracellular signal-regulated
kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogenactivated protein kinase pathways. Cigarette smoke has been
shown 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 its

inhibitory protein IB. Many stimuli, including cytokines and
oxidants, result in activation of IB kinase, resulting in phosphorylation of IB, cleaving of IB from NF-B, and the destruction
of IB in the proteasome. This critical event in the inflammatory
response is redox-sensitive (83, 84). Studies in macrophage cell
lines and alveolar and bronchial epithelial cells show that oxidants cause the release of inflammatory mediators, such as IL-8,
IL-1, and NO, and that these events are associated with increased
expression of the genes for these inflammatory mediators and
increased nuclear binding or activation of NF-B (85, 86). The
linking of NF-B to its consensus site in the nucleus leads to
enhanced transcription of proinflammatory genes and therefore
inflammation, which itself will produce more oxidative stress,
creating a vicious circle of enhanced inflammation resulting from
the increased oxidative stress (Figure 9). Animal models of
smoke exposure show that neutrophil influx in the lungs is associated with increased IL-8 gene expression and protein release
and with NF-B activation (87). All of these events are associated
with oxidative stress because they can be abrogated by antioxidant therapy (Figure 10). NF-B can be activated and translocated to the nucleus in lung tissue in smokers and in patients with
COPD compared with healthy subjects (88). NF-B activation in
lung tissue has been shown to correlate with the FEV1 (89).
A further event induced by oxidative stress, which may enhance lung inflammation, is chromatin remodeling. Under normal circumstances, DNA is wound tightly around a core of histones, and in this configuration prevents excessive transcription
by factors such as NF-B, as well as reduced access of RNA
polymerase to DNA, thereby resulting in transcriptional repression and gene silencing (Figure 11). Under the influence of histone acetyltransferases, histone residues are acetylated, causing
a change in their charge and the unwinding of DNA. This then
allows access for transcription factors such as NF-B and/or RNA
polymerase to the transcriptional machinery, therefore enhancing gene expression. This process is reversed by histone deacetylases (HDAC), enzymes that deacetylate histone residues, resulting in the rewinding of DNA and gene silencing. These
processes are known to be redox-sensitive. Histone acetylation
can be shown to occur after cigarette smoke exposure of epithelial 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 shown
to result in increased acetylated histone in lung and decreased
HDAC activity, and both of these events would enhance gene
expression (91). HDAC activity in alveolar macrophages, specifically HDAC2 activity, has also been shown to be downregulated

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

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

in alveolar macrophages obtained from cigarette smokers, a process that would enhance gene expression (Figure 12) (92). Recent
studies suggest that acetylated histone residues, specifically histone
4 (H4), are present to a greater extent in smokers and in patients
with COPD who smoke, with an associated decrease in HDAC2,
specifically in patients with COPD who smoke and in patients
with severe COPD (88). Acetylated histone 3 appears to be
elevated in lung tissue in ex-smoking patients with COPD and
may be a mechanism for persistent inflammation in COPD after
smoking cessation (88). A correlation has also been shown between decreased HDAC activity in lung tissue and FEV1 in
patients with COPD (88, 93).
Many of the markers of oxidative stress do not respond to
therapy with corticosteroids, and it is believed that oxidative
stress may be involved in the relative resistance to these drugs
in COPD. HDAC recruitment is believed to be required for
the antiinflammatory action of corticosteroids in smokers and in
patients with COPD. A mechanism involving nitration of HDACs,
and therefore downregulation of HDACs in lung tissue, may
prevent the action of corticosteroids (94).
Recent evidence suggests that latent adenoviral infection may
be associated with the pathogenesis of COPD by enhancing lung
inflammation (95). E1A protein derived from latent adenoviral

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

infection interacts with transcription factor cofactors and enhances nuclear binding of transcription factors (95). E1A has
been noted to be more prevalent in the lungs of smokers who
develop COPD than in smokers who do not develop the disease
(96). Cells transfected with the E1A protein have also been
shown to have increased IL-8 release in response to oxidants,
such as H2O2, and enhanced NF-B activation (97). Latent adenoviral infection may, therefore, render lung cells more susceptible to oxidative stress, thus enhancing the release of proinflammatory mediators. The presence of E1A also enhances smokeinduced inflammation and emphysema in animal models (98).
The pathogenesis of emphysema has been proposed recently
to result from loss of alveolar endothelial cells via apoptosis,
and this may be as an initial event in the development of emphysema (99). Apoptosis occurs to a greater extent in emphysematous lungs than in lungs of nonsmokers (100). The process of
endothelial apoptosis is believed to be under the influence of
vascular endothelial growth factor (VEGF) R2 receptors. Downregulation of VEGF-R2 has been shown in animals to produce
emphysema (100), and reduced expression of VEGF-R2 is evident in emphysematous human lungs (100). Studies have also
shown that the apoptosis/emphysema produced by VEGF inhibition in animal models is associated with increased markers
of oxidative stress and prevented by antioxidants (101).
Ablation in mice of nuclear factor, erythroid-derived 2, like
2 (Nrf2), a redox-sensitive transcription factor that is involved
in the regulation of many antioxidant genes results in more
extensive cigarette smokeinduced emphysema than occurs in
wild-type mice (102), and this was associated with increased
markers of oxidative stress and increased apoptosis in the lungs,
suggesting a role for the Nrf2 pathway in determining the susceptibility to tobacco smokeinduced emphysema through a mechanism involving upregulation of antioxidant defenses.
Other events involving oxidative stress and epithelial cells
may be relevant to the development of COPD. For example,
TGF-1 expression is increased in small airway epithelial cells
of smokers compared with nonsmokers and increased even more
in patients with COPD (101, 102). TGF- mRNA levels correlated positively with smoking history and the degree of airway
obstruction (103, 104).
Furthermore, TGF-1 itself may increase oxidative stress because this substance produces profound changes in endothelial
and epithelial cell glutathione (105, 106) by a mechanism involving the downregulation of -glutamylcysteine synthetase (GCS)
RNA in alveolar epithelial cells (39, 106). In addition, oxidative
stress has been shown to activate TGF-.
Studies in vitro show that the lipid peroxidation product

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Figure 12. Histone deacetylase

(HDAC) activity (left) and immunoreactivity (right) in alveolar
macrophages from smokers and
nonsmokers. Modified by permission from Reference 90.

4-HNE is present in increased amounts in lung tissue in patients

with COPD (36). 4-HNE increases TGF- expression by a mechanism 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 in
the lungs of patients with COPD (107). Thus, it has been suggested that 4-HNE can act as a second messenger in the regulation of protective antioxidant genes, such as GCS, and a variety
of other genes, such as TGF- (107).
Thus, as a response to oxidative stress induced by cigarette
smoke, there appears to be upregulation of protective antioxidant genes. Glutathione is concentrated in epithelial lining fluid
compared with plasma (108). Human studies have shown that
glutathione is elevated in the epithelial lining fluid in chronic
cigarette smokers compared with nonsmokers, an increase that
does not occur during acute cigarette smoking (61). The effects
of chronic cigarette smoking can be mimicked by exposure of
airspace 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). This
effect is mimicked by a similar change in glutathione in rat lungs
in vivo after exposure to cigarette smoke (109), associated with
an increase in the oxidized form of glutathione (GSSG). The
increase in glutathione after cigarette smoke exposure is caused
by transcriptional upregulation of GCS mRNA, the rate-limiting
enzyme in GSH synthesis (109). The mechanism of the upregulation of GCS mRNA is by the activation by cigarette smoke of
the redox-sensitive transcription factor AP-1 (108, 110). These
events likely account for the increased glutathione levels seen
in epithelial lining fluid in chronic cigarette smokers, a protective
mechanism. The injurious effects of cigarette smoke may occur
repeatedly during and immediately after cigarette smoking when
the lung is depleted of antioxidants, including glutathione.
TNF-, which is believed to have a role in the lung inflammation
of COPD, also decreases intracellular glutathione levels initially
in epithelial cells by a mechanism involving intracellular oxidative stress, which is followed 24 hours thereafter by a rebound
increase in intracellular glutathione as a result of AP-1 activation
and increased GCS expression (108). Animals exposed to whole
cigarette smoke for up to 14 days also show increased expression
of a number of antioxidant genes, including manganese superoxide dismutase, metallothionine, and glutathione peroxidase (111).

Sensitivity to oxidative stress may also be a cofactor in the

development of a protease/antiprotease imbalance and consequent emphysema. Strains of mice that decreased their lung antioxidant defenses developed emphysema when exposed chronically to cigarette smoke, whereas emphysema did not develop
in strains of mice that showed upregulation of antioxidants in
response to smoke (112).

SYSTEMIC OXIDATIVE STRESS

An increased systemic oxidative burden has been shown to occur
in smokers. In COPD, peripheral blood neutrophils have been
shown to release more ROS than in normal subjects and this is
enhanced still further in exacerbation, being associated with
marked depletion of the plasma antioxidant capacity, indicating
increased systemic oxidative stress (Figure 8) (113). Products of
lipid peroxidation are also increased in plasma in smokers with
COPD, particularly during exacerbations (113). Increased levels
of nitrotyrosine have also been shown to occur in the plasma of
patients with COPD (25).
Exacerbations of COPD result from increased levels of air
pollutants, specifically particulate air pollution (see article in this
issue by van Eeden and coworkers, pp. 6167) (114). Particulate
air pollution causes oxidative stress in the airways (115), and
enhanced inflammation by mechanisms similar to those described previously, through oxidative stressinduced NF-B activation, decreased histone deacetylation, and increased histone
acetylation (72, 86). Air pollution has also been associated with
increased cardiovascular deaths. The mechanism of this effect
may involve oxidative stressinduced changes in fibrinolytic balance, resulting in enhanced plasminogen activator inhibitor type
1 and decreased tissue-type plasminogen activator, both of which
would reduce fibrinolysis of clots forming on ruptured atherosclerotic plaques and therefore lead to acute cardiac events, such
as myocardial infarction and death (discussed in detail in the
articles in this issue by MacCallum, pp. 3443, and Tapson, pp.
7177) (116). Furthermore, COPD is an independent risk factor
for the development of ischemic heart disease and death from
myocardial infarction, as reviewed by Sin and Man in this issue,
pp. 811 (117).
It is now recognized that COPD is not only a disease that
affects the lungs but has important systemic consequences, such

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Proc Am Thorac Soc 2005.2:50-60.

MacNee: Systemic Oxidative Stress in COPD

Figure 13. Altered glutamate metabolism associated with reduced muscle glutathione levels in patients with emphysema. **p 0.01; ***p
0.001. Adapted from Reference 125.

as cachexia of skeletal muscle function (118). Increasing evidence

suggests that similar mechanisms involving oxidative stress and
inflammation in the lungs may also be responsible for many of
the systemic effects of COPD (5, 118).
Patients with COPD often display weight loss, which is seen
as an independent predictor of outcome (119, 120). Loss of fatfree mass also results in peripheral muscle dysfunction, decreased exercise capacity, and reduced health status (121123).
There are several factors that influence the loss of weight and
fat-free mass in patients with COPD, including malnutrition,
imbalance in overall protein turnover and hormones involved
in this process, tissue hypoxia, and pulmonary inflammation (118,
122, 124, 125). The roles of body weight and fat-free mass in
the pathogenesis and morbidity of COPD are reviewed by Wouters in this issue (pp. 2633).
The cachexia and loss of fat-free mass that occur in COPD
may involve oxidative stress. Skeletal muscle is exposed continuously to changes in the redox environment as occurs during
exercise. There is increased ROS production by the mitochondrial respiratory chain after exercise in patients with COPD. It
has been shown that lipid peroxidation products increase in
serum during exercise accompanied by an increase in GSSG/
GSH ratio (125128). The increased oxidative stress was much
greater than in healthy individuals. Thus, although redox homeostasis seems to be conserved in COPD, it requires only a little
stress to disturb this balance. Skeletal muscle cells adapt to
oxidative stress by upregulating antioxidant enzymes, such as
manganese, copper, and zinc superoxide dismutase (SOD), catalase, and glutathione peroxidase (129). In patients with COPD
submitted 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 muscle
to oxidative stress may be impaired in COPD. There is also
evidence of disturbed redox homeostasis in the phenotype of
COPD associated with emphysema, in which glutathione levels
in the vastus lateralis muscle were decreased. This was associated
with reduced concentrations of glutamate, an important substrate in the synthesis of glutamine and glutathione (Figure 13)
(131). This suggests that glutathione metabolism may be impaired in COPD, which is supported further by studies that
demonstrate a decrease in glutathione peroxidase activity, elevated glutathione reductase activity, and increased lipid peroxidation indicative of oxidative damage in skeletal muscle of experimental emphysema in hamsters (132).
The mitochondrial electron transport chain may be responsible for the increased ROS production in skeletal muscle during
exercise and the mitochondrial electron transport chain may be
stimulated by TNF (133), which is elevated in the circulation in

57

patients with COPD who lose weight (134). Leukocytes that

infiltrate muscles in patients with COPD may be another source
of ROS (135). Moreover, exercise also increases xanthine/xanthine oxidase activity, another source of ROS (136). Reactive
nitrogen species may also contribute to oxidative stress. Inducible NO expression in skeletal muscle in response to inflammatory cytokines has been reported and depends on NF-B activation (135). Muscle function may be directly compromised by
oxidative stress, which has been shown in vitro to decrease contractility and ex vivo to increase fragility of muscle to oxidants
(136, 137). Proteins in the contractile apparatus of muscle may be
oxidized by ROS, critically sulfhydryl residues in the contractile
proteins, which may impair force development (138). In addition
to impairing muscle contractility and muscle fatigue, oxidative
stress may also compromise muscle function directly and induce
muscle atrophy. Muscle mass can be reduced as a result of an
imbalance in muscle protein metabolism, which has been described in studies of oxidative stressinduced inhibition of muscle-specific protein expression (139, 140). In addition, oxidative
stress may result in apoptosis, which has also been described for
skeletal monocytes and may contribute to oxidative stress
dependent muscle atrophy (141, 142).
In conclusion, there is now considerable evidence of both
local and systemic oxidative stress in patients with COPD. There
is also increasing evidence that oxidative stress is involved in
the pathogenesis of local lung inflammation as well as in systemic
phenomena, such as skeletal muscle dysfunction, and perhaps
even the increased cardiovascular risk of mortality that results
from COPD.
Conflict of Interest Statement : W.M. has been reimbursed for travel by GlaxoSmithKline, Zambon, AstraZeneca, Boehringer Ingelheim, Pfizer, and Micromet for
attending conferences and has received honoraria from GlaxoSmtihKline, AstraZeneca, 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, and
research grants to support work carried out in his laboratory come from SMB, Pfizer,
Ceremedix, GlaxoSmithKline, Chugi, and Novartis.

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