American Journal of Respiratory and Critical Care Medicine

There has been increasing interest in using pulmonary biomarkers to understand and monitor the inflammation in the respiratory tract of patients with chronic obstructive pulmonary disease (COPD). In this Pulmonary Perspective we discuss the merits of the various approaches by reviewing the current literature on pulmonary biomarkers in COPD and underscore the need for more systematic studies in the future. Bronchial biopsies and bronchoalveolar lavage provide valuable information about inflammatory cells and mediators, but are invasive, so that repeated measurements have to be very limited in assessing any interventions. Induced sputum has provided considerable information about the inflammatory process, including mediators and proteinases in COPD, but selectively samples proximal airways and may not closely reflect distal inflammatory processes. Exhaled gases and breath condensate are noninvasive procedures, so repeated measurements are possible, but for some assays the variability is relatively high. There is relatively little information about how any of these biomarkers relate to other clinical outcomes, such as progression of the disease, severity of disease, clinical subtypes, or response to therapy. More information is also needed about the variability in these measurements. In the future, pulmonary biomarkers may be useful in predicting disease progression, indicating disease instability, and in predicting response to current therapies and novel therapies, many of which are now in development.

A biomarker refers to the measurement of any molecule or material (cells, tissue) that reflects the disease process. In chronic obstructive pulmonary disease (COPD), several types of biomarker have been measured that are related to disease pathophysiology and the inflammatory and destructive process in the lung. Here we consider biomarkers in bronchial biopsies, sputum, bronchoalveolar lavage (BAL), and exhaled breath. A recent review of over 600 published studies suggests that few of these biomarkers have been validated, and there is little information about reproducibility and the relationship to disease development, severity, or progression (1). This metaanalysis covered almost 150,000 patients with COPD, and revealed the poor sensitivity of current biomarkers to define clinical status and quantify the effect of treatment. Only sputum neutrophils and IL-8, as well as serum tumor necrosis factor (TNF)-α and C-reactive protein, showed any trend toward separating different stages of COPD. There is, therefore, clearly a need for more research in this area with repeated measurements in carefully phenotyped patients. With the development of many new drugs that target inflammation in COPD, there is a pressing need to identify reliable biomarkers that may indicate whether an antiinflammatory therapy is likely to have clinical benefit. A major problem is the lack of any gold standard antiinflammatory therapy that is effective in COPD, such as inhaled corticosteroids in asthma, as a yardstick to compare potential therapies.

Many inflammatory cells, mediators, and enzymes are involved in the complex pathophysiology of COPD, so that there are many possible biomarkers to study and there is a high degree of redundancy (2, 3). The pulmonary inflammation in COPD appears to increase with disease progression and increases during exacerbations. It is likely that some biomarkers will prove to be much more useful than others in terms of reproducibility of measurement, ease of assay, relationship to disease severity, and predictability for assessing therapeutic efficacy. Some biomarkers are more easily measurable and reliable than others and are more easily applied in clinical studies, especially when they are multicenter studies involving large numbers of patients. New assays may have greater sensitivity and assay reproducibility, so that this is a constantly evolving field. In addition, many novel biomarkers may be identified in the future by genomic and proteomic analyses of COPD samples.

Although the inflammation in COPD involves predominately lung parenchyma and small airways, bronchial biopsies appear to reflect the cellular abnormalities seen in the peripheral lung (4, 5). Bronchial biopsies have been useful for documenting the structural changes, cellular patterns, and expression of inflammatory proteins in patients with COPD. In stable COPD, there is increased infiltration of macrophages and activated T lymphocytes, particularly of CD8+ T lymphocytes (4, 6), which express IFN-γ, CXCL10 (IFN-γ–inducible 10-kD protein [IP-10]), and IL-9 (7, 8). Moreover, these lymphocytes express chemokine receptors associated with a type-1 response, such as CXCR3, in contrast to lymphocytes in asthma which express chemokine receptors typical of a type-2 response (CCR4) (9) (as shown in Table E1 in the online supplement). There is also a reduction in T cells expressing CCR5 (10). Although a prominent neutrophilia is present in the airway lumen of patients with COPD in stable conditions, it is not observed at the tissue level, except in patients with severe airflow limitation (11). Finally, during exacerbations of the disease, an increased recruitment of eosinophils and neutrophils has been described, which is associated with upregulation of specific chemoattractants, such as CCL5 (regulated on activation, normal T-cell expressed and secreted) and CXCL5 (epithelial neutrophil-activating peptide-78) (1214).

Bronchial biopsies may give some insights into disease pathogenesis. For example, there is increased activation of the transcription factor, nuclear factor–κB (NF-κB), in bronchial epithelial cells of patients with COPD, increasing with disease severity (15). There is also a reduction in histone deacetylase (HDAC) activity and HDAC2 expression in bronchial biopsies of patients with COPD compared with normal smokers and nonsmokers, and this is correlated with a reduction in NF-κB activity and increased expression of inflammatory genes (16). These changes in bronchial biopsies reflect the changes in NF-κB and HDAC found in lung parenchyma.

Several studies have assessed the potential antiinflammatory effects of treatments in bronchial biopsies of patients with COPD (Table E2). These studies usually involve either a baseline biopsy and then a second biopsy after a defined period of treatment, or a single biopsy at the end of active treatment with a biopsy in a parallel group of patients taking placebo therapy. Overall, inhaled corticosteroids seem to have little effect on the airway inflammation typical of COPD, whereas they are able to reduce mast cells, and this effect is associated with a reduction in the number of exacerbations (17, 18). Greater antiinflammatory effects have been obtained after treatment with either a phosphodiesterase-4 inhibitor or with the combination of corticosteroid and a long-acting β2-agonist, but these changes have not been related to functional or clinical improvements (19, 20). However, further studies are required to establish whether the airway inflammation in COPD can be successfully eradicated and whether this would result in a significant clinical improvement.


The main advantage of endobronchial biopsies is that they directly sample airway tissue, maintaining the spatial relationships of structural components that may be important to functional changes (21). At variance with sputum and BAL, bronchial biopsies can provide an assessment of structural components of the airway wall, such as epithelium, basement membrane, vessels, connective tissue deposition, and, sometimes, smooth muscle and submucosal glands. Therefore, biomarkers of structural damage, such as apoptosis or uncontrolled proliferation, can be measured. Moreover, the different inflammatory cell subtypes can be identified by immunostaining in their microenvironment, thus allowing investigation of the interaction between inflammatory and resident cells. Finally, individual structural components can be dissected from the biopsies and studied in isolation, using new techniques recently developed, such as laser microdissection (22).


There are, however, several limitations to bronchial biopsies as an outcome measurement in COPD. Because this is an invasive procedure, it may be difficult to recruit patients, especially in the studies investigating treatment effects, which require two biopsies (pre- and post-treatment). The biopsy of proximal airways may not closely reflect all the pathologic changes present in peripheral airways and lung parenchyma, which are the sites responsible for airflow limitation in COPD. Moreover, it may not be possible to apply this procedure to patients with more severe disease, complicated by cardiac comorbid conditions, and often associated with significant oxygen desaturation and hypercapnia (23). There is also a relatively high variability in baseline measurements of inflammatory cells, which necessitates multiple biopsies. Finally, because studies evaluating the effect of treatment should be designed to provide a power of at least 80%, a large numbers of patients for each treatment group is usually required.

BAL, unlike bronchial biopsies, has the advantage of sampling inflammation in the lung periphery. BAL can generally be safely performed (23), provided careful assessment is performed and guidelines are adhered to. In general, fluid recovery is greater in patients with less extensive emphysema as assessed by diffusion capacity (24). BAL may be performed in the same patients in which bronchial biopsy is performed, thus providing additional and complementary information.

Cellular Composition

The cellular composition in individuals with COPD is predominantly (> 80%) alveolar macrophages, with some neutrophils and T lymphocytes, and some patients having increased numbers of eosinophils (25). In general, percentage of macrophages and neutrophils are significantly higher than those reported in healthy nonsmokers and, frequently, in healthy smokers. Studies investigating individuals with COPD, healthy smokers, and ex-smokers show that, generally, smoking is associated with increased numbers of neutrophils (Table E3). Lymphocytes are generally higher in ex-smokers than in smokers, whether with or without COPD. Moreover, some patients with COPD have higher eosinophil percentages than healthy smokers, a finding that is not consistently shown in the literature. Alveolar macrophages may be separated by adhesion and cultured in vitro for functional studies. Macrophages from patients with COPD behave abnormally in tissue culture, with increased expression of inflammatory proteins, such as TNF-α, IL-8, and matrix metalloproteinase (MMP)-9 (26, 27). Alveolar macrophages also show a reduction in expression and activity of HDAC2, which modulates the expression of inflammatory genes, with progressive reduction associated with increased disease severity (16). The reduction in HDAC2 is associated with increased activation of the NF-κB. In the future, it may be possible to study, in vitro, the effects of treatments on cellular behavior in patients.

Inflammatory Mediators

Several mediators can be measured in BAL fluid. Levels of eosinophil cationic protein, myeloperoxidase, and IL-8 are frequently increased in patients with COPD and in healthy smokers compared with healthy nonsmokers, an observation suggesting that smoking, rather COPD itself, induces the changes (Table E4). Two studies investigated tryptase and histamine levels, and showed that patients with COPD had higher levels of both mediators, suggesting mast cell activation in COPD (28, 29). However, data were not compared with healthy smokers, and thus the increase in mast cell mediators may be completely attributed to smoking itself. This is also suggested by findings that AMP responsiveness decreases after smoking cessation (30). Studies investigating other mediators have not been replicated and are not discussed here.

Proteases and antiproteases are also detectable in BAL fluid. There is an increase in total elastase activity and a decrease in antielastase activity in patients with COPD compared with normal smokers, confirming the imbalance between proteases and antiproteases (31).

Effect of Smoking and Disease Severity

In one study, ex-smokers with COPD had lower mast cell numbers in BAL than ex-smokers without COPD. No other studies have compared smokers and ex-smokers with COPD (32). Only one study has investigated the association between the severity of COPD and BAL inflammation (33), and shows that healthy smoking men with a near-normal FEV1 show signs of inflammation in the lower airways that are related to a decrease in diffusing capacity of carbon monoxide (DlCO) and to emphysematous lesions seen on high-resolution computer tomography. This inflammation seems to be the result of macrophage and neutrophil activation, as assessed by mediators measured in BAL. In contrast, in a healthy population, the number of inflammatory cells did not correlate with lung function decline over a 4-yr follow up. However, higher levels of neutrophil elastase-α1 protease inhibitor complexes in BAL fluid were significantly associated with accelerated decline in FEV1 (34). This also suggests that the number or percentage of cells is not a prerequisite for development or progression of emphysema, but that the activation state of these cells, with accompanying mediator release, is important.

Effects of Interventions

There are few published studies of the effects of treatments on BAL cellular and mediator components (Table E5). Three studies, one open label, and two double blind, assessed the effect of different types of inhaled corticosteroid, for various periods of treatment, on inflammatory cell counts and mediators in BAL. Although numbers of patients involved were small, precluding firm conclusions, these studies suggest that there may be a reduction in percent neutrophils and percent lymphocytes with inhaled corticosteroid treatment; however, long-term studies in larger populations are needed. Some studies have investigated the effects of smoking cessation on BAL composition, showing inconsistent decreases in cell numbers, particularly macrophages (35, 36).


BAL is an invasive procedure, and may cause more discomfort to the patients than bronchial biopsy. It may also cause transient fever (23). The return of fluid is often reduced in patients with COPD, resulting in samples that are inadequate for analysis. Quantification of biomarkers in supernatant is a problem, as there is no satisfactory marker for the dilution of the saline lavage. This is one of the factors that may contribute to the variability in measurements and the necessity for relatively large numbers of patients.

Many patients with COPD produce suitable sputum spontaneously, but spontaneous sputum may contain a high proportion of dead cells (37), which can potentially give misleading cell counts and mediator measurements (38, 39). For this reason, induced sputum has usually been the procedure of choice. It should be recognized that “sputum” obtained after inhaling nebulized hypertonic saline may have a different composition than mucus, and may be more similar to a washing of proximal airways. The procedure is tolerated by patients with FEV1 greater than 30% predicted. However, airflow obstruction is often observed (40, 41) and cannot be totally prevented by premedication with β2-agonists (42).

Cellular Composition

There is an abnormal pattern of inflammatory cells in patients with COPD, with an increase in number of total inflammatory cells in the percentage of neutrophils and, in some patients, eosinophils (the latter predicting a greater response to corticosteroids [43, 44]). CD8+ T cells are increased in induced sputum of patients with COPD (45). An increased number of eosinophils may indicate concomitant asthma, and appears to predict the patients who show a larger bronchodilator response and improvement with corticosteroids (46). There is little information about the reproducibility of differential cell counts in induced sputum of patients with COPD, but there appears to be a reasonably good reproducibility of cell and mediator measurements in long-term repeatability studies (47). Neutrophils have been studied most extensively, and are increased in number compared with matched smokers with normal lung function (48). Several studies have reported the effects of drugs on sputum neutrophils. Most studies have shown no change in inflammatory cells with inhaled or oral corticosteroids (4951); however, a reduction with oral theophylline has been reported (52).

Inflammatory Mediators

Many mediators have been reported to be increased in the supernatant of patients with COPD, and most show a greater increase in COPD than in normal smokers, with a further increase during exacerbations; however, few mediators have been related to disease severity or progression (Table E6). Sputum IL-8 has been studied most extensively, and is increased in patients with COPD compared with smokers, is related to disease severity (FEV1% predicted), and further increased with exacerbations (48, 53, 54). Sputum concentrations are unaffected by corticosteroids, but reduced by theophylline (4951, 55). Increased concentrations of TNF-α and soluble TNF receptors are found in sputum of patients with COPD compared with normal smokers (48, 56). Higher concentrations of inflammatory cytokines, including TNF-α, IL-8, and IL-6, are reported in patients with more severe COPD compared with those with less severe COPD (57). Leptin is detectable in induced sputum of patients with COPD, and is correlated with other inflammatory markers, including TNF-α and C-reactive protein (58).

Increased proteases, including neutrophil elastase (59), MMP-8 and -9 (6062), and MMP-12 (63), have been reported in sputum of patients with COPD. Sputum markers of structural changes in the airways have been difficult to identify. Hyaluronan, a component of extracellular matrix, is found in higher concentration in sputum of patients with COPD than in that of normal smokers and nonsmokers, especially in the patients with the lowest FEV1 values (64). This might indicate increased breakdown of extracellular matrix in COPD.

No differences in the concentrations of the tachykinins, substance P and neurokinin A, were found between patients with COPD, normal smokers, and nonsmokers, although there was a reduction in tachykinins during exacerbations of COPD (65).


Although induced sputum samples are relatively easy to obtain in patients with COPD, and provide much information about inflammatory cells and mediators, there are several problems that need to be addressed. Induced sputum is sampled predominantly from large airways (66), and may not reflect the peripheral inflammation that may be important for clinical outcomes in COPD. Sputum induction with hypertonic saline induces neutrophilic inflammation, which persists for 24 h, so repeated sampling within this period is not possible (66, 67). Solubilization of sputum with dithiothreitol, which disrupts sulphydryl bonds, may alter proteins so that they are not recognized by antibodies (68). This is a particular problem with several cytokines and chemokines. Furthermore, proteases in sputum, particularly in COPD, may degrade certain protein mediators. Recent studies using dialysis to remove dithiothreitol and protease inhibitors show that it is possible to markedly increase the concentrations of several cytokines in induced sputum of patients with COPD (69). More work is needed on long-term reproducibility in patients with COPD, studying the effect and duration of exacerbations, and correlating individual biomarkers with severity and progression.

Measuring biomarkers in the breath is a very attractive approach to monitoring COPD inflammation, as it is noninvasive and makes repeated sampling possible (70, 71). However, there are important issues about reproducibility and sensitivity that need to be addressed before this approach can be recommended as an outcome measurement.

Nitric Oxide

Fraction of nitric oxide (NO) in exhaled air (FeNO) has been extensively investigated in asthma, and has been shown to correlate with airway inflammation and to be reduced by corticosteroid therapy. There are European Respiratory Society and American Thoracic Society recommendations for measuring FeNO (72, 73). The measurement is highly reproducible in normal subjects and in subjects with asthma if careful attention is paid to technique (74). However, in COPD, conventionally measured FeNO is less useful, as the levels are usually normal or only slightly elevated, except during exacerbations (7578). This is likely due to the increase in oxidative stress, resulting in formation of peroxynitrite and then nitrate, so that NO is removed from the gaseous phase. This also explains why FeNO is reduced in normal smokers (79). An increase in FeNO in patients with COPD is correlated with increased numbers of eosinophils, an increased bronchodilator response, and steroid responsiveness, and so may be useful in detecting associated asthma (46).

Recently, the measurement of FeNO has been extended by making measurements of exhaled NO at different flows, so that it is possible to partition airway-derived NO, which is flow-independent, and peripheral NO derived from alveoli and, probably, small airways. Using this technique, it is possible to show that, although airway NO is low or normal in COPD, there is an increase in peripheral NO that is related to disease severity (80). This may reflect the increase in inducible NO synthase in the lung periphery of patients with COPD (81). This peripheral NO may prove to be a useful noninvasive biomarker of COPD inflammation, but further studies on reproducibility, relationship to disease severity, and the effects of treatments are now needed.

Carbon Monoxide

Although it is easy to measure carbon monoxide (CO) in the breath, this has not turned out to be as useful a measurement as FeNO. Exhaled CO is elevated in patients with COPD, but it is also elevated in normal smokers, due to the high CO content in cigarette smoke (78, 82). However, exhaled CO is elevated to a greater extent in COPD than in matched normal smokers, and remains elevated in sustained ex-smokers. However, the signal is small, and the measurement is also confounded by highly variable environmental CO levels and the effects of passive smoking, so further evaluation is not warranted.


Volatile hydrocarbons, such as ethane and pentane, have been detected in exhaled breath, and are biomarkers of lipid peroxidation as a result of oxidative stress. Concentrations of ethane are elevated in patients with COPD and correlated with disease severity (82). Measurement of ethane by gas chromatography–mass spectrometry offline is difficult, so this measurement is unlikely to be useful in clinical trials; however, smaller and more sensitive detectors for hydrocarbons are now in development.

Many mediators have now been detected in exhaled breath condensate (EBC), which has the advantage of being easy to perform and completely noninvasive (83) (Table E7). Several factors affect the measurement, and recommendations have recently been formulated by a European Respiratory Society/American Thoracic Society taskforce (84). A limitation of the technique is the variability of the measurement and the low concentrations of mediators (often close to the limits of detection) measured.

Oxidative/Nitrative Stress

Hydrogen peroxide (H2O2) is increased in EBC of patients with COPD, is further increased during exacerbations (85), and is related to disease severity (86). Exhaled H2O2 is reported to be reproducible in repeated measurements over 3 d (87). 8-Isoprostane is a stable marker of oxidative stress, and is also increased in EBC of patients with COPD. Concentrations of 8-isoprostane are greater in patients with COPD than in normal smokers, are related to disease severity (8891), and are further increased during exacerbations (92). Certain aldehydes resulting from lipid peroxidation are also increased in patients with COPD, but only malondialdehyde is increased in patients with COPD compared with normal smokers (93). Increased nitrative stress in COPD is indicated by increased concentration of nitrite and nitrosothiols in EBC (94).

Inflammatory Mediators

Inflammation is associated with tissue acidification, and there is a decrease in pH in EBC of patients with COPD (95). There is considerable variability in exhaled pH in patients with COPD that is greater than in normal subjects (96), and the lower pH has been ascribed to increased acidity of salivary contaminants (97). There is an increase in the concentration of leukotriene B4 in patients with COPD, which is further increased during exacerbations (92, 98, 99). Increases in prostaglandin E2 and IL-6 have also been reported in patients with COPD (98, 100). It is not yet clear how most of these biomarkers relate to disease severity. Most proteins, including cytokines and enzymes, cannot reliably be measured in EBC. A recent study reported increases in the concentrations of proinflammatory cytokines, including IL-1β, IL-6, and TNF-α, during exacerbations of COPD, but reproducibility was not reported (101). Chemokines cannot be reliably measured in EBC (91).


There is relatively high variability in repeated measurements of EBC biomarkers, and this may relate to the extensive variable dilution that occurs from water vapor during condensation and the low concentrations that may be near the detection limits of the assays used (102). Further work is needed to optimize these measurements and to determine the causes of variability. Correction for the variable dilution is one approach (103). Assays are usually performed using ELISA, and these assays have been validated for some mediators using gas chromatography–mass spectrometry (104, 105).

Many drugs are now in development as potential antiinflammatory therapies for COPD (106). Because no effective antiinflammatory treatments for COPD currently exist, it is not certain how much and how rapidly clinical parameters will change in patients. This makes it important to develop reliable biomarkers to quantify inflammation in patients with COPD and to validate these against some other measure of disease activity and progression. For assessment of antiinflammatory treatments, it is important to identify biomarkers that indicate the efficacy of the drug on components of the inflammatory process before proceeding to large and prolonged clinical trials. Biomarkers can facilitate drug development in a number of ways, such as providing evidence that a drug can reach its target and modify that target in some positive way, identifying criteria for dose selection for phase-2 and phase-3 studies, providing “go-no-go” decisions at early stages of the drug development process, identifying populations that are more likely to benefit from a drug, and predicting safety problems.

There are several types of drugs that can be developed for COPD based on whether the drug is intended to improve airflow obstruction, provide symptom relief, modify or prevent exacerbations, alter disease progression, or modify lung structure. The efficacy endpoints that are currently used in phase-3 studies to support registration of a drug for COPD are based on measures that translate to direct benefit of some aspects of the disease that is clinically meaningful to patients, such as improvement of symptoms, functional capacity, or survival. Examples of such endpoints include pulmonary function tests, tests of exercise capacity (e.g., treadmill or cycle ergometry), activity scales (e.g., Medical Research Council dyspnea score, Borg Scale, Mahler baseline dyspnea index/transitional dyspnea index), health- related quality-of-life instruments (e.g., St. George's Respiratory Questionnaire, Chronic Respiratory Disease Questionnaire), scores based on patient- or physician-reported symptom severity, and death. With the possible exception of a drug that is intended to improve airflow obstruction, the efficacy of which can be relatively easily assessed by measuring FEV1 in short-term studies, drugs of other types will likely require prolonged studies, often extending to many years. These studies become rather risky, expensive endeavors, and this further underscores the need for development of biomarkers.

The biomarkers described elsewhere in this article are, as yet, not sufficiently validated for use as evidence of efficacy in phase-3 studies, or for supporting specific labeling claims. Nevertheless, these biomarkers are reflective of the disease, and have potential use for regulatory purposes. Carefully selected biomarkers, with or without a patient-centered, clinically meaningful endpoint, can be used in early-phase studies, such as proof-of-action or proof-of-concept studies, based on which a rational decision can be made on further development of the drug. Biomarkers can also be used in either early-phase or phase-3 studies to support the drug's putative mode of action. Also, use of the biomarkers in phase-3 studies in conjunction with clinically meaningful endpoints may help validate the use of the biomarker, or even help elevate a biomarker to a surrogate endpoint status.

Many biomarkers of inflammation and oxidative/nitrative stress have now been documented in the airways of patients with COPD using a variety of techniques of differing invasiveness. Bronchial biopsies provide valuable information about inflammatory cells and mediators, as well as the spatial relationships between the inflammatory processes in the airway wall. However, they may not reflect all pathologic changes in the periphery of the lung that appear to be more important in COPD, and the invasiveness of bronchoscopy precludes repeated measurements. BAL may provide more information about peripheral inflammation, but there are problems of quantification of mediators because of variable dilution and the same problems as biopsies in reproducibility. Induced sputum is a valuable procedure, providing information about cells, mediators, and markers of oxidative/nitrative stress, but standardization of the technique is important for reducing the high variability in the biomarkers. The technique probably samples more proximal airways, and so may not reflect the inflammatory process in the lung periphery. Exhaled biomarkers are noninvasive and may be repeated, but are technique dependent and have a relatively high variability. For all of these biomarkers, there is a relative lack of information about how they relate to diseased severity, how reproducible they are, and how they may be affected by concurrent therapies. In addition, there is little information at present about how they relate to other outcome measurements in COPD, such as rate of decline in FEV1, exacerbation frequency, and mortality. There is a need for comparison of all pulmonary biomarkers in patients with COPD with those in smokers without airflow limitation but matched for smoke exposure (pack-years), and also with age-matched, nonsmoking control subjects. The effect of smoking itself is rarely documented, and ex-smokers may have a different profile of biomarkers from that of active smokers. Patients with mixed asthma and COPD, and patients who have COPD without smoking, also need to be characterized.

COPD involves small airway inflammation and fibrosis as well as alveolar destruction. It is not yet clear whether pulmonary biomarkers will be able to discriminate these two pathophysiologic processes. Various indicators of tissue destruction (such as proteases) and of the inflammatory process (cytokines, chemokines, and lipid mediators) have been measured, but these have not yet been correlated with the extent of emphysema or small airway disease.

Clinical Perspectives

Although many pulmonary biomarkers have been described in patients with COPD, their clinical relevance is far from certain. None of the approaches described in this article are in routine use for the diagnosis of COPD, for predicting disease progression, or for predicting response to therapy. However, progress is now being made in asthma, where monitoring sputum eosinophils and FeNO appears to improve control of asthma, and at the same time reduce steroid requirements (107109). Similar studies have not been done in patients with COPD, as these patients do not respond well to corticosteroids. However, measurement of sputum eosinophils and FeNO may be very useful in clinical practice in identifying the patients with COPD who have concomitant asthma, and who may respond better to bronchodilators and inhaled corticosteroids (46, 110). When more effective antiinflammatory treatments become available for patients with COPD, it is possible that inflammatory cells in sputum may be used to monitor the response to treatment, which may be difficult using physiologic parameters that are likely to improve only very slowly. What is of critical importance is to ensure that there are carefully matched control groups (smokers and nonsmokers), and that the patients with COPD are phenotyped in as much detail as possible; ideally, with detailed lung function assessments (including lung volumes and gas transfer), exercise performance, measurement of free fat mass, and high-resolution computer tomography scanning.

Choice of Biomarker

The choice of which pulmonary biomarker is measured will depend on the research question posed or the clinical problem that is being addressed. Bronchial biopsies and BAL provide important information about cellular composition, but cannot be repeated, whereas induced sputum and exhaled markers are repeatable. The biomarkers selected for measurement will depend on the nature of the study. For example, assessment of anti-inflammatory drugs will require the measurement of inflammatory cells and specific inflammatory mediators, whereas assessment of an antioxidant may require measurements of oxidative stress, and that of an antiprotease will require measurement of protease activity. Prediction of steroid responsiveness may be provided by increased FeNO and sputum eosinophils. In the future, it is possible that patterns of pulmonary biomarkers may predict exacerbations, as they do in asthma, and may reflect different mechanisms of exacerbations, discriminating between bacterial, viral, and noninfective mechanisms.

This is a very active field of research, and further studies addressing several of the issues raised in this article are already in progress. Correlation of pulmonary biomarkers with other outcome measures is essential for the future assessment of the inflammatory destructive process and the effects of the new antiinflammatory drugs that are now in development for the treatment of COPD, as well as for understanding how disease mechanisms relate to clinical outcomes.

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133 Rutgers SR, Postma DS, ten Hacken NH, Kauffman HF, Van der Mark TW, Koeter GH, Timens W. Ongoing airway inflammation in patients with COPD who do not currently smoke. Thorax 2000;55:12–18.

134 Metso T, Rytila P, Peterson C, Haahtela T. Granulocyte markers in induced sputum in patients with respiratory disorders and healthy persons obtained by two sputum-processing methods. Respir Med 2001;95:48–55.

135 Chalmers GW, Macleod KJ, Sriram S, Thomson LJ, McSharry C, Stack BH, Thomson NC. Sputum endothelin-1 is increased in cystic fibrosis and chronic obstructive pulmonary disease. Eur Respir J 1999;13:1288–1292.

136 Traves SL, Culpitt S, Russell REK, Barnes PJ, Donnelly LE. Elevated levels of the chemokines GRO- and MCP-1 in sputum samples from COPD patients. Thorax 2002;57:590–595.

137 Beeh KM, Beier J, Koppenhoefer N, Buhl R. Increased glutathione disulfide and nitrosothiols in sputum supernatant of patients with stable COPD. Chest 2004;126:1116–1122.

138 Takanashi S, Hasegawa Y, Kanehira Y, Yamamoto K, Fujimoto K, Satoh K, Okamura K. Interleukin-10 level in sputum is reduced in bronchial asthma, COPD and in smokers. Eur Respir J 1999;14:309–314.

139 Bhowmik A, Seemungal TA, Sapsford RJ, Wedzicha JA. Relation of sputum inflammatory markers to symptoms and lung function changes in COPD exacerbations. Thorax 2000;55:114–120.

140 Kelly MG, Brown V, Martin SL, Ennis M, Elborn JS. Comparison of sputum induction using high-output and low-output ultrasonic nebulizers in normal subjects and patients with COPD. Chest 2002;122:955–959.

141 Silkoff PE, Martin D, Pak J, Westcott JY, Martin RJ. Exhaled nitric oxide correlated with induced sputum findings in COPD. Chest 2001;119:1049–1055.

142 Banerjee D, Khair OA, Honeybourne D. Impact of sputum bacteria on airway inflammation and health status in clinical stable COPD. Eur Respir J 2004;23:685–691.

143 Ferreira IM, Hazari MS, Gutierrez C, Zamel N, Chapman KR. Exhaled nitric oxide and hydrogen peroxide in patients with chronic obstructive pulmonary disease: effects of inhaled beclomethasone. Am J Respir Crit Care Med 2001;164:1012–1015.

144 Carpagnano GE, Kharitonov SA, Foschino-Barbaro MP, Resta O, Gramiccioni E, Barnes PJ. Increased inflammatory markers in the exhaled breath condensate of cigarette smokers. Eur Respir J 2003;21:589–593.

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50. Culpitt SV, Nightingale JA, Barnes PJ. Effect of high dose inhaled steroid on cells, cytokines and proteases in induced sputum in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1999;160:1635–1639.
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67. Nightingale JA, Rogers DF, Barnes PJ. Effect of repeated sputum induction on cell counts in normal volunteers. Thorax 1998;53:87–90.
68. Kelly MM, Keatings V, Leigh R, Peterson C, Shute J, Venge P, Djukanovic R. Analysis of fluid-phase mediators. Eur Respir J Suppl 2002;37:24s–39s.
69. Erin EM, Barnes PJ, Hansel TT. Optimizing sputum methodology. Clin Exp Allergy 2002;32:653–657.
70. Kharitonov SA, Barnes PJ. Exhaled markers of pulmonary disease. Am J Respir Crit Care Med 2001;163:1693–1772.
71. Kharitonov SA, Barnes PJ. Biomarkers of some pulmonary diseases in exhaled breath. Biomarkers 2002;7:1–32.
72. Kharitonov SA, Alving K, Barnes PJ. Exhaled and nasal nitric oxide measurement: recommendations. Eur Respir J 1997;10:1683–1693.
73. ATS/ERS. Recommendations for standardized procedures for the online and offline measurement of exhaled lower respiratory nitric oxide and nasal nitric oxide, 2005. Am J Respir Crit Care Med 2005;171:912–930.
74. Kharitonov SA, Gonio F, Kelly C, Meah S, Barnes PJ. Reproducibility of exhaled nitric oxide measurements in healthy and asthmatic adults and children. Eur Respir J 2003;21:433–438.
75. Maziak W, Loukides S, Culpitt S, Sullivan P, Kharitonov SA, Barnes PJ. Exhaled nitric oxide in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1998;157:998–1002.
76. Rutgers SR, Van der Mark TW, Coers W, Moshage H, Timens W, Kauffman HF, Koeter GH, Postma DS. Markers of nitric oxide metabolism in sputum and exhaled air are not increased in chronic obstructive pulmonary disease. Thorax 1999;54:576–580.
77. Agusti AG, Villaverde JM, Togores B, Bosch M. Serial measurements of exhaled nitric oxide during exacerbations of chronic obstructive pulmonary disease. Eur Respir J 1999;14:523–528.
78. Montuschi P, Kharitonov SA, Barnes PJ. Exhaled carbon monoxide and nitric oxide in COPD. Chest 2001;120:496–501.
79. Kharitonov SA, Robbins RA, Yates D, Keatings V, Barnes PJ. Acute and chronic effects of cigarette smoking on exhaled nitric oxide. Am J Respir Crit Care Med 1995;152:609–612.
80. Brindicci C, Ito K, Resta O, Pride NB, Barnes PJ, Kharitonov SA. Exhaled nitric oxide from lung periphery is increased in COPD. Eur Respir J 2005;26:52–59.
81. Maestrelli P, Paska A, Saetta M, Turato G, Nowicki Y, Monti S, Formichi B, Miniati M, Fabbri LM. Decreased haem oxygenase-1 and increased inducible nitric oxide synthase in the lung of severe COPD patients. Eur Respir J 2003;21:971–976.
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83. Montuschi P, Barnes PJ. Analysis of exhaled breath condensate for monitoring airway inflammation. Trends Pharmacol Sci 2002;23:232–237.
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85. Dekhuijzen PNR, Aben KHH, Dekker I, Aarts LPHJ, Wielders PLM, van Herwarden CLA, Bast A. Increased exhalation of hydrogen peroxide in patients with stable and unstable chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1996;154:813–816.
86. Kostikas K, Papatheodorou G, Psathakis K, Panagou P, Loukides S. Oxidative stress in expired breath condensate of patients with COPD. Chest 2003;124:1373–1380.
87. Gerritsen WB, Asin J, Zanen P, van den Bosch JM, Haas FJ. Markers of inflammation and oxidative stress in exacerbated chronic obstructive pulmonary disease patients. Respir Med 2005;99:84–90.
88. Montuschi P, Collins JV, Ciabattoni G, Lazzeri N, Corradi M, Kharitonov SA, Barnes PJ. Exhaled 8-isoprostane as an in vivo biomarker of lung oxidative stress in patients with COPD and healthy smokers. Am J Respir Crit Care Med 2000;162:1175–1177.
89. Carpagnano GE, Resta O, Foschino-Barbaro MP, Spanevello A, Stefano A, Di Gioia G, Serviddio G, Gramiccioni E. Exhaled Interleukine-6 and 8-isoprostane in chronic obstructive pulmonary disease: effect of carbocysteine lysine salt monohydrate (SCMC-Lys). Eur J Pharmacol 2004;505:169–175.
90. Carpagnano GE, Kharitonov SA, Foschino-Barbaro MP, Resta O, Gramiccioni E, Barnes PJ. Supplementary oxygen in healthy subjects and those with COPD increases oxidative stress and airway inflammation. Thorax 2004;59:1016–1019.
91. Ko FW, Lau CY, Leung TF, Wong GW, Lam CW, Hui DS. Exhaled breath condensate levels of 8-isoprostane, growth related oncogene alpha and monocyte chemoattractant protein-1 in patients with chronic obstructive pulmonary disease. Respir Med 2006;100:630–638.
92. Biernacki WA, Kharitonov SA, Barnes PJ. Increased leukotriene B4 and 8-isoprostane in exhaled breath condensate of patients with exacerbations of COPD. Thorax 2003;58:294–298.
93. Corradi M, Rubinstein I, Andreoli R, Manini P, Caglieri A, Poli D, Alinovi R, Mutti A. Aldehydes in exhaled breath condensate of patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2003;167:1380–1386.
94. Corradi M, Montuschi P, Donnelly LE, Pesci A, Kharitonov SA, Barnes PJ. Increased nitrosothiols in exhaled breath condensate in inflammatory airway diseases. Am J Respir Crit Care Med 2001;163:854–858.
95. Kostikas K, Papatheodorou G, Ganas K, Psathakis K, Panagou P, Loukides S. pH in expired breath condensate of patients with inflammatory airway diseases. Am J Respir Crit Care Med 2002;165:1364–1370.
96. Borrill Z, Starkey C, Vestbo J, Singh D. Reproducibility of exhaled breath condensate pH in chronic obstructive pulmonary disease. Eur Respir J 2005;25:269–274.
97. Effros RM, Casaburi R, Su J, Dunning M, Torday J, Biller J, Shaker R. The effects of volatile salivary acids and bases upon exhaled breath condensate pH. Am J Respir Crit Care Med 2006;173:386–392.
98. Montuschi P, Kharitonov SA, Ciabattoni G, Barnes PJ. Exhaled leukotrienes and prostaglandins in COPD. Thorax 2003;58:585–588.
99. Kostikas K, Gaga M, Papatheodorou G, Karamanis T, Orphanidou D, Loukides S. Leukotriene B4 in exhaled breath condensate and sputum supernatant in patients with COPD and asthma. Chest 2005;127:1553–1559.
100. Bucchioni E, Kharitonov SA, Allegra L, Barnes PJ. High levels of interleukin-6 in the exhaled breath condensate of patients with COPD. Respir Med 2003;97:1299–1302.
101. Gessner C, Scheibe R, Wotzel M, Hammerschmidt S, Kuhn H, Engelmann L, Hoheisel G, Gillissen A, Sack U, Wirtz H. Exhaled breath condensate cytokine patterns in chronic obstructive pulmonary disease. Respir Med 2005;99:1229–1240.
102. Effros RM, Su J, Casaburi R, Shaker R, Biller J, Dunning M. Utility of exhaled breath condensates in chronic obstructive pulmonary disease: a critical review. Curr Opin Pulm Med 2005;11:135–139.
103. Effros RM, Peterson B, Casaburi R, Su J, Dunning M, Torday J, Biller J, Shakir R. Epithelial lining fluid concentrations in chronic obstructive lung disease patients and normal subjects. J Appl Physiol 2005;99:1286–1292.
104. Cap P, Chladek J, Pehal F, Maly M, Petru V, Barnes PJ, Montuschi P. Gas chromatography/mass spectrometry analysis of exhaled leukotrienes in asthmatic patients. Thorax 2004;59:465–470.
105. Montuschi P, Ragazzoni E, Valente S, Corbo G, Mondino C, Ciappi G, Barnes PJ, Ciabattoni G. Validation of leukotriene B4 measurements in exhaled breath condensate. Inflamm Res 2003;52:69–73.
106. Barnes PJ, Hansel TT. Prospects for new drugs for chronic obstructive pulmonary disease. Lancet 2004;364:985–996.
107. Green RH, Brightling CE, McKenna S, Hargadon B, Parker D, Bradding P, Wardlaw AJ, Pavord ID. Asthma exacerbations and sputum eosinophil counts: a randomised controlled trial. Lancet 2002;360:1715–1721.
108. Smith AD, Cowan JO, Brassett KP, Herbison GP, Taylor DR. Use of exhaled nitric oxide measurements to guide treatment in chronic asthma. N Engl J Med 2005;352:2163–2173.
109. Pijnenburg MW, Bakker EM, Hop WC, de Jongste JC. Titrating steroids on exhaled nitric oxide in children with asthma: a randomized controlled trial. Am J Respir Crit Care Med 2005;172:831–836.
110. Fabbri LM, Romagnoli M, Corbetta L, Casoni G, Busljetic K, Turato G, Ligabue G, Ciaccia A, Saetta M, Papi A. Differences in airway inflammation in patients with fixed airflow obstruction due to asthma or chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2003;167:418–424.
Correspondence and requests for reprints should be addressed to Professor Peter J. Barnes, National Heart and Lung Institute, Imperial College London, Dovehouse Street, London SW3 6LY, UK. E-mail:


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