Ken M. Kunisaki, Kathryn L. Rice, Edward N. Janoff, Thomas S. Rector and Dennis E. Niewoehner

Original Research Exhaled nitric oxide, systemic inflammation, and the spirometric response to inhaled fluticasone propionate in severe chronic obstructive pulmonary disease: A prospective study Therapeutic Advances in Respiratory Disease (2008) 2(2) 55 64 DOI: 10.1177/ 1753465808088902 SAGE Publications 2008 Los Angeles, London, New Delhi and Singapore Ken M. Kunisaki, Kathryn L. Rice, Edward N. Janoff, Thomas S. Rector and Dennis E. Niewoehner Abstract Background: A subset of patients with chronic obstructive pulmonary disease (COPD) may respond more favorably to inhaled corticosteroids (ICS), but no simple method is currently utilized to predict the presence or absence of ICS responses in patients with COPD. We evaluated the ability of exhaled nitric oxide (F E NO) and serum inflammatory markers (C-reactive protein [CRP], interleukin-6 [IL-6], and interleukin-8 [IL-8]) to independently predict spirometric responses to ICS in patients with COPD. Methods: Among 60 ex-smokers with severe COPD (mean FEV 1 1.07 L, 36% of predicted), we conducted a single-arm, open-label study. Participants spent four weeks free of any ICS, followed by four weeks of ICS use (fluticasone propionate 500 mcg twice daily). F E NO, CRP, IL-6, IL-8, and pre-bronchodilator spirometry were measured immediately before and after the four weeks of ICS use. Results: Baseline F E NO, CRP, IL-6, and IL-8 showed no correlations to FEV 1 responses to ICS. ICS responders (increase in FEV 1 200 ml after four weeks of ICS) did have significantly higher baseline F E NO levels compared with non-responders (46.5 parts per billion [ppb] vs. 25 ppb, p = 0.028). The receiver operating characteristic curve for F E NO to discriminate responders from non-responders had an area under curve of 0.72. Baseline serum inflammatory markers did not differ between responders and non-responders. Conclusion: In ex-smokers with severe COPD, a measure of local pulmonary inflammation, F E NO, may be more closely associated with FEV 1 responses to four weeks of ICS than are standard markers of systemic inflammation, serum CRP, IL-6, and IL-8 Keywords: Androstadienes / tu (therapeutic use), anti-inflammatory agents / tu (therapeutic use), breath tests, C-reactive protein, inflammation / bl (blood), interleukin-6 / bl (blood), interleukin-8 / bl (blood), nitric oxide / du (diagnostic use), pulmonary disease, chronic obstructive Abbreviations ATS = American Thoracic Society AUC = area under curve COPD = chronic obstructive pulmonary disease CRP = C-reactive protein F E NO = fraction of exhaled nitric oxide FEV 1 = forced expiratory volume in one second FVC = forced vital capacity ICS = inhaled corticosteroid IL-6 = interleukin-6 IL-8 = interluekin-8 IQR = interquartile range ppb = parts per billion ROC = receiver operating characteristic Introduction Patients with chronic obstructive pulmonary disease (COPD) exhibit persistent abnormal pulmonary inflammation. When compared to persons without COPD, patients with COPD have increased numbers of inflammatory cells in bronchoalveolar lavage and lung biopsy samples [Baraldo et al. 2004; Hogg et al. 2004; Turato et al. 2002], elevated cytokine levels in Correspondence to: Ken M. Kunisaki, MD Division of Pulmonary, Allergy, Critical Care and Sleep, University of Minnesota, USA. Minneapolis Veterans Affairs Medical Center, Pulmonary, 111N, One Veterans Drive, Minneapolis, MN 55417, kunis001@umn.edu Kathryn L. Rice, MD Dennis E. Niewoehner, MD Pulmonary Section, Minneapolis Veterans Affairs Medical Center, USA, Division of Pulmonary, Allergy, Critical Care and Sleep Medicine, University of Minnesota, USA Edward N. Janoff, MD Division of Infectious Diseases, Colorado Center for AIDS Research, University of Colorado Health Sciences Center, USA Thomas S. Rector, PharmD, PhD Center for Chronic Disease Outcomes Research and Center for Epidemiological and Clinical Research, Minneapolis Veterans Affairs Medical Center, USA, Department of Medicine, University of Minnesota, USA http://tar.sagepub.com 55

induced sputum [Vernooy et al. 2002; Keatings et al. 1996], and elevated fractions of nitric oxide (F E NO) in exhaled breath [Montuschi et al. 2001; Corradi et al. 1999; Kanazawa et al. 1998; Maziak et al. 1998]. Many patients with COPD also show evidence of systemic inflammation, as evidenced by elevated serum levels of proinflammatory cytokines and C-reactive protein (CRP) [Mannino et al. 2003; Dentener et al. 2001; Eid et al. 2001; Mendall et al. 2000; Takabatake et al. 2000; Schols et al. 1996]. One study in COPD patients also showed that inhaled corticosteroid (ICS) therapy for two weeks lowered serum CRP levels by 50% [Sin et al. 2004].While this potentially could have resulted from systemic absorption of ICS, it also potentially suggests that the systemic inflammation seen in COPD may be caused by local pulmonary inflammation. The presence of pulmonary inflammation serves as the principal rationale for ICS therapy in COPD patients. Several large trials failed to demonstrate that ICS slow lung function decline over time in COPD patients [Burge et al. 2000; Lung Health Study Research Group, 2000; Pauwels et al. 1999; Vestbo et al. 1999; Paggiaro et al. 1998]. Secondary outcomes indicated some reduction in the number and severity of exacerbations and some improvement in overall health status. The use of ICS therapy in COPD is currently recommended for patients with severe COPD, particularly in patients with frequent exacerbations [Rabe et al. 2007]. However, the clinical benefits are modest, the drugs are costly, and concerns persist about long-term safety. Responses to ICS in COPD patients are not uniform. A simple and reliable method for predicting responsiveness to ICS (or lack thereof) in COPD patients could have useful clinical applications, as it might allow targeted therapy to patients most likely to benefit, while limiting exposure to those unlikely to derive benefit. In one small study (n = 30), ICS were more likely to improve lung function in COPD patients with a positive inhaled mannitol challenge, but these results have not been confirmed and the test is somewhat complex [Leuppi et al. 2005]. The presence of sputum eosinophilia may also predict greater spirometric responses to both inhaled and systemic steroids [Brightling et al. 2005; Brightling et al. 2000; Pizzichini et al. 1998], but the technique requires sputum induction and specialized training to perform reliably. In contrast, measurement of F E NO is noninvasive, simple to perform in a clinic setting, and provides instant results. In patients with asthma, F E NO appears to correlate with sputum eosinophilia [Berry et al. 2005; Jatakanon et al. 1998], and higher levels are associated with better responses to both inhaled [Szefler et al. 2002] and oral corticosteroids [Little et al. 2000]. In addition, if systemic inflammation does reflect lung inflammation in COPD, then measurement of inflammatory markers in serum may also predict subsequent ICS responses. Therefore, we determined the ability of F E NO, as well as serum CRP, interkeukin-6 (IL-6), and interleukin-8 (IL-8), to independently predict spirometric responses to ICS in patients with severe COPD. Methods and materials Subjects: The study was performed in accordance with recommendations in the Helsinki Declaration of 1975 [World Medical Association, 1997]. The institutional review board of the Minneapolis Veterans Affairs Medical Center approved this study. All subjects provided written informed consent. Subjects were recruited from the Minneapolis Veterans Affairs Medical Center between May 2005 and February 2006. Inclusion criteria were: (1) a clinical diagnosis of COPD, with a FEV 1 /FVC ratio <70%, and FEV 1 <60% of predicted; (2) age >45 years; (3) cigarette smoking history of >10 pack-years; (4) abstinence from cigarette smoking of at least 6 months, as active smoking can suppress F E NO levels [Robbins et al. 1996; Kharitonov et al. 1995]; (5) stable clinical status, as evidenced by the lack of hospitalizations, urgent care visits, antibiotics, or changes in medications within four weeks prior to enrollment; and (6) ability to provide informed consent. Exclusion criteria were: (1) a clinical diagnosis of asthma; (2) leukotriene inhibitor use; (3) severe or uncompensated heart failure; (4) the presence of conditions known to elevate CRP levels (such as collagen vascular disease and chronic infection); (5) malignancy requiring active treatment with chemotherapy or radiation therapy, or any co-morbidity making survival greater than one year unlikely; (6) women who were pregnant or lactating; (7) oral corticosteroid use within four weeks prior to enrollment; and (8) participation in another investigational trial within four weeks of enrollment and for the duration of this study. 56 http://tar.sagepub.com

Original Research Study design: Our primary goal was to determine if correlations existed between potential predictor variables and within-subject changes in spirometry. Because analyses of correlations do not require a control group, we designed this study as a single-arm, open-label study. Subjects who met study criteria and agreed to participate entered a four-week run-in period. During the run-in, subjects were treated with salmeterol, 50 micrograms inhalation twice daily (Serevent Diskus, GlaxoSmithKline, Research Triangle Park, NC). The use of ICS was not allowed during the run-in. Tiotropium use was not allowed for the duration of the study. Subjects were allowed to continue use of all other respiratory medications, including short-acting beta agonists and ipratropium. After the run-in, subjects returned to the study center for baseline measurements of F E NO and pre-bronchodilator spirometry. All visits were in the morning to minimize diurnal variation in measures and subjects were fasting as food intake rich in L-arginine or nitrates may affect F E NO results for several hours after intake [Olin et al. 2001; Kharitonov et al. 1995]. Subjects withheld use of angiotensin-converting enzyme inhibitors on mornings of study visits, as this class of medication has also been reported to affect F E NO measurements [Sumino et al. 2000]. Before each study visit, subjects withheld use of short-acting bronchodilators for 6 hours and withheld use of salmeterol for 12 hours. Blood samples were collected for assessment of baseline serum inflammatory markers CRP, IL-6, and IL-8. For the next four weeks, subjects were treated twice daily with 500 micrograms of fluticasone propionate and 50 micrograms of salmeterol (Advair Diskus 500/50, GlaxoSmithKline, Research Triangle Park, NC, USA). After four weeks, subjects returned to the study center for repeat measurement of F E NO, pre-bronchodilator spirometry, and serum inflammatory marker blood sample collection. Protocols: F E NO was measured online (realtime) with a chemiluminescence device (Sievers NOA 280i, GE Analytical Instruments, Boulder, CO). Two-point nitric oxide calibration was performed daily, flow calibration was performed weekly, and F E NO measurements were performed at a single expiratory flow rate of 50 ml/second. Published American Thoracic Society (ATS) recommendations were followed for F E NO test procedures and interpretation [American Thoracic Society & European Respiratory Society, 2005]. Spirometry was performed after F E NO and in accordance to ATS standards [American Thoracic Society, 1995] (MicroLab 3500, MicroMedical, Kent, UK). Technicians performing spirometry were blinded to subjects F E NO results. Third National Health and Nutrition Examination Survey spirometric reference values were used as reference values [Hankinson et al. 1999]. Serum blood samples were allowed to clot at room temperature, centrifuged, and frozen at 80 C in aliquots. All blood samples were analyzed in paired (pre-ics and post-ics) fashion. Serum high-sensitivity CRP was measured by immunoturbidimetry (Kit#474630, Beckman Coulter, Inc., Brea, CA). Serum IL-6 and IL-8 were measured by chemiluminescent enzyme-linked immunosorbent assay (Q6000B and Q8000B kits, R&D Systems, Minneapolis, MN). All values were calculated from standard curves with control samples on each plate to assure reproducibility of the assays. The analytic sensitivities of CRP, IL-6, and IL-8 were 0.2 mg/l, 0.16 pg/ml, and 0.28 pg/ml, respectively. Laboratory technicians were blinded to F E NO and spirometry results. Statistical methods: The primary outcomes of interest were correlations between potential predictor variables (F E NO, CRP, IL-6, and IL-8) and the outcome variable pre-bronchodilator change in FEV 1 from baseline to after four weeks of ICS. The study was powered (alpha of 0.05 and beta of 0.20) for potential predictor variables and the change in FEV 1 after four weeks of ICS to show a correlation coefficient of 0.35. This resulted in a sample size calculation of 62 patients. We consented 78 subjects, allowing for 20% of consented subjects to either fail spirometry screening or not complete the full protocol. Because the data were non-normally distributed, analyses were conducted with non-parametric statistical tests. For the primary correlation analyses, Spearman s rank-correlation testing (reported as Spearman s rho) was used. Withinsubject changes in FEV 1, FVC, F E NO, CRP, IL-6, and IL-8 were analyzed using Wilcoxon signed-rank testing. http://tar.sagepub.com 57

For post-hoc analyses, we dichotomized subjects into responders and non-responders to ICS, using a FEV 1 improvement of 200 ml after four weeks of ICS to define responders. There is no consensus on a meaningful FEV 1 response to ICS in COPD. We thus extrapolated from ATS guidelines which require 200 ml improvement in FEV 1 as a component of defining a significant bronchodilator response [American Thoracic Society, 1995]. A similar study in COPD patients dichotomized responders and non-responders to oral prednisolone using a cutoff of 200 ml, as well [Chanez et al. 1997]. Distributions of potential predictor variables among responders and non-responders were compared using the Wilcoxon rank-sum. Receiver operating characteristic (ROC) analyses were also conducted, using FEV 1 improvement of 200 ml after four weeks of ICS as the outcome of interest. ROC curves for potential predictor variables were constructed with calculation of ROC area under curve (AUC). All statistical analyses were performed using Stata 8.2 (StataCorp LP, College Station, TX, USA). Results Enrollment and subject flow 76 subjects consented to study participation. Of these, 73 met spirometry criteria for enrollment, and 60 subjects completed the full protocol and were thus analyzed (Figure 1). 2 subjects were unable to perform F E NO to ATS standards, but both provided serum samples and completed all other study procedures, and were thus not excluded from study participation. Subjects who withdrew from the study were similar in COPD severity, but were more likely to have been prescribed antibiotics and prednisone in the previous 12 months, and were more likely to have received Consented = 76 Enrolled = 73 Completed visit 2 = 62 Completed visit 3 = 60 Failed screening = 3 Withdrawn from study = 11 Exacerbations = 6 Withdrawal of consent = 5 Withdrawn from study = 2 Exacerbation = 1 Withdrawal of consent = 1 Figure 1. Diagram of study subject flow. more inhaled medications, including inhaled corticosteroids, before study participation (Table 1). Baseline values Baseline (pre-ics) F E NO did not significantly correlate with any of the serum inflammatory markers (CRP, IL-6, and IL-8), nor with FEV 1 or FVC (Table 2). Statistically significant correlations were present between baseline CRP and IL-6 and between baseline IL-6 and IL-8, though the strength of these correlations was only modest (Spearman s rho = 0.46 and 0.31, respectively). Spirometry and biomarker responses to ICS Use of ICS for four weeks was associated with statistically significant changes in prebronchodilator spirometry (Table 3). 11 of 60 subjects (18%) experienced an increase in FEV 1 of 200 ml and were thus classified as responders to ICS. After four weeks of ICS therapy, F E NO significantly decreased, whereas none of the systemic inflammatory markers showed a significant change (Table 3). Relationships between F E NO and FEV 1 responses to ICS For the primary correlation analysis, baseline F E NO did not correlate with the change in FEV 1 after four weeks of ICS (Spearman s rho = 0.04, p = 0.75) (Figure 2). Post-hoc comparison of ICS responders and nonresponders demonstrated a significant difference in baseline F E NO levels between responders and non-responders (46.5 ppb vs. 25 ppb, p = 0.028) (Figure 3). For the ROC analysis, the AUC was 0.72 (95% CI: 0.53 to 0.91) (Figure 4). The ROC curve did not identify an ideal cut-point for both optimal sensitivity and specificity. For example, evaluating 60 ppb as a cut-point yielded a specificity of 94% of 48 non-responders to ICS, 45 had a F E NO <60 ppb. Sensitivity, however, was only 50% of 10 responders to ICS, 5 had a F E NO 60 ppb. The positive predictive value of 60 ppb was 63%. The negative predictive value of <60 ppb was 88%. Relationships between serum inflammatory markers and FEV 1 responses to ICS We found no statistically significant relationships between the serum inflammatory markers 58 http://tar.sagepub.com

Original Research Table 1. Characteristics of study sample at time of enrollment. Data are presented as mean ± standard deviation for continuous variables or as number (%) for categorical variables. FEV 1 = forced expiratory volume in one second, FVC = forced vital capacity. Completed study Withdrawn from study Baseline characteristics at screening (n = 60) (n = 13) Demographics and smoking history Male 59 (98%) 13 (100%) Female 1 (2%) 0 (0%) Age, years 71 ± 7.3 70 ± 7.9 Smoking history, pack-years 57 ± 30.6 53 ± 26.1 Duration of smoking abstinence, years 13 ± 10 13 ± 9.4 Spirometry Pre-bronchodilator FEV 1 (L) 1.07 ± 0.36 1.09 ± 0.28 Pre-bronchodilator FEV 1 (% predicted) 35.6 ± 10.6 33.0 ± 8.5 Pre-bronchodilator FVC (L) 2.44 ± 0.72 2.54 ± 0.62 Pre-bronchodilator FVC (% predicted) 58.5 ± 14.6 56.8 ± 12.9 Medication use Prescribed one or more courses of 11 (18%) 6 (46%) antibiotics in the 12 months prior to enrollment Prescribed one or more courses of 7 (12%) 6 (46%) prednisone in the 12 months prior to enrollment Prescribed chronic oxygen 10 (16%) 3 (23%) Prescribed albuterol 59 (98%) 12 (92%) Prescribed ipratropium 35 (58%) 11 (84%) Prescribed long-acting beta agonist 30 (50%) 9 (69%) Prescribed long-acting anticholinergic 1 (2%) 0 (0%) Prescribed inhaled corticosteroid 28 (47%) 9 (69%) Prescribed theophylline 2 (3%) 0 (0%) Co-morbid conditions Coronary artery disease 16 (27%) 1 (8%) Heart failure 6 (12%) 1 (8%) Hypertension 36 (60%) 8 (62%) Atrial fibrillation/flutter 5 (8%) 0 (0%) Hyperlipidemia 37 (62%) 8 (62%) Diabetes mellitus 14 (23%) 1 (8%) Peripheral vascular disease 9 (15%) 1 (8%) History of stroke 2 (3%) 0 (0%) Chronic kidney disease 5 (8%) 0 (0%) Obstructive sleep apnea 4 (7%) 0 (0%) Gastroesophageal reflux disease 12 (20%) 1 (8%) Chronic rhinosinusitis 5 (8%) 1 (8%) Table 2. Correlation matrix of baseline F E NO, serum CRP, serum IL-6, serum IL-8, FEV 1, and FVC. Data are presented as Spearman s rho (p-value). F E NO = fraction of exhaled nitric oxide, ppb = parts per billion, CRP = C-reactive protein, IL-6 = interleukin-6, IL-8 = interleukin-8, FEV 1 = forced expiratory volume in one second, FVC = forced vital capacity. F E NO CRP IL-6 IL-8 FEV 1 FVC F E NO 1.00 CRP 0.19 (0.14) 1.00 IL-6 0.09 (0.48) 0.46 (<0.001) 1.00 IL-8 0.08 (0.52) 0.24 (0.06) 0.31 (0.01) 1.00 FEV 1 0.04 (0.76) 0.13 (0.33) 0.07 (0.56) 0.02 (0.87) 1.00 FVC 0.07 (0.60) 0.06 (0.63) 0.05 (0.70) 0.14 (0.29) 0.74 (<0.001) 1.00 http://tar.sagepub.com 59

Table 3. Median change in measures after four weeks of inhaled corticosteroid. Wilcoxon signed rank test used for calculation of p-values. FEV 1 = forced expiratory volume in one second, FVC = forced vital capacity, F E NO = fraction of exhaled nitric oxide, ppb = parts per billion, CRP = C-reactive protein, IL-6 = interleukin-6, IL-8 = interleukin-8. 180 150 p=0.028 Median Interquartile Measurement (units) Change range p-value FEV 1 (ml) 65 20 to 140 0.002 FVC (ml) 200 90 to 340 0.003 F E NO (ppb) 7 17 to 2 <0.001 CRP (mg/l) 0.14 1.12 to 0.83 0.87 IL-6 (pg/ml) 0.30 0.90 to 1.65 0.26 IL-8 (pg/ml) 1.85 5.40 to 5.30 0.52 Baseline FeNo (ppb) 120 90 60 30 0 25.0 Non-responders (n=48) Responders (n=10) 46.5 Change in FEV 1 (L) 0.8 0.6 0.4 0.2 0 0 0.2 50 100 150 200 0.4 Spearman s rho = 0.04 (p=0.75) 0.6 Baseline F E NO (ppb) Figure 3. Boxplots of F E NO distributions in responders and non-responders to four weeks of ICS. Responders were defined as having 200 ml FEV 1 improvement following four weeks of ICS. The median value for each group is presented numerically, with interquartile ranges, ranges, and outliers plotted in boxplot format. Two-sample Wilcoxon rank-sum used for calculation of p-value. F E NO = fraction of exhaled nitric oxide, FEV 1 = forced expiratory volume in one second, ICS = inhaled corticosteroid, ppb = parts per billion. Figure 2. Scatterplot of baseline (pre-ics) F E NO and change in pre-bronchodilator FEV 1 following four weeks of ICS. Horizontal dashed line represents cutoff for dividing subjects into responders ( 200 ml FEV 1 improvement following four weeks of ICS) and non-responders. Spearman s rank-correlation test used for calculation of Spearman s rho and corresponding p-value. F E NO = fraction of exhaled nitric oxide, FEV 1 = forced expiratory volume in one second, ICS = inhaled corticosteroid, ppb = parts per billion. (CRP, IL-6, and IL-8) and changes in FEV 1 following four weeks of ICS when analyzing for correlations, responder/non-responder comparisons, and ROC analyses. Discussion The pre-specified primary correlation analyses in our study were negative, with no overall correlations between the studied predictor variables F E NO, serum CRP, IL-6, and IL-8 and the spirometric response to four weeks of ICS in ex-smokers with severe COPD. Post-hoc analyses showed significant differences in F E NO between responders and nonresponders to ICS, with higher F E NO in responders and lower F E NO in non-responders. Sensitivity 1 0.8 0.6 0.4 0.2 60 ppb 19 ppb AUC = 0.722 (95% CI: 0.531 0.913) 0 0 0.2 0.4 0.6 0.8 1 1-specificity Figure 4. Receiver operating characteristic (ROC) curve for the performance of baseline F E NO to discriminate the presence or absence of a 200 ml FEV 1 improvement following four weeks of inhaled fluticasone propionate. Two F E NO levels (19 ppb and 60 ppb) are labeled with arrows for illustrative purposes. AUC = area under curve, CI = confidence interval, F E NO = fraction of exhaled nitric oxide, FEV 1 = forced expiratory volume in one second, ppb = parts per billion. 60 http://tar.sagepub.com

Original Research Although we identified no optimal F E NO cutpoint to discriminate responders from nonresponders, we found good specificity for a F E NO cut-point of 60 ppb, along with good negative predictive value for a level <60 ppb. We would caution, however, that predictive values are influenced by the prevalence of the condition in the population. Our study sample may have been biased towards non-responders, given the ICS wash-out period. Those patients with previous good clinical responses to ICS may have been either less likely to enroll in this study or more likely to withdraw during the study, as was suggested by the higher percent of previous ICS use in the group that did not complete the study. The availability of F E NO results at the point of care and its ease of measurement are distinct advantages of this test compared with others proposed in the literature, such as sputum eosinophilia and mannitol challenge. Exhaled nitric oxide levels may reflect the presence of local steroid-responsive inflammation in pulmonary airspaces [Smith et al. 2005] and the absence of such inflammation appears to be associated with a lack of spirometric response to ICS therapy. Thus, F E NO testing might potentially provide useful clinical guidance to both focus and limit the use of ICS and their associated costs and complications. Further studies would be required, however, before advocating for ICS treatment decisions in COPD based on F E NO testing. Because of the exploratory nature of our study, we limited treatment duration to only four weeks of ICS and evaluated a surrogate endpoint change in FEV 1 after four weeks of ICS. Studies which might actually influence clinical practice should involve a longer duration of ICS treatment (to mimic current clinical practice) and evaluate clinically relevant endpoints such as acute exacerbations of COPD. Compared with COPD, F E NO has been more extensively studied in patients with asthma, among whom F E NO levels are more commonly elevated. As such, it might be argued that those COPD patients with high F E NO or significant FEV 1 responses to ICS actually have asthma. In this study, we excluded patients who carried a clinical diagnosis of asthma or were being treated with a leukotriene inhibitor, but we did not formally exclude asthma with testing such as methacholine challenge. Nevertheless, our subjects met the global initiative for chronic obstructive lung disease definition of COPD [Rabe et al. 2007] and represent the population for whom clinical decisions about the need for ICS are made. Considerable overlap may exist between asthma and COPD, and choosing the appropriate disease label may be less important than identification of appropriate treatments tailored to the individual patient. Our F E NO findings are consistent with those of Zietkowski et al. [2005]. In a sample of 47 smokers and ex-smokers with severe COPD, they demonstrated a similar decrease in F E NO after use of ICS and an association between baseline F E NO and FEV 1 responses to ICS. We add to their work by having studied a larger sample of ex-smokers (19 ex-smokers in their study vs. 60 in ours, which may be significant given that active smoking is associated with lower F E NO levels), by presenting ROC data, and by also investigating serum inflammatory markers. We demonstrated that serum CRP, IL-6, and IL-8 did not relate to changes in FEV 1 after four weeks of ICS in patients with severe COPD. This suggests that spirometric responses to ICS are associated primarily with local lung inflammation (as measured by F E NO) and not systemic inflammation. One limitation of our study was our lack of data on other markers of inflammation in the airway and lung compartment, such as IL-6 or IL-8 levels in bronchoalveolar lavage, induced sputum, or breath condensate. While such data may have been interesting from a scientific standpoint, our primary intent was to explore tests that are simple and feasible for a busy clinical practice to implement. Thus, we focused on single-flow F E NO and serum analysis as tests meeting these criteria. We did not confirm the work of Sin and colleagues, who demonstrated a 50% decrease in serum CRP following only two weeks of the same dose of inhaled fluticasone [Sin et al. 2004]. Our subjects had substantially more advanced COPD (FEV 1 of 1.07 L vs. 1.67 L, respectively) and were slightly older (71 years vs. 64 years, respectively) than those in Sin and colleagues study. A comparison of the frequency and severity of co-morbidities could not be assessed. In conclusion, we demonstrated that in ex-smokers with severe COPD, neither F E NO nor serum inflammatory markers (CRP, IL-6, and IL-8) correlate to changes in FEV 1 following four weeks of ICS treatment. In post-hoc analysis, however, http://tar.sagepub.com 61

low F E NO was associated with a lack of significant FEV 1 responses to four weeks of ICS. While we identified no ideal F E NO cut-point for discriminating the presence or absence of a FEV 1 response to ICS, this measure of local inflammation may have a stronger relationship with spirometric outcome than do standard markers of systemic inflammation, serum CRP, IL-6, and IL-8. Acknowledgements The study was supported by the CHEST Foundation of the American College of Chest Physicians (Dr. Kunisaki), NIH/NHLBI 2 T32 HL07741 (Dr. Kunisaki), Veterans Affairs Clinical Science and Health Services Research and Development Grants 04S-CRCOE-001 and HFP-98-001 (Dr. Rector), the Veterans Affairs Research Service and NIH AI-48796 (Dr. Janoff). The study was also supported in part by an investigator-initiated research grant from GlaxoSmithKline (Drs. Kunisaki and Niewoehner). Study conception and execution, as well as all study data, were in the exclusive control of the authors. We thank Fran Lebahn and Carrie Beaner for assistance with spirometry, Claudine Fasching for laboratory technical support, and the veterans who made this work possible. Study conducted at the Minneapolis Veterans Affairs Medical Center Potential conflicts of interest Study supported in part by an investigatorinitiated research grant from GlaxoSmithKline (Drs. Kunisaki and Niewoehner). Study conception and execution, as well as all study data, were in the exclusive control of the authors. Dr. Kunisaki has received the grant from GlaxoSmithKline as described above. Dr. Rice has received grants, honoraria, or advisory fees from Boehringer Ingelheim, Pfizer, and sanofi-aventis. Dr. Niewoehner has received grants, honoraria, or advisory fees from GlaxoSmithKline, Boehringer Ingelheim, Pfizer, AstraZeneca, Adams Respiratory Therapeutics, and sanofi-aventis. Drs. Janoff and Rector have no potential conflicts of interest to disclose. The views expressed in this article are those of the authors and do not necessarily represent the views of the University of Minnesota or the U.S. Department of Veterans Affairs. References American Thoracic Society. (1995) Standardization of spirometry, 1994 update. Am J Respir Crit Care Med 152: 1107 1136. American Thoracic Society and European Respiratory Society. (2005) 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 171: 912 930. Baraldo, S., Turato, G., Badin, C., Bazzan, E., Beghe, B., Zuin, R. et al. (2004) Neutrophilic infiltration within the airway smooth muscle in patients with COPD. Thorax 59: 308 312. Berry, M.A., Shaw, D.E., Green, R.H., Brightling, C.E., Wardlaw, A.J. and Pavord, I.D. (2005) The use of exhaled nitric oxide concentration to identify eosinophilic airway inflammation: An observational study in adults with asthma. Clin Exp Allergy 35: 1175 1179. Brightling, C.E., McKenna, S., Hargadon, B., Birring, S., Green, R., Siva, R. et al. (2005) Sputum eosinophilia and the short term response to inhaled mometasone in chronic obstructive pulmonary disease. Thorax 60: 193 198. Brightling, C.E., Monteiro, W., Ward, R., Parker, D., Morgan, M.D., Wardlaw, A.J. et al. (2000) Sputum eosinophilia and short-term response to prednisolone in chronic obstructive pulmonary disease: A randomised controlled trial. Lancet 356: 1480 1485. Burge, P.S., Calverley, P.M., Jones, P.W., Spencer, S., Anderson, J.A. and Maslen, T.K. (2000) Randomised, double blind, placebo controlled study of fluticasone propionate in patients with moderate to severe chronic obstructive pulmonary disease: The ISOLDE trial. BMJ 320: 1297 1303. Chanez, P., Vignola, A.M., O Shaugnessy, T., Enander, I., Li, D., Jeffery, P.K. et al. (1997) Corticosteroid reversibility in COPD is related to features of asthma. Am J Respir Crit Care Med 155: 1529 1534. Corradi, M., Majori, M., Cacciani, G.C., Consigli, G.F., de Munari, E. and Pesci, A. (1999) Increased exhaled nitric oxide in patients with stable chronic obstructive pulmonary disease. Thorax 54: 572 575. Dentener, M.A., Creutzberg, E.C., Schols, A.M., Mantovani, A., van t Veer, C., Buurman, W.A. et al. (2001) Systemic anti-inflammatory mediators in COPD: Increase in soluble interleukin 1 receptor II 62 http://tar.sagepub.com

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