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Exhaled Nitric Oxide Correlated With Induced Sputum Findings in COPD
Exhaled Nitric Oxide Correlated With Induced Sputum Findings in COPD* Philip E. Silkoff, MD; Dan Martin, MD; Juno Pak, BS; Jay Y. Westcott, PhD; and Richard J. Martin, MD, FCCP
Study objectives: Neutrophilic airway inflammation may underlie the pathogenesis of COPD. We examined repeated measurements of the fractional concentration of exhaled nitric oxide (FENO) and the correlation with cells and mediators in induced sputum (IS) from patients with COPD. Participants: Eleven COPD subjects (9 men and 2 women, aged 46 to 69 years) with predicted FEV1 of 45 to 70%. Setting: A hospital research laboratory. Design: Single-cohort, prospective study with four visits at two weekly intervals. Interventions: FENO and spirometry were assessed at all visits, and IS for differential cell count, leukotriene-B4 (LTB4) and interleukin (IL)-8, nitrite, and nitrate at visit 1, visit 3, and visit 4. Results: During the study, there were significant declines in mean percent predicted FEV1, from 55.2 to 51.6% (p ⴝ 0.029), and mean FEV1/FVC ratio, from 50.4 to 45.4% (p ⴝ 0.001), accompanied by a significant increase in FENO geometric mean (95% confidence limits), from 15.2 (10.9 to 21.2) to 23.6 (17.1 to 32.4) parts per billion (p ⴝ 0.037), and sputum LTB4, from 1.79 (1.03 to 3.11) to 3.57 (1.95 to 6.53) ng/mL (p ⴝ 0.033), but no significant change in other sputum parameters. From visits 1 to 4, the change in percent neutrophils correlated with the changes in FENO and IL-8 (r ⴝ 0.648, p ⴝ 0.028; r ⴝ 0.60, p ⴝ 0.05, respectively). Hypertonic saline solution induction of sputum caused a fall in FEV1, from 1.83 ⴞ 0.44 to 1.46 ⴞ 0.44 L (p ⴝ 0.049). Conclusions: The worsening spirometry results were accompanied by significant increases in FENO and sputum LTB4. FENO may be related to neutrophilic inflammation driven by the chemoattractant IL-8. FENO and IS may be useful markers of airway inflammation in COPD patients. Sputum induction with hypertonic saline solution causes a significant fall in FEV1 requiring appropriate caution. (CHEST 2001; 119:1049 –1055) Key words: COPD; inflammation; nitric oxide; sputum Abbreviations: FENO ⫽ fractional concentration of exhaled nitric oxide; IL ⫽ interleukin; iNOS ⫽ inducible form of nitric oxide synthase; IS ⫽ induced sputum; LTB4 ⫽ leukotriene-B4; NO nitric oxide; ppb ⫽ parts per billion
A
irway inflammation, a central feature of asthma,1 is increasingly recognized as an important pathogenic mechanism in COPD.2,3 Airway inflammation has been assessed noninvasively by the analysis of spontaneous or induced sputum (IS),4 the measurement of endogenous exhaled gaseous mediators such as nitric oxide (NO),5 and the measurement of substances such as hydrogen peroxide in breath condensate, the fluid phase of exhaled breath.6 The latter may reflect oxidative stress in the airway. The fractional concentration of exhaled nitric oxide (FENO) has been extensively studied as a non*From the Department of Medicine, National Jewish Medical and Research Center, Denver, CO. Manuscript received June 19, 2000; revision accepted November 28, 2000. Correspondence to: Philip E. Silkoff, MD, National Jewish Medical and Research Center, 1400 Jackson St, Denver, CO 80206; e-mail: [email protected]
invasive marker of airway inflammation, particularly in asthma, where levels are high in nonsteroidtreated subjects and fall rapidly after inhaled corticosteroid treatment.7 There is induction of the inducible form of NO synthase (iNOS) in asthmatic bronchial epithelium8 by inflammatory cytokines such as interferon-␥, tumor necrosis factor-␣, and interleukin (IL)-1.9 In COPD, abnormalities of FENO are less clear-cut than in asthma. Some studies10 suggest that FENO is increased in patients with stable COPD, others11 only in patients with unstable COPD, while some12 report no increase. The discrepancies between studies may be related to differences in FENO measurement techniques. Alternatively, viral infections, which increase FENO in normal subjects,13 may underlie the increases in FENO seen in patients with unstable COPD, while current smoking depresses FENO values.14 Although the airway inflammation and the accompanyCHEST / 119 / 4 / APRIL, 2001
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ing cytokine production are different in COPD from that found in asthma, expression of both constitutive and inducible NO synthase isoforms have been reported to be elevated in macrophages and other cell lines in COPD.15 The analysis of spontaneous sputum or IS in COPD also provides much information regarding the airway cells and mediators in the lower airway inflammatory milieu.16 Many studies17–20 of COPD have examined sputum cell differential and mediators such as IL-8. The influence of medications on sputum cell differential and mediators has also been assessed using IS.19 In this study, we performed serial measurements of FENO together with IS analysis over a 6-week period in a cohort of subjects with moderate-tosevere COPD, to ascertain patient acceptability, safety, correlation, and short-term reproducibility. Materials and Methods Eleven subjects (9 men and 2 women, aged 46 to 69 years) with COPD and FEV1 45 to 70% predicted, carbon monoxide diffusion coefficient ⬍ 70% predicted, and ⬎ 10 pack-years cigarette use were recruited. Current smokers were included (n ⫽ 6). The diagnosis of COPD was confirmed by information available in the subjects’ hospital records. Exclusion factors were other pulmonary diseases, including lung cancer and asthma; atopy; unstable systemic diseases; myocardial infarction or cardiac arrhythmias in preceding 6 months; use of oral steroids or continuous oxygen; and subjects within 6 weeks of enrollment who had an upperrespiratory-tract infection or hospitalization. The use of inhaled corticosteroids (n ⫽ 6) and/or theophylline (n ⫽ 2) was not an exclusion criterion. The study was approved by our institutional review board, and subjects signed an informed consent form. Study Design The study consisted of four visits (visits 1 to 4) every 2 weeks over a 6-week period. FEV1 and FENO were determined at each visit. Induction of sputum with hypertonic saline solution was performed at visit 1, visit 3, and visit 4. Exhaled NO Measurement A rapid-response chemiluminescent NO analyzer (model 280; Sievers Instruments; Boulder, CO) was used for NO analysis. Daily two-point calibrations were performed using a 5.2-parts per million calibration gas. FENO was measured using a previously described restricted breath technique, which employed expiratory resistance and positive mouth pressure to close the velum and exclude nasal NO, and a constant expiratory flow of 45 mL/s.21 The inhaled gas was ambient air that was passed through a filter to reduce inhaled NO concentrations to ⬍ 5 parts per billion (ppb). Subjects inhaled to total lung capacity, and exhaled while targeting a constant pressure of 20 mm Hg. Exhalations proceeded until a clear NO plateau of at least 3-s duration was achieved. Repeated exhalations were performed until three plateaus agreed at the 5% level. Induction of Sputum Induction of sputum was achieved by ultrasonic nebulization of 3% hypertonic saline solution. Spirometry was performed to 1050
establish the baseline FEV1 followed by the administration of four activations of albuterol (90 g per activation) via spacer device. Repeat spirometry was performed after 15 min to establish preinduction baseline FEV1. Subjects then performed tidal breathing while inhaling the hypertonic saline solution aerosol. At 2-min intervals, subjects removed the mouthpiece and spat saliva into a container. Then subjects inhaled twice to total lung capacity and were asked to expectorate into a sputum container. Nasal blowing was encouraged if needed to prevent contamination by nasal secretion. FEV1 was monitored at 2-min intervals. The induction procedure was continued until ⬎ 1 mL of sputum was obtained or FEV1 had fallen by ⬎ 20% from preinduction values. After the induction procedure, two activations of albuterol were administered and the subject remained in the laboratory until FEV1 had returned to ⬎ 90% of baseline. Processing of Sputum Processing of sputum was performed within 1 h of collection according to the method of Fahy et al,22 in which the entire expectorate is processed. Samples were refrigerated until processing. Samples were liquefied to disperse the cells by adding 0.1% dithiothreitol in a fixed proportion to the volume of sputum obtained and then centrifuged to separate supernatant, which was stored at ⫺ 70°C for future analysis. Cytospins for differential cell count were prepared. Assays Sputum supernatant was assayed for the following substances: IL-8, leukotriene-B4 (LTB4), nitrite, and nitrate. Sputum nitrite and nitrate were analyzed (after deproteinization with cold ethanol) by reduction chemiluminesence using sodium iodide in ascetic acid (nitrite) at room temperature, and vanadium chloride at 95°C (nitrate), using a Radical Purger (Sievers Instruments). IL-8 was measured by a sandwich enzyme-linked immunosorbent assay using matched antibody pairs (R&D Systems; Minneapolis, MN). LTB4 was measured by a competitive immunoassay, using reagents (Cayman Chemical; Ann Arbor, MI). Processed sputum was analyzed without purification, and results were extrapolated back to levels in the original sputum volumes. Statistical Methods The following 13 end points were analyzed: FEV1 (percent predicted), FVC (percent predicted), FEV1/FVC ratio (percent), FENO (ppb), sputum total cell count, neutrophil count, eosinophil count, macrophage count, lymphocyte count, LTB4, IL-8, nitrite, and nitrate. Preliminary analysis suggested that data variability was not the same at different visits, for FENO, cell count, eosinophil count, lymphocyte count, IL-8, and LTB4, which were therefore log transformed (log 10) for all statistical analyses. Eosinophil count and lymphocyte count could not be log transformed because some subjects had values of 0. For these two end points, models that required equal variance were not used. The data were summarized using means and SEs; for logtransformed variables, geometric means were used. A mixedeffects model with the best-fitting covariance structure was used to determine if there were differences between the means for each pair of visits for each end point. The mixed-effects model accounts for the repeated measures for subjects across visits. All statistical tests were two sided and conducted using a significance level of 5% (ie, 0.05). Clinical Investigations
Results Eleven subjects completed the study, which took place between September and November 1997. Correlation Between Parameters At baseline, there was a significant correlation between sputum percent neutrophils and sputum IL-8 (r ⫽ 0.727, p ⫽ 0.011, Fig 1), but not LTB4. Additionally, percent predicted FEV1 showed a significant negative correlation with percent sputum neutrophil count (r ⫽ ⫺ 0.63, p ⫽ 0.037; Fig 2). There was no significant correlation between FENO and sputum NO metabolites (nitrite or nitrate) at baseline. Change in Pulmonary Function There was a progressive decline in pulmonary function in the cohort over the period of the study (Fig 3). Comparing visit 1 to visit 4, percent predicted FEV1 fell from 55.2 ⫾ 1.93 to 51.6 ⫾ 1.97 (p ⫽ 0.029), FEV1/FVC percentage ratio fell from 50.4 ⫾ 4.02 to 45.4 ⫾ 3.7 (p ⫽ 0.004), but percent predicted FVC was not significantly changed (93.7 ⫾ 6.86 to 95.1 ⫾ 5.4). Change in FENO
Figure 2. The correlation between sputum percent neutrophil count and FEV1 percent predicted at baseline.
1.18 ⫾ 0.07 to 1.37 ⫾ 0.06 ppb (p ⫽ 0.037). Looking at the change in spirometry and FENO from visit 1 to visit 4, there was a significant correlation between the change in FENO and that of FVC (r ⫽ 0.47, p ⫽ 0.01; Fig 5), but not with FEV1.
Exhaled NO showed a progressive significant rise over the period of the study in the study cohort (Fig 4), but remained within the previously determined range of our laboratory for normal subjects (24.2 ⫾ 12.8 ppb) using the same FENO measurement technique and flow rate. Comparing visit 1 to visit 4, log 10 FENO (mean ⫾ SE) rose from
Change in Sputum Variables
Figure 1. The correlation between sputum percent neutrophil count and sputum IL-8 at baseline.
Figure 3. The decline in FEV1/FVC ratio over the course of the study. The p value compares visit 1 to visit 4.
Comparing visit 1 and visit 4, log 10 LTB4 (mean ⫾ SE) rose significantly from 0.25 ⫾ 0.11 to 0.55 ⫾ 0.12 ng/mL (p ⫽ 0.033; Fig 6). However, total and differential cell count (neutrophils, eosinophils, macrophages, lymphocytes), IL-8, nitrite, and nitrate showed no significant change over the period of the study (Table 1), although there was a significant correlation between the changes in both FENO and sputum IL-8 from visit 1 to visit 4 with the
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Figure 4. The increase in FENO over the course of the study. The p value compares visit 1 to visit 4.
Figure 6. The increase in LTB4 from visit 3 to visit 4. The p value compares visit 1 to visit 4.
change in sputum neutrophils (r ⫽ 0.684, p ⫽ 0.028; r ⫽ 0.60, p ⫽ 0.05, respectively; Fig 7, 8).
Discussion
Acceptability and Safety of Sputum Induction and FENO Measurement All subjects tolerated the sputum induction procedure well. However, sputum induction with hypertonic saline solution was associated with a significant transient fall in FEV1 from 1.83 ⫾ 0.44 to 1.46 ⫾ 0.44 L (p ⫽ 0.049; data from all three inductions combined). The administration of albuterol resulted in return of FEV1 to ⬎ 90% of baseline levels, and no subject reported prolonged exacerbation of COPD as a result of the induction. Exhaled NO measurement was acceptable to all subjects despite significant airflow limitation. There were no complications of the procedure.
Figure 5. The correlation between the changes in FENO and FVC from visit 1 to visit 4. 1052
In this 6-week study, we examined repeated exhaled NO and IS parameters and their correlation in a cohort of subjects with COPD. We are unaware of any published studies in COPD that have looked at the relationship between IS cells and mediators and FENO on a repeated basis. There was a progressive decline in FEV1 and FEV1/FVC ratio over the period accompanied by a significant progressive rise in FENO. Sputum LTB4 also significantly rose from visit 3 to visit 4. Sputum differential cell counts showed no significant change in the group as a whole despite the worsening lung function, but the changes in percent neutrophils and in FVC both correlated with the change in FENO. The level of neutrophilic inflammation correlated positively with IL-8, a product of and chemoattractant for neutrophils, and negatively with FEV1. The decline in pulmonary function occurred during the study period from September to November. This may be related to the known worsening of COPD during the winter, perhaps related in turn to climatic change and/or increased air pollution. The rise in FENO accompanying the declining pulmonary function could signify worsening airway inflammation in this cohort of subjects, or could even be a direct result of increasing air pollution, as ambient air pollution can cause increases in endogenous FENO even in normal subjects.23,24 It is also possible that the sputum induction, performed repeatedly, had some deleterious effect. The increase in FENO could originate from two NO synthase isoforms. There could be induction of the iNOS in intrapulmonary cells, such as the bronchial epithelium or in intraluminal inflammatory cells. This is the currently accepted mechanism underlying the increased FENO in asthma, as reported by Saleh et al,25 where FENO was high and iNOS was expressed in the bronchial epithelium in Clinical Investigations
Table 1—Mean Values of Measured Parameters Over the Study Time Period* Parameters
Visit 1 (Baseline)
Visit 2 (Week 2)
Visit 3 (Week 4)
Visit 4 (Week 6)
FEV1, % predicted FVC, % predicted* FEV1/FVC, % FENO, ppb Total cell count, 106/mL Sputum eosinophils, % Sputum macrophages, % Sputum lymphocytes, % Sputum neutrophils, % IL-8, pg/mL LTB4, pg/mL Sputum nitrite, mol/L Sputum nitrate, mol/L
55.1 ⫾ 1.9 93.7 ⫾ 6.8 50.3 ⫾ 4.0 15.2 (10.9–21.2) 3.2 (1.7–5.8) 2.7 ⫾ 1.3 38.4 ⫾ 6.8 1.1 ⫾ 0.2 51.1 ⫾ 6.6 3.7 (1.9–7.24) 1.8 (1.0–3.1) 5.6 (3.0–11.0) 18.9 (6.4–56.2)
54.5 ⫾ 2.8 92.2 ⫾ 5.3 49.2 ⫾ 4.4 18.6 (12.3–28.1) NM NM NM NM NM NM NM NM NM
54.4 ⫾ 2.5 95.6 ⫾ 5.8 48.1 ⫾ 4.5 19.7 (12.8–30.5) 3.3 (2.0–5.6) 1.5 ⫾ 0.7 34.6 ⫾ 5.7 1.3 ⫾ 0.3 57.1 ⫾ 5.8 2.7 (1.4–5.4) 1.8 (0.9–3.4) 4.1 (2.2–7.4) 34.6 (21.1–56.8)
51.6 ⫾ 1.9† 95.1 ⫾ 5.4 45.4 ⫾ 3.7† 23.6 (17.1–32.4)† 2.9 (1.6–5.1) 4.2 ⫾ 2.2 33.9 ⫾ 6.6 0.8 ⫾ 0.2 52.7 ⫾ 7.5 2.8 (1.4–5.7) 3.6 (2.0–6.5)† 6.02 (3.6–10.1) 26.4 (15.7–44.3)
*Data are expressed as mean ⫾ SEM or geometric mean (95% confidence limits). NM ⫽ not measured. †Significant change compared to baseline.
nonsteroid-treated asthma, but was significantly reduced after inhaled budesonide therapy. However, in asthma, using the same FENO measurement technique as that used in the present study, we have observed much higher FENO levels (in some subjects up to 450 ppb). Perhaps the NO formed in the COPD airway is reacting with other free radicals, such as superoxide, produced by neutrophils and/or undergoing denitrification by bacteria, thereby decreasing the observed levels.26 Indeed, the observed increase in FENO may not be iNOS-related at all. In support of this, Rutgers et al12 did not find increased iNOS expression in COPD sputum macrophages. Alternatively, there may be upregulation of constitutive NO synthase isoforms, such as neuronal NO synthase in airway nonadrenergic noncholinergic inhibitory bronchodilating nerves, which might serve to overcome the worsening airway obstruction.27
Certainly, the airway inflammation in COPD is different from asthma, and perhaps it is unreasonable to expect the same changes in NO formation and disposition. In patients with cystic fibrosis, which has a similar neutrophilic predominance and bacterial colonization as COPD, FENO is in fact lower than in healthy control subjects.28 The relatively low levels of FENO found in COPD patients, together with differing reports as to whether FENO is raised in patients with COPD and/or in patients with worsening COPD, have cast aspersions on the use of this marker in the assessment of COPD airway inflammation,29 in marked contrast to asthma, where there is general consensus that FENO is high, increases during exacerbation, and falls after anti-inflammatory therapy. Agusti et al30 reported recently that FENO was markedly elevated during COPD exacerbation, did not fall during therapy with IV steroids (unlike asthma), but
Figure 7. The correlation between the changes in FENO and percent neutrophils from visit 1 to visit 4.
Figure 8. The correlation between the changes in IL-8 and percent neutrophils from visit 1 to visit 4. CHEST / 119 / 4 / APRIL, 2001
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had normalized several months after hospital discharge. Delen et al31 reported that FENO in COPD with chronic bronchitis was elevated to levels comparable to those in asthma, in contrast to COPD without bronchitis, in which levels were normal. This raises the possibility that subsets of COPD with bronchitis, or perhaps eosinophils in sputum, may have different FENO profiles. Further studies will be necessary to clarify the role of FENO in COPD, including biopsy studies to ascertain if and where NO synthase isoforms are present in the airways; studies before, during, and after COPD exacerbation; and studies of the effects of anti-inflammatory interventions. While sputum differential cell count did not change over the study period in the group as a whole, the changes in percent neutrophils and FENO showed a mild correlation, suggesting that FENO is a marker of neutrophilic inflammation, or may arise from neutrophils, in keeping with reports of iNOS expression in these cells.32 Alternatively, increased proinflammatory cytokines that are chemoattractants for neutrophils, such as IL-8 and LTB4, might also result in augmented iNOS expression in other cells such as the bronchial epithelium. The use of IS analysis in COPD is a promising technique that has been applied by several investigators. In a placebo-controlled trial, Confalonieri et al33 studied the effects of 2 months of treatment with inhaled beclomethasone diproprionate on IS in COPD. There were no differences in sputum cell counts between the groups at baseline. After 2 months of treatment, IS samples from patients in the treated group showed a reduction in both neutrophils (⫺ 27%) and total cells (⫺ 42%) with respect to baseline, while the control group did not (neutrophils ⫹ 9%, total cells ⫹ 7%). In contrast, standard outcome measures, spirometry and blood gas data, did not change from baseline in either patient group. In our study, IL-8 and percent sputum neutrophils at baseline, and also the changes in percent sputum neutrophils and change in IL-8 from visit 1 to visit 4 were significantly correlated, in keeping with the role of IL-8 as a neutrophil chemoattractant. Of great interest is the correlation between percent neutrophils and FEV1, suggesting that neutrophilic inflammation is of pathogenic significance to worsening lung function. The safety of sputum induction is an important issue if we are to apply the technique to COPD, where significant degrees of airway obstruction are common. In this study, 3% saline solution caused a significant fall in FEV1 that returned to near baseline values after albuterol. A recent publication34 showed that oxygen saturation, which we did not monitor, fell by a mean value of 6.0% in smokers during 1054
sputum induction. These findings led us to recommend precautionary measures when inducing sputum in COPD subjects. These might include the use of isotonic saline solution to initiate induction in COPD subjects with percent predicted FEV1 ⬍ 50%, with gradual increases in saline solution concentration until sputum is produced, the close attendance of a physician, monitoring of pulse oximetry, and oxygen supplementation if needed. The use of low-output ultrasonic nebulizers may also reduce the degree of bronchospasm as demonstrated recently in severe asthma.35 In summary, FENO and IS are an acceptable technique to investigate airway inflammation in COPD patients. Exhaled NO may reflect neutrophilic inflammation in this disease. Caution with sputum induction is recommended. References 1 Shelhamer JH, Levine SJ, Wu T, et al. NIH conference: airway inflammation. Ann Intern Med 1995; 123:288 –304 2 Cosio MG, Guerassimov A. Chronic obstructive pulmonary disease: inflammation of small airways and lung parenchyma. Am J Respir Crit Care Med 1999; 160:S21–S25 3 Saetta M. Airway inflammation in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1999; 160:S17–S20 4 Hargreave FE, Leigh R. Induced sputum, eosinophilic bronchitis, and chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1999; 160:S53–S57 5 Alving K, Weitzberg E, Lundberg JM. Increased amount of nitric oxide in exhaled air of asthmatics. Eur Respir J 1993; 6:1368 –1370 6 Dekhuijzen PN, Aben KK, Dekker I, et al. 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 7 Silkoff PE, McClean PA, Slutsky AS, et al. Exhaled nitric oxide and bronchial reactivity during and after inhaled beclomethasone in mild asthma. J Asthma 1998; 35:473– 479 8 Hamid Q, Springall DR, Riveros-Moreno V, et al. Induction of nitric oxide synthase in asthma. Lancet 1993; 342:1510 – 1513 9 Robbins RA, Springall DR, Warren JB, et al. Inducible nitric oxide synthase is increased in murine lung epithelial cells by cytokine stimulation. Biochem Biophys Res Commun 1994; 198:835– 843 10 Corradi M, Majori M, Cacciani GC, et al. Increased exhaled nitric oxide in patients with stable chronic obstructive pulmonary disease. Thorax 1999; 54:572–575 11 Maziak W, Loukides S, Culpitt S, et al. Exhaled nitric oxide in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1998; 157:998 –1002 12 Rutgers SR, van der Mark TW, Coers W, et al. Markers of nitric oxide metabolism in sputum and exhaled air are not increased in chronic obstructive pulmonary disease. Thorax 1999; 54:576 –580 13 Kharitonov SA, Yates D, Barnes PJ. Increased nitric oxide in exhaled air of normal human subjects with upper respiratory tract infections. Eur Respir J 1995; 8:295–297 14 Persson MG, Zetterstrom O, Agrenius V, et al. Single-breath nitric oxide measurements in asthmatic patients and smokers. Lancet 1994; 343:146 –147 Clinical Investigations
15 van Straaten JF, Postma DS, Coers W, et al. Macrophages in lung tissue from patients with pulmonary emphysema express both inducible and endothelial nitric oxide synthase. Mod Pathol 1998; 11:648 – 655 16 Pizzichini MM, Popov TA, Efthimiadis A, et al. Spontaneous and induced sputum to measure indices of airway inflammation in asthma. Am J Respir Crit Care Med 1996; 154:866 – 869 17 Peleman RA, Rytila PH, Kips JC, et al. The cellular composition of induced sputum in chronic obstructive pulmonary disease. Eur Respir J 1999; 13:839 – 843 18 Keatings VM, Collins PD, Scott DM, et al. Differences in interleukin-8 and tumor necrosis factor-␣ in induced sputum from patients with chronic obstructive pulmonary disease or asthma. Am J Respir Crit Care Med 1996; 153:530 –534 19 Culpitt SV, Maziak W, Loukidis S, et al. 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 20 Balzano G, Stefanelli F, Iorio C, et al. Eosinophilic inflammation in stable chronic obstructive pulmonary disease: relationship with neutrophils and airway function. Am J Respir Crit Care Med 1999; 160:1486 –1492 21 Silkoff PE, McClean PA, Slutsky AS, et al. Marked flowdependence of exhaled nitric oxide using a new technique to exclude nasal nitric oxide. Am J Respir Crit Care Med 1997; 155:260 –267 22 Fahy JV, Liu J, Wong H, et al. Analysis of cellular and biochemical constituents of induced sputum after allergen challenge: a method for studying allergic airway inflammation. J Allergy Clin Immunol 1994; 93:1031–1039 23 Steerenberg PA, Snelder JB, Fischer PH, et al. Increased exhaled nitric oxide on days with high outdoor air pollution is of endogenous origin. Eur Respir J 1999; 13:334 –337 24 van Amsterdam JG, Verlaan BP, van Loveren H, et al. Air pollution is associated with increased level of exhaled nitric oxide in nonsmoking healthy subjects. Arch Environ Health 1999; 54:331–335
25 Saleh D, Ernst P, Lim S, et al. Increased formation of the potent oxidant peroxynitrite in the airways of asthmatic patients is associated with induction of nitric oxide synthase: effect of inhaled glucocorticoid. FASEB J 1998; 12:929 –937 26 Jones KL, Bryan TW, Jinkins PA, et al. Superoxide released from neutrophils causes a reduction in nitric oxide gas. Am J Physiol 1998; 275:L1120 –L1126 27 Belvisi MG, Stretton CD, Yacoub M, et al. Nitric oxide is the endogenous neurotransmitter of bronchodilator nerves in humans. Eur J Pharmacol 1992; 210:221–222 28 Lundberg JO, Nordvall SL, Weitzberg E, et al. Exhaled nitric oxide in paediatric asthma and cystic fibrosis. Arch Dis Child 1996; 75:323–326 29 Sterk PJ, de Gouw HW, Ricciardolo FL, et al. Exhaled nitric oxide in COPD: glancing through a smoke screen. Thorax 1999; 54:565–567 30 Agusti AG, Villaverde JM, Togores B, et al. Serial measurements of exhaled nitric oxide during exacerbations of chronic obstructive pulmonary disease. Eur Respir J 1999; 14:523– 528 31 Delen FM, Sippel JM, Osborne ML, et al. Increased exhaled nitric oxide in chronic bronchitis: comparison with asthma and COPD. Chest 2000; 117:695–701 32 Curran AD. The role of nitric oxide in the development of asthma. Int Arch Allergy Immunol 1996; 111:1– 4 33 Confalonieri M, Mainardi E, Della Porta R, et al. Inhaled corticosteroids reduce neutrophilic bronchial inflammation in patients with chronic obstructive pulmonary disease. Thorax 1998; 53:583–585 34 Castagnaro A, Chetta A, Foresi A, et al. Effect of sputum induction on spirometric measurements and arterial oxygen saturation in asthmatic patients, smokers, and healthy subjects. Chest 1999; 116:941–945 35 Hunter CJ, Ward R, Woltmann G, et al. The safety and success rate of sputum induction using a low output ultrasonic nebuliser. Respir Med 1999; 93:345–348
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Study objectives: Neutrophilic airway inflammation may underlie the pathogenesis of COPD. We examined repeated measurements of the fractional concentration of exhaled nitric oxide (FENO) and the correlation with cells and mediators in induced sputum (IS) from patients with COPD. Participants: Eleven COPD subjects (9 men and 2 women, aged 46 to 69 years) with predicted FEV1 of 45 to 70%. Setting: A hospital research laboratory. Design: Single-cohort, prospective study with four visits at two weekly intervals. Interventions: FENO and spirometry were assessed at all visits, and IS for differential cell count, leukotriene-B4 (LTB4) and interleukin (IL)-8, nitrite, and nitrate at visit 1, visit 3, and visit 4. Results: During the study, there were significant declines in mean percent predicted FEV1, from 55.2 to 51.6% (p ⴝ 0.029), and mean FEV1/FVC ratio, from 50.4 to 45.4% (p ⴝ 0.001), accompanied by a significant increase in FENO geometric mean (95% confidence limits), from 15.2 (10.9 to 21.2) to 23.6 (17.1 to 32.4) parts per billion (p ⴝ 0.037), and sputum LTB4, from 1.79 (1.03 to 3.11) to 3.57 (1.95 to 6.53) ng/mL (p ⴝ 0.033), but no significant change in other sputum parameters. From visits 1 to 4, the change in percent neutrophils correlated with the changes in FENO and IL-8 (r ⴝ 0.648, p ⴝ 0.028; r ⴝ 0.60, p ⴝ 0.05, respectively). Hypertonic saline solution induction of sputum caused a fall in FEV1, from 1.83 ⴞ 0.44 to 1.46 ⴞ 0.44 L (p ⴝ 0.049). Conclusions: The worsening spirometry results were accompanied by significant increases in FENO and sputum LTB4. FENO may be related to neutrophilic inflammation driven by the chemoattractant IL-8. FENO and IS may be useful markers of airway inflammation in COPD patients. Sputum induction with hypertonic saline solution causes a significant fall in FEV1 requiring appropriate caution. (CHEST 2001; 119:1049 –1055) Key words: COPD; inflammation; nitric oxide; sputum Abbreviations: FENO ⫽ fractional concentration of exhaled nitric oxide; IL ⫽ interleukin; iNOS ⫽ inducible form of nitric oxide synthase; IS ⫽ induced sputum; LTB4 ⫽ leukotriene-B4; NO nitric oxide; ppb ⫽ parts per billion
A
irway inflammation, a central feature of asthma,1 is increasingly recognized as an important pathogenic mechanism in COPD.2,3 Airway inflammation has been assessed noninvasively by the analysis of spontaneous or induced sputum (IS),4 the measurement of endogenous exhaled gaseous mediators such as nitric oxide (NO),5 and the measurement of substances such as hydrogen peroxide in breath condensate, the fluid phase of exhaled breath.6 The latter may reflect oxidative stress in the airway. The fractional concentration of exhaled nitric oxide (FENO) has been extensively studied as a non*From the Department of Medicine, National Jewish Medical and Research Center, Denver, CO. Manuscript received June 19, 2000; revision accepted November 28, 2000. Correspondence to: Philip E. Silkoff, MD, National Jewish Medical and Research Center, 1400 Jackson St, Denver, CO 80206; e-mail: [email protected]
invasive marker of airway inflammation, particularly in asthma, where levels are high in nonsteroidtreated subjects and fall rapidly after inhaled corticosteroid treatment.7 There is induction of the inducible form of NO synthase (iNOS) in asthmatic bronchial epithelium8 by inflammatory cytokines such as interferon-␥, tumor necrosis factor-␣, and interleukin (IL)-1.9 In COPD, abnormalities of FENO are less clear-cut than in asthma. Some studies10 suggest that FENO is increased in patients with stable COPD, others11 only in patients with unstable COPD, while some12 report no increase. The discrepancies between studies may be related to differences in FENO measurement techniques. Alternatively, viral infections, which increase FENO in normal subjects,13 may underlie the increases in FENO seen in patients with unstable COPD, while current smoking depresses FENO values.14 Although the airway inflammation and the accompanyCHEST / 119 / 4 / APRIL, 2001
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ing cytokine production are different in COPD from that found in asthma, expression of both constitutive and inducible NO synthase isoforms have been reported to be elevated in macrophages and other cell lines in COPD.15 The analysis of spontaneous sputum or IS in COPD also provides much information regarding the airway cells and mediators in the lower airway inflammatory milieu.16 Many studies17–20 of COPD have examined sputum cell differential and mediators such as IL-8. The influence of medications on sputum cell differential and mediators has also been assessed using IS.19 In this study, we performed serial measurements of FENO together with IS analysis over a 6-week period in a cohort of subjects with moderate-tosevere COPD, to ascertain patient acceptability, safety, correlation, and short-term reproducibility. Materials and Methods Eleven subjects (9 men and 2 women, aged 46 to 69 years) with COPD and FEV1 45 to 70% predicted, carbon monoxide diffusion coefficient ⬍ 70% predicted, and ⬎ 10 pack-years cigarette use were recruited. Current smokers were included (n ⫽ 6). The diagnosis of COPD was confirmed by information available in the subjects’ hospital records. Exclusion factors were other pulmonary diseases, including lung cancer and asthma; atopy; unstable systemic diseases; myocardial infarction or cardiac arrhythmias in preceding 6 months; use of oral steroids or continuous oxygen; and subjects within 6 weeks of enrollment who had an upperrespiratory-tract infection or hospitalization. The use of inhaled corticosteroids (n ⫽ 6) and/or theophylline (n ⫽ 2) was not an exclusion criterion. The study was approved by our institutional review board, and subjects signed an informed consent form. Study Design The study consisted of four visits (visits 1 to 4) every 2 weeks over a 6-week period. FEV1 and FENO were determined at each visit. Induction of sputum with hypertonic saline solution was performed at visit 1, visit 3, and visit 4. Exhaled NO Measurement A rapid-response chemiluminescent NO analyzer (model 280; Sievers Instruments; Boulder, CO) was used for NO analysis. Daily two-point calibrations were performed using a 5.2-parts per million calibration gas. FENO was measured using a previously described restricted breath technique, which employed expiratory resistance and positive mouth pressure to close the velum and exclude nasal NO, and a constant expiratory flow of 45 mL/s.21 The inhaled gas was ambient air that was passed through a filter to reduce inhaled NO concentrations to ⬍ 5 parts per billion (ppb). Subjects inhaled to total lung capacity, and exhaled while targeting a constant pressure of 20 mm Hg. Exhalations proceeded until a clear NO plateau of at least 3-s duration was achieved. Repeated exhalations were performed until three plateaus agreed at the 5% level. Induction of Sputum Induction of sputum was achieved by ultrasonic nebulization of 3% hypertonic saline solution. Spirometry was performed to 1050
establish the baseline FEV1 followed by the administration of four activations of albuterol (90 g per activation) via spacer device. Repeat spirometry was performed after 15 min to establish preinduction baseline FEV1. Subjects then performed tidal breathing while inhaling the hypertonic saline solution aerosol. At 2-min intervals, subjects removed the mouthpiece and spat saliva into a container. Then subjects inhaled twice to total lung capacity and were asked to expectorate into a sputum container. Nasal blowing was encouraged if needed to prevent contamination by nasal secretion. FEV1 was monitored at 2-min intervals. The induction procedure was continued until ⬎ 1 mL of sputum was obtained or FEV1 had fallen by ⬎ 20% from preinduction values. After the induction procedure, two activations of albuterol were administered and the subject remained in the laboratory until FEV1 had returned to ⬎ 90% of baseline. Processing of Sputum Processing of sputum was performed within 1 h of collection according to the method of Fahy et al,22 in which the entire expectorate is processed. Samples were refrigerated until processing. Samples were liquefied to disperse the cells by adding 0.1% dithiothreitol in a fixed proportion to the volume of sputum obtained and then centrifuged to separate supernatant, which was stored at ⫺ 70°C for future analysis. Cytospins for differential cell count were prepared. Assays Sputum supernatant was assayed for the following substances: IL-8, leukotriene-B4 (LTB4), nitrite, and nitrate. Sputum nitrite and nitrate were analyzed (after deproteinization with cold ethanol) by reduction chemiluminesence using sodium iodide in ascetic acid (nitrite) at room temperature, and vanadium chloride at 95°C (nitrate), using a Radical Purger (Sievers Instruments). IL-8 was measured by a sandwich enzyme-linked immunosorbent assay using matched antibody pairs (R&D Systems; Minneapolis, MN). LTB4 was measured by a competitive immunoassay, using reagents (Cayman Chemical; Ann Arbor, MI). Processed sputum was analyzed without purification, and results were extrapolated back to levels in the original sputum volumes. Statistical Methods The following 13 end points were analyzed: FEV1 (percent predicted), FVC (percent predicted), FEV1/FVC ratio (percent), FENO (ppb), sputum total cell count, neutrophil count, eosinophil count, macrophage count, lymphocyte count, LTB4, IL-8, nitrite, and nitrate. Preliminary analysis suggested that data variability was not the same at different visits, for FENO, cell count, eosinophil count, lymphocyte count, IL-8, and LTB4, which were therefore log transformed (log 10) for all statistical analyses. Eosinophil count and lymphocyte count could not be log transformed because some subjects had values of 0. For these two end points, models that required equal variance were not used. The data were summarized using means and SEs; for logtransformed variables, geometric means were used. A mixedeffects model with the best-fitting covariance structure was used to determine if there were differences between the means for each pair of visits for each end point. The mixed-effects model accounts for the repeated measures for subjects across visits. All statistical tests were two sided and conducted using a significance level of 5% (ie, 0.05). Clinical Investigations
Results Eleven subjects completed the study, which took place between September and November 1997. Correlation Between Parameters At baseline, there was a significant correlation between sputum percent neutrophils and sputum IL-8 (r ⫽ 0.727, p ⫽ 0.011, Fig 1), but not LTB4. Additionally, percent predicted FEV1 showed a significant negative correlation with percent sputum neutrophil count (r ⫽ ⫺ 0.63, p ⫽ 0.037; Fig 2). There was no significant correlation between FENO and sputum NO metabolites (nitrite or nitrate) at baseline. Change in Pulmonary Function There was a progressive decline in pulmonary function in the cohort over the period of the study (Fig 3). Comparing visit 1 to visit 4, percent predicted FEV1 fell from 55.2 ⫾ 1.93 to 51.6 ⫾ 1.97 (p ⫽ 0.029), FEV1/FVC percentage ratio fell from 50.4 ⫾ 4.02 to 45.4 ⫾ 3.7 (p ⫽ 0.004), but percent predicted FVC was not significantly changed (93.7 ⫾ 6.86 to 95.1 ⫾ 5.4). Change in FENO
Figure 2. The correlation between sputum percent neutrophil count and FEV1 percent predicted at baseline.
1.18 ⫾ 0.07 to 1.37 ⫾ 0.06 ppb (p ⫽ 0.037). Looking at the change in spirometry and FENO from visit 1 to visit 4, there was a significant correlation between the change in FENO and that of FVC (r ⫽ 0.47, p ⫽ 0.01; Fig 5), but not with FEV1.
Exhaled NO showed a progressive significant rise over the period of the study in the study cohort (Fig 4), but remained within the previously determined range of our laboratory for normal subjects (24.2 ⫾ 12.8 ppb) using the same FENO measurement technique and flow rate. Comparing visit 1 to visit 4, log 10 FENO (mean ⫾ SE) rose from
Change in Sputum Variables
Figure 1. The correlation between sputum percent neutrophil count and sputum IL-8 at baseline.
Figure 3. The decline in FEV1/FVC ratio over the course of the study. The p value compares visit 1 to visit 4.
Comparing visit 1 and visit 4, log 10 LTB4 (mean ⫾ SE) rose significantly from 0.25 ⫾ 0.11 to 0.55 ⫾ 0.12 ng/mL (p ⫽ 0.033; Fig 6). However, total and differential cell count (neutrophils, eosinophils, macrophages, lymphocytes), IL-8, nitrite, and nitrate showed no significant change over the period of the study (Table 1), although there was a significant correlation between the changes in both FENO and sputum IL-8 from visit 1 to visit 4 with the
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Figure 4. The increase in FENO over the course of the study. The p value compares visit 1 to visit 4.
Figure 6. The increase in LTB4 from visit 3 to visit 4. The p value compares visit 1 to visit 4.
change in sputum neutrophils (r ⫽ 0.684, p ⫽ 0.028; r ⫽ 0.60, p ⫽ 0.05, respectively; Fig 7, 8).
Discussion
Acceptability and Safety of Sputum Induction and FENO Measurement All subjects tolerated the sputum induction procedure well. However, sputum induction with hypertonic saline solution was associated with a significant transient fall in FEV1 from 1.83 ⫾ 0.44 to 1.46 ⫾ 0.44 L (p ⫽ 0.049; data from all three inductions combined). The administration of albuterol resulted in return of FEV1 to ⬎ 90% of baseline levels, and no subject reported prolonged exacerbation of COPD as a result of the induction. Exhaled NO measurement was acceptable to all subjects despite significant airflow limitation. There were no complications of the procedure.
Figure 5. The correlation between the changes in FENO and FVC from visit 1 to visit 4. 1052
In this 6-week study, we examined repeated exhaled NO and IS parameters and their correlation in a cohort of subjects with COPD. We are unaware of any published studies in COPD that have looked at the relationship between IS cells and mediators and FENO on a repeated basis. There was a progressive decline in FEV1 and FEV1/FVC ratio over the period accompanied by a significant progressive rise in FENO. Sputum LTB4 also significantly rose from visit 3 to visit 4. Sputum differential cell counts showed no significant change in the group as a whole despite the worsening lung function, but the changes in percent neutrophils and in FVC both correlated with the change in FENO. The level of neutrophilic inflammation correlated positively with IL-8, a product of and chemoattractant for neutrophils, and negatively with FEV1. The decline in pulmonary function occurred during the study period from September to November. This may be related to the known worsening of COPD during the winter, perhaps related in turn to climatic change and/or increased air pollution. The rise in FENO accompanying the declining pulmonary function could signify worsening airway inflammation in this cohort of subjects, or could even be a direct result of increasing air pollution, as ambient air pollution can cause increases in endogenous FENO even in normal subjects.23,24 It is also possible that the sputum induction, performed repeatedly, had some deleterious effect. The increase in FENO could originate from two NO synthase isoforms. There could be induction of the iNOS in intrapulmonary cells, such as the bronchial epithelium or in intraluminal inflammatory cells. This is the currently accepted mechanism underlying the increased FENO in asthma, as reported by Saleh et al,25 where FENO was high and iNOS was expressed in the bronchial epithelium in Clinical Investigations
Table 1—Mean Values of Measured Parameters Over the Study Time Period* Parameters
Visit 1 (Baseline)
Visit 2 (Week 2)
Visit 3 (Week 4)
Visit 4 (Week 6)
FEV1, % predicted FVC, % predicted* FEV1/FVC, % FENO, ppb Total cell count, 106/mL Sputum eosinophils, % Sputum macrophages, % Sputum lymphocytes, % Sputum neutrophils, % IL-8, pg/mL LTB4, pg/mL Sputum nitrite, mol/L Sputum nitrate, mol/L
55.1 ⫾ 1.9 93.7 ⫾ 6.8 50.3 ⫾ 4.0 15.2 (10.9–21.2) 3.2 (1.7–5.8) 2.7 ⫾ 1.3 38.4 ⫾ 6.8 1.1 ⫾ 0.2 51.1 ⫾ 6.6 3.7 (1.9–7.24) 1.8 (1.0–3.1) 5.6 (3.0–11.0) 18.9 (6.4–56.2)
54.5 ⫾ 2.8 92.2 ⫾ 5.3 49.2 ⫾ 4.4 18.6 (12.3–28.1) NM NM NM NM NM NM NM NM NM
54.4 ⫾ 2.5 95.6 ⫾ 5.8 48.1 ⫾ 4.5 19.7 (12.8–30.5) 3.3 (2.0–5.6) 1.5 ⫾ 0.7 34.6 ⫾ 5.7 1.3 ⫾ 0.3 57.1 ⫾ 5.8 2.7 (1.4–5.4) 1.8 (0.9–3.4) 4.1 (2.2–7.4) 34.6 (21.1–56.8)
51.6 ⫾ 1.9† 95.1 ⫾ 5.4 45.4 ⫾ 3.7† 23.6 (17.1–32.4)† 2.9 (1.6–5.1) 4.2 ⫾ 2.2 33.9 ⫾ 6.6 0.8 ⫾ 0.2 52.7 ⫾ 7.5 2.8 (1.4–5.7) 3.6 (2.0–6.5)† 6.02 (3.6–10.1) 26.4 (15.7–44.3)
*Data are expressed as mean ⫾ SEM or geometric mean (95% confidence limits). NM ⫽ not measured. †Significant change compared to baseline.
nonsteroid-treated asthma, but was significantly reduced after inhaled budesonide therapy. However, in asthma, using the same FENO measurement technique as that used in the present study, we have observed much higher FENO levels (in some subjects up to 450 ppb). Perhaps the NO formed in the COPD airway is reacting with other free radicals, such as superoxide, produced by neutrophils and/or undergoing denitrification by bacteria, thereby decreasing the observed levels.26 Indeed, the observed increase in FENO may not be iNOS-related at all. In support of this, Rutgers et al12 did not find increased iNOS expression in COPD sputum macrophages. Alternatively, there may be upregulation of constitutive NO synthase isoforms, such as neuronal NO synthase in airway nonadrenergic noncholinergic inhibitory bronchodilating nerves, which might serve to overcome the worsening airway obstruction.27
Certainly, the airway inflammation in COPD is different from asthma, and perhaps it is unreasonable to expect the same changes in NO formation and disposition. In patients with cystic fibrosis, which has a similar neutrophilic predominance and bacterial colonization as COPD, FENO is in fact lower than in healthy control subjects.28 The relatively low levels of FENO found in COPD patients, together with differing reports as to whether FENO is raised in patients with COPD and/or in patients with worsening COPD, have cast aspersions on the use of this marker in the assessment of COPD airway inflammation,29 in marked contrast to asthma, where there is general consensus that FENO is high, increases during exacerbation, and falls after anti-inflammatory therapy. Agusti et al30 reported recently that FENO was markedly elevated during COPD exacerbation, did not fall during therapy with IV steroids (unlike asthma), but
Figure 7. The correlation between the changes in FENO and percent neutrophils from visit 1 to visit 4.
Figure 8. The correlation between the changes in IL-8 and percent neutrophils from visit 1 to visit 4. CHEST / 119 / 4 / APRIL, 2001
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had normalized several months after hospital discharge. Delen et al31 reported that FENO in COPD with chronic bronchitis was elevated to levels comparable to those in asthma, in contrast to COPD without bronchitis, in which levels were normal. This raises the possibility that subsets of COPD with bronchitis, or perhaps eosinophils in sputum, may have different FENO profiles. Further studies will be necessary to clarify the role of FENO in COPD, including biopsy studies to ascertain if and where NO synthase isoforms are present in the airways; studies before, during, and after COPD exacerbation; and studies of the effects of anti-inflammatory interventions. While sputum differential cell count did not change over the study period in the group as a whole, the changes in percent neutrophils and FENO showed a mild correlation, suggesting that FENO is a marker of neutrophilic inflammation, or may arise from neutrophils, in keeping with reports of iNOS expression in these cells.32 Alternatively, increased proinflammatory cytokines that are chemoattractants for neutrophils, such as IL-8 and LTB4, might also result in augmented iNOS expression in other cells such as the bronchial epithelium. The use of IS analysis in COPD is a promising technique that has been applied by several investigators. In a placebo-controlled trial, Confalonieri et al33 studied the effects of 2 months of treatment with inhaled beclomethasone diproprionate on IS in COPD. There were no differences in sputum cell counts between the groups at baseline. After 2 months of treatment, IS samples from patients in the treated group showed a reduction in both neutrophils (⫺ 27%) and total cells (⫺ 42%) with respect to baseline, while the control group did not (neutrophils ⫹ 9%, total cells ⫹ 7%). In contrast, standard outcome measures, spirometry and blood gas data, did not change from baseline in either patient group. In our study, IL-8 and percent sputum neutrophils at baseline, and also the changes in percent sputum neutrophils and change in IL-8 from visit 1 to visit 4 were significantly correlated, in keeping with the role of IL-8 as a neutrophil chemoattractant. Of great interest is the correlation between percent neutrophils and FEV1, suggesting that neutrophilic inflammation is of pathogenic significance to worsening lung function. The safety of sputum induction is an important issue if we are to apply the technique to COPD, where significant degrees of airway obstruction are common. In this study, 3% saline solution caused a significant fall in FEV1 that returned to near baseline values after albuterol. A recent publication34 showed that oxygen saturation, which we did not monitor, fell by a mean value of 6.0% in smokers during 1054
sputum induction. These findings led us to recommend precautionary measures when inducing sputum in COPD subjects. These might include the use of isotonic saline solution to initiate induction in COPD subjects with percent predicted FEV1 ⬍ 50%, with gradual increases in saline solution concentration until sputum is produced, the close attendance of a physician, monitoring of pulse oximetry, and oxygen supplementation if needed. The use of low-output ultrasonic nebulizers may also reduce the degree of bronchospasm as demonstrated recently in severe asthma.35 In summary, FENO and IS are an acceptable technique to investigate airway inflammation in COPD patients. Exhaled NO may reflect neutrophilic inflammation in this disease. Caution with sputum induction is recommended. References 1 Shelhamer JH, Levine SJ, Wu T, et al. NIH conference: airway inflammation. Ann Intern Med 1995; 123:288 –304 2 Cosio MG, Guerassimov A. Chronic obstructive pulmonary disease: inflammation of small airways and lung parenchyma. Am J Respir Crit Care Med 1999; 160:S21–S25 3 Saetta M. Airway inflammation in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1999; 160:S17–S20 4 Hargreave FE, Leigh R. Induced sputum, eosinophilic bronchitis, and chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1999; 160:S53–S57 5 Alving K, Weitzberg E, Lundberg JM. Increased amount of nitric oxide in exhaled air of asthmatics. Eur Respir J 1993; 6:1368 –1370 6 Dekhuijzen PN, Aben KK, Dekker I, et al. 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 7 Silkoff PE, McClean PA, Slutsky AS, et al. Exhaled nitric oxide and bronchial reactivity during and after inhaled beclomethasone in mild asthma. J Asthma 1998; 35:473– 479 8 Hamid Q, Springall DR, Riveros-Moreno V, et al. Induction of nitric oxide synthase in asthma. Lancet 1993; 342:1510 – 1513 9 Robbins RA, Springall DR, Warren JB, et al. Inducible nitric oxide synthase is increased in murine lung epithelial cells by cytokine stimulation. Biochem Biophys Res Commun 1994; 198:835– 843 10 Corradi M, Majori M, Cacciani GC, et al. Increased exhaled nitric oxide in patients with stable chronic obstructive pulmonary disease. Thorax 1999; 54:572–575 11 Maziak W, Loukides S, Culpitt S, et al. Exhaled nitric oxide in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1998; 157:998 –1002 12 Rutgers SR, van der Mark TW, Coers W, et al. Markers of nitric oxide metabolism in sputum and exhaled air are not increased in chronic obstructive pulmonary disease. Thorax 1999; 54:576 –580 13 Kharitonov SA, Yates D, Barnes PJ. Increased nitric oxide in exhaled air of normal human subjects with upper respiratory tract infections. Eur Respir J 1995; 8:295–297 14 Persson MG, Zetterstrom O, Agrenius V, et al. Single-breath nitric oxide measurements in asthmatic patients and smokers. Lancet 1994; 343:146 –147 Clinical Investigations
15 van Straaten JF, Postma DS, Coers W, et al. Macrophages in lung tissue from patients with pulmonary emphysema express both inducible and endothelial nitric oxide synthase. Mod Pathol 1998; 11:648 – 655 16 Pizzichini MM, Popov TA, Efthimiadis A, et al. Spontaneous and induced sputum to measure indices of airway inflammation in asthma. Am J Respir Crit Care Med 1996; 154:866 – 869 17 Peleman RA, Rytila PH, Kips JC, et al. The cellular composition of induced sputum in chronic obstructive pulmonary disease. Eur Respir J 1999; 13:839 – 843 18 Keatings VM, Collins PD, Scott DM, et al. Differences in interleukin-8 and tumor necrosis factor-␣ in induced sputum from patients with chronic obstructive pulmonary disease or asthma. Am J Respir Crit Care Med 1996; 153:530 –534 19 Culpitt SV, Maziak W, Loukidis S, et al. 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 20 Balzano G, Stefanelli F, Iorio C, et al. Eosinophilic inflammation in stable chronic obstructive pulmonary disease: relationship with neutrophils and airway function. Am J Respir Crit Care Med 1999; 160:1486 –1492 21 Silkoff PE, McClean PA, Slutsky AS, et al. Marked flowdependence of exhaled nitric oxide using a new technique to exclude nasal nitric oxide. Am J Respir Crit Care Med 1997; 155:260 –267 22 Fahy JV, Liu J, Wong H, et al. Analysis of cellular and biochemical constituents of induced sputum after allergen challenge: a method for studying allergic airway inflammation. J Allergy Clin Immunol 1994; 93:1031–1039 23 Steerenberg PA, Snelder JB, Fischer PH, et al. Increased exhaled nitric oxide on days with high outdoor air pollution is of endogenous origin. Eur Respir J 1999; 13:334 –337 24 van Amsterdam JG, Verlaan BP, van Loveren H, et al. Air pollution is associated with increased level of exhaled nitric oxide in nonsmoking healthy subjects. Arch Environ Health 1999; 54:331–335
25 Saleh D, Ernst P, Lim S, et al. Increased formation of the potent oxidant peroxynitrite in the airways of asthmatic patients is associated with induction of nitric oxide synthase: effect of inhaled glucocorticoid. FASEB J 1998; 12:929 –937 26 Jones KL, Bryan TW, Jinkins PA, et al. Superoxide released from neutrophils causes a reduction in nitric oxide gas. Am J Physiol 1998; 275:L1120 –L1126 27 Belvisi MG, Stretton CD, Yacoub M, et al. Nitric oxide is the endogenous neurotransmitter of bronchodilator nerves in humans. Eur J Pharmacol 1992; 210:221–222 28 Lundberg JO, Nordvall SL, Weitzberg E, et al. Exhaled nitric oxide in paediatric asthma and cystic fibrosis. Arch Dis Child 1996; 75:323–326 29 Sterk PJ, de Gouw HW, Ricciardolo FL, et al. Exhaled nitric oxide in COPD: glancing through a smoke screen. Thorax 1999; 54:565–567 30 Agusti AG, Villaverde JM, Togores B, et al. Serial measurements of exhaled nitric oxide during exacerbations of chronic obstructive pulmonary disease. Eur Respir J 1999; 14:523– 528 31 Delen FM, Sippel JM, Osborne ML, et al. Increased exhaled nitric oxide in chronic bronchitis: comparison with asthma and COPD. Chest 2000; 117:695–701 32 Curran AD. The role of nitric oxide in the development of asthma. Int Arch Allergy Immunol 1996; 111:1– 4 33 Confalonieri M, Mainardi E, Della Porta R, et al. Inhaled corticosteroids reduce neutrophilic bronchial inflammation in patients with chronic obstructive pulmonary disease. Thorax 1998; 53:583–585 34 Castagnaro A, Chetta A, Foresi A, et al. Effect of sputum induction on spirometric measurements and arterial oxygen saturation in asthmatic patients, smokers, and healthy subjects. Chest 1999; 116:941–945 35 Hunter CJ, Ward R, Woltmann G, et al. The safety and success rate of sputum induction using a low output ultrasonic nebuliser. Respir Med 1999; 93:345–348
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