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Academic Unit of Respiratory Medicine, Royal Free and University College Medical School, London, United Kingdom
ABSTRACT
Rationale: In addition to pulmonary involvement, stable chronic obstructive pulmonary disease (COPD) is associated with nasal and systemic inflammation. Although exacerbations of COPD are associated with increased pulmonary and systemic inflammation, determinants of the systemic response remain obscure, and nor is it known whether there is nasal involvement.
Objectives: To investigate upper airway, lower airway, and systemic inflammation at exacerbation of COPD.
Methods: We sampled sputum, nasal wash, and serum from 41 exacerbations (East London cohort) for analysis of pathogenic microorganisms and inflammatory indices (sputum/nasal wash leukocytes, interleukin [IL]-6, IL-8, and myeloperoxidase; serum IL-6 and C-reactive protein). Values were compared with stable COPD.
Measurements and Main Results: Exacerbation of COPD is associated with greater nasal, sputum, and serum inflammation than the stable state. At exacerbation, inflammatory markers were highly correlated within nasal wash and serum (all r 0.62, p < 0.001), but not sputum. The degree of upper airway inflammation correlated with the degree of lower airway inflammation (e.g., nasal wash/sputum myeloperoxidase; r = 0.50, p = 0.001). The degree of systemic inflammation correlated with the degree of lower airway inflammation (e.g., serum IL-6/sputum IL-8; r = 0.35, p = 0.026), and was greater in the presence of a sputum bacterial pathogen (29.0 g/dl C-reactive protein difference, p = 0.002). We did not find relationships between the upper airway and systemic compartments.
Conclusions: Exacerbation of COPD is associated with pan-airway inflammation; the systemic inflammatory response is proportional to that occurring in the lower airway and greater in the presence of a bacterial pathogen.
Key Words: cytokine nose pathogen
Chronic obstructive pulmonary disease (COPD) is a condition characterized by an abnormal inflammatory response in the lung to noxious particles or gases (1). However, this focus on the lung ignores accumulating evidence of extrapulmonary manifestations in COPD including, for example, cachexia (2) and systemic inflammation (3). The presence of systemic inflammation may be particularly important given the recognized association between raised systemic inflammatory markers and increased cardiovascular mortality (4), a common cause of death in patients with COPD (5).
We have recently reported a further extrapulmonary manifestation of COPD: nasal (upper airway) involvement (6–8). Upper airway symptoms are common in COPD (6, 9) and cause impairment to quality of life (7). Moreover, COPD is associated with the presence of nasal neutrophilic inflammation (8, 10, 11), the severity of which reflects that occurring in the lung (8).
Much of the morbidity, mortality, hospital admission, and health care cost in COPD relate to episodes of acute deterioration in respiratory health termed "exacerbations." Exacerbations are therefore important events, yet little is known about the underlying inflammatory mechanisms. Although several studies have reported increased lower airway (12–16) and systemic (13, 17) inflammation at exacerbation, such changes have been inconsistent, even for markers of the neutrophilic inflammation that is characteristic of COPD. There are, furthermore, no studies specifically examining interrelationships between sputum inflammatory markers at exacerbation, and nor is it known whether the magnitude of the systemic inflammatory response reflects that occurring in the lung. Also, to date, there have been no reports investigating upper airway inflammation and exacerbation of COPD.
In addition to reported increases in bronchial and systemic inflammation, we hypothesized that exacerbation of COPD may also be associated with increased upper airway inflammation. Furthermore, given the considerable cardiovascular mortality in the periexacerbation period and the relationships between systemic inflammation and cardiovascular disease, we hypothesized that the magnitude of the systemic response at exacerbation may relate to the degree of lower airway involvement. We have performed a study to examine upper airway, lower airway, and systemic inflammation at exacerbation of COPD, including an analysis of pathogenic microorganisms, with the aim of testing these hypotheses and investigating determinants of the local and systemic inflammatory responses. We have compared these exacerbation data with that recently published from the same cohort in the baseline state (8). This is the first study to sample these three compartments at exacerbation of COPD, with results that inform on the mechanisms underlying such events. Some of the results of this study have been previously published in the form of abstracts (18–20).
METHODS
The following is an abridged version of METHODS; the full text appears in the online supplement.
Study Subjects and Protocol
Forty-one exacerbations were sampled from 41 patients with COPD enrolled in the East London cohort. After the diagnosis of exacerbation (see the following section), samples of sputum, nasal wash, and serum were obtained at a single clinic visit before the initiation of any additional therapy, and processed as described. Solely for the assessment of change in inflammatory markers and bacteria between the baseline state and exacerbation, data are provided on these variables from 47 individuals in the same cohort, which has been the subject of a prior publication (8). In 21 cases, paired baseline-exacerbation data was available. The methodology for these baseline samples is as previously described (8).
Entry criteria to the East London cohort consisted of a postbronchodilator FEV1 < 80% predicted, FEV1 to FVC ratio < 70%, 2-agonist reversibility on baseline FEV1 < 200 ml or 15%, and the absence of clinical asthma or other significant respiratory pathology. In particular, given the recognized association between bronchiectasis and sinusitis, none of the patients had clinical findings suggestive of bronchiectasis (such as the production of large volumes of purulent sputum or coarse inspiratory crepitations). Patients recorded daily change in symptoms on diary cards allowing the confirmation of exacerbation as described in the following section, and attended for 3-mo reviews. Preexacerbation clinical data are reported for all 41 patients, representing that obtained at a routine visit during clinical stability up to 3 mo preceding the exacerbation. Although these patients have been the subject of previous publications, referenced throughout the text, and with the exception of the baseline samples referred to previously, the data included in this analysis are entirely original.
Nasal symptoms were assessed at exacerbation using a 6-point nasal score, developed in this department, which we have previously shown to correlate with more complex assessments of rhinosinusitis (7). Each of the five principal nasal symptoms of rhinorrhea, postnasal drip, sneezing, impaired sense of smell, and nasal blockage were binary coded as present/increased over baseline (1) or absent/not (0) and summed to yield a total nasal score between 0 and 5.
Definition of Exacerbation
An exacerbation was defined according to our previous work (12, 13, 17, 21, 22), now validated against important outcome measures in COPD, including lung function decline (21) and quality of life (22), and based on that first described by Anthonisen and colleagues (23). Patients were instructed to record any increase in daily symptoms on diary cards. The symptoms recorded were termed major (dyspnea, sputum volume, or sputum purulence) and minor (cough, wheeze, sore throat, or coryza). An exacerbation was defined as the onset of two or more new or worsening symptoms, on 2 or more consecutive d, at least one symptom of which must be major.
Sputum Samples
A single sample of spontaneous sputum was obtained and divided into three aliquots. One sample was processed with dithiothreitol and centrifuged to produce a cell pellet for leukocyte count. A second sample was homogenized in phosphate-buffered saline with glass beads. A portion of this preparation was frozen at –70°C and used for later detection of rhinovirus by polymerase chain reaction as described in the following section; the remainder was centrifuged with aliquots of supernatant stored at –70°C for subsequent analysis of inflammatory mediators. The third portion was used for quantitative bacterial culture according to methods that we have previously described (8).
Nasal Wash Procedure and Samples
Nasal wash was performed using a technique adapted from that of Hilding (24). Briefly, a 12-F Foley catheter (Bard, Crawley, UK), modified by removal of the tip distal to the balloon, was inserted into the nostril and inflated with sufficient air to form a comfortable seal (typically 7–10 ml). With the patients head flexed 45° forward, 7 ml of warmed 0.9% saline was instilled through the catheter and washed in and out of the nasal cavity three times. A portion of the pooled wash from both nostrils was processed for quantitative bacteriology and an aliquot stored for later detection of rhinovirus by polymerase chain reaction as described for sputum. The remainder was centrifuged to yield a cell pellet for leukocyte count and a supernatant for analysis of inflammatory cytokines, also as described for sputum.
Serum Samples
A 5-ml sample of venous blood was collected into a sterile Vacutainer, centrifuged, and the serum stored at –70°C for later analysis of inflammatory mediators.
Analysis of Inflammatory Mediators
The inflammatory mediators interleukin (IL)-6, IL-8, myeloperoxidase (MPO) and C-reactive protein (CRP) were quantified using commercial sandwich ELISA kits. IL-6, IL-8, and MPO were assayed in sputum and nasal wash. Serum was assayed for IL-6 and CRP. Concentrations of mediators in sputum samples represent a 10-fold dilution by weight of the original sample. The limits of detection were 0.7 pg/ml (IL-6), 10 pg/ml (IL-8), 1.5 ng/ml (MPO), and 0.1 g/dl (CRP).
Reporting of Bacteriology Data
Bacteriology data are expressed as both total bacterial load (cfu/ml, of nasal wash or sputum supernatant, in log10 units) and the presence or absence of a range of potentially pathogenic microorganisms (PPMs) associated with exacerbations of COPD. For the purposes of this study, we defined PPMs as including Streptococcus pneumoniae, Haemophilus influenzae, Branhamella catarrhalis, Klebsiella pneumoniae, and Pseudomonas aeruginosa.
Rhinovirus Polymerase Chain Reaction
Viral RNA was extracted using commercial kits before reverse transcription and polymerase chain reaction.
Statistical Analysis
Data were analyzed using STATA-5 software (Stata Corporation, College Station, TX). The Kolmogorov-Smirnov test of normality was applied. Exacerbation inflammatory indices not normally distributed were rendered so by log10 transformation. Data are expressed as mean and SD, differences between groups employed t tests, and relationships between groups used Pearson correlations. Comparisons between baseline and exacerbation data used paired and unpaired t tests and Mann-Whitney U tests as appropriate. A probability of error of 5% or less was considered statistically significant.
RESULTS
Clinical Characteristics
The baseline clinical characteristics of the 41 patients studied at exacerbation are reported in Table 1, which demonstrates that the cohort have COPD of moderate severity with a mean baseline FEV1 of 40% predicted. Clinical data from the 47 patients studied at baseline, previously reported (8), are also included in Table 1. There were no statistically significant differences between these two populations.
Comparison of Inflammatory Markers and Microorganisms between Baseline and Exacerbation
The results of the leukocyte count, inflammatory protein, and microbiological analysis of the 41 exacerbation sputum, nasal wash, and serum samples are reported in Table 2. Table 2 also includes the previously reported baseline data from 47 patients in the stable state (8). There were significant rises in selected inflammatory markers from all three compartments at the time of exacerbation. The analysis of 21 paired baseline exacerbation samples confirmed significant rises in the sputum leukocyte count (p = 0.003), sputum MPO concentration (p < 0.001), sputum PPM prevalence (p = 0.028), nasal leukocyte count (p = 0.035), and both systemic inflammatory markers (IL-6, p = 0.010; CRP, p = 0.004). As in the baseline state (8), we did not detect differences in airway inflammatory markers at exacerbation between the 13 patients who continued to smoke and the 28 ex-smokers.
Relationships at Exacerbation between Inflammatory Markers and Microorganisms in Individual Compartments: Sputum, Nasal Wash, and Serum
With the exception of MPO, present at relatively high concentrations in nasal wash, the concentrations of other inflammatory markers at exacerbation were more than 10-fold greater in the sputum than the nasal samples. Concentrations of all the markers, including MPO, were significantly higher in sputum than the nasal wash samples (all p 0.001).
The results of correlations between inflammatory markers in individual compartments at exacerbation are illustrated in Figures 1A (nasal wash), 1B (sputum), and 1C (serum). Although inflammatory markers were highly correlated with each other in nasal wash (all r > 0.69, p < 0.001) and serum (r = 0.62, p < 0.001), relationships in sputum were more variable with a significant correlation only between the sputum leukocyte count and MPO concentration (r = 0.33, p = 0.035).
The presence of microorganisms had different associations when isolated in the upper and lower airway samples. The presence of a sputum PPM was associated with a higher sputum IL-8 concentration (3.59 vs. 3.46 log10 pg/ml, p = 0.027) and there was a relationship between sputum IL-8 concentration and sputum bacterial load that just failed to achieve statistical significance (r = 0.29, p = 0.064). The presence of rhinovirus in sputum was not associated with significant changes in sputum inflammatory markers. In the nose, the presence of nasal rhinovirus was associated with a significantly higher nasal IL-6 concentration (1.5 vs. 0.8 log10 pg/ml, p = 0.015), but there was no association between nasal bacterial load and nasal inflammatory markers. Relationships between the presence of a nasal PPM and both higher nasal leukocyte count (4.9 vs. 4.2 log10 cells/ml, p = 0.069) and nasal IL-6 concentration (1.3 vs. 0.7 log10 pg/ml, p = 0.083) also just failed to achieve statistical significance.
Relationships between Upper Airway, Lower Airway, and Systemic Inflammation at Exacerbation of COPD
The degree of both systemic and upper airway inflammation was related to the degree of lower airway inflammation, but we did not find significant relationships between the degree of inflammation in the upper airway and systemic compartments. These data are presented in Table 3. Illustrating the relationship between lower airway and systemic inflammation, the correlation between sputum IL-8 and serum IL-6 concentrations is presented as Figure 2. Illustrating the relationship between lower and upper airway inflammation, the correlation between MPO concentrations in sputum and nasal wash is presented as Figure 3.
Relationships between Upper and Lower Airway Microorganisms at Exacerbation of COPD
Pathogenic microorganisms were identified in 34 of the 41 exacerbation sputum samples (83%) and 17 of the 41 nasal wash specimens (41%). The prevalence of individual pathogens is illustrated in Figure 4.
Rhinovirus was more commonly isolated in sputum than nasal wash samples, and, when isolated in nasal wash, was always present in sputum. Seven of the 10 exacerbations in which rhinovirus was isolated in sputum were also associated with the presence of a sputum PPM, most commonly H. influenzae.
H. influenzae was the commonest PPM isolated in sputum, but only 2 of 18 (11%) of these exacerbations were associated with simultaneous nasal carriage. The corresponding percentages for S. pneumoniae, B. catarrhalis, and gram-negative rods were 50, 40, and 100%, respectively. There were no cases in which a PPM was isolated in nasal wash without a PPM also being present in sputum. In addition to the relationship between the presence of a sputum PPM and greater sputum inflammation described previously, exacerbations in which a PPM was identified in sputum were also associated with significantly greater systemic inflammation (CRP: 55.2 vs. 26.2 g/dl, p = 0.002, Figure 5, and IL-6: 1.2 vs. 0.8 log10 pg/ml, p = 0.043).
Relationship between Nasal Symptoms and Nasal Inflammation at Exacerbation of COPD
The median (interquartile range) nasal score at exacerbation was 2 (1–3). The nasal score correlated with the degree of nasal inflammation as assessed by the nasal IL-6 or IL-8 concentrations (r = 0.415, p = 0.010, and r = 0.356, p = 0.028, respectively).
DISCUSSION
This is the first study to simultaneously sample the upper airway, lower airway, and systemic compartments at exacerbation of COPD. The new and major findings of this study are, first, that the degree of systemic inflammation at exacerbation of COPD is related to the degree of cellular lower airway inflammation and the presence of a sputum PPM. Second, exacerbation of COPD is associated with pan-airway inflammation, in which selected upper airway inflammatory markers are increased in relation to stable COPD, and to a degree that correlates with the lower airway involvement. We have also reported on the comparative prevalence of pathogenic microorganisms in upper and lower airway samples at exacerbation and their association with selected markers of inflammation at these sites.
The correlations between both sputum leukocyte count and IL-8 concentration with the systemic markers IL-6 and CRP suggest, for the first time, that there is a significant relationship between neutrophilic lower airway inflammation and the systemic inflammatory response at exacerbation of COPD. Although there are reports of both increased pulmonary (12–16) and systemic (13, 17) inflammation during exacerbations, the only previous study describing a direct relationship between these two compartments reported the sputum and plasma concentrations of the broncho- and vasoconstrictor peptide endothelin-1 (13). Indeed, in the stable state, we (8) and others (25) have been unable to demonstrate such a relationship suggesting that the pulmonary and systemic responses may be modulated separately at this time. In some studies (26), though not in our previous work (8), the presence of a sputum PPM in stable COPD has been associated with greater systemic inflammation (26). The present study suggests that such a relationship is present at exacerbation, building on previous work describing an association between sputum purulence and increased serum CRP at exacerbation (27).
The presence of a relationship between pulmonary and systemic inflammation at exacerbation of COPD has a number of important implications for clinical practice. First, it suggests that the assessment of serum CRP at exacerbation, a simple and readily available assay in many health care settings, may inform not only on the systemic response, but also the inflammatory load in the lower airway at that time. This is of relevance given the considerable problems in reliably assessing lower airway inflammatory markers. Further longitudinal studies would be required, however, to determine whether the time course of changes in CRP reflect that occurring in the lung and therefore whether CRP may be useful in monitoring exacerbation resolution and response to therapy. Second, that the serum IL-6 concentration was related to the degree of lower airway inflammation may be of particular importance given that IL-6 is the primary stimulus to hepatic production of fibrinogen. A raised plasma fibrinogen concentration is an independent risk factor for cardiovascular disease (4), and one could speculate that the present study provides a link between pulmonary inflammation and increased cardiovascular morbidity in the periexacerbation period. We hypothesize that the magnitude of excess cardiovascular risk may be related to the severity of lower airway inflammation, and this suggestion warrants further investigation. In addition, the associations observed between the upper and lower airway suggest that upper airway sampling also deserves further study as a noninvasive tool for the assessment of airway inflammatory markers at exacerbation of COPD.
Investigating relationships between inflammatory markers may aid in understanding the mechanisms of exacerbation. Although it is generally accepted that COPD is characterized by neutrophilic inflammation, and that exacerbations are associated with increased airway inflammation, the published data are conflicting. IL-8, for example, is a potent neutrophil chemoattractant; yet, although some studies have reported increases in sputum IL-8 concentration at exacerbation (14–16), others have not (12, 13). In addition, there are no studies reporting a significant association between sputum IL-8 concentration and leukocyte count in which the IL-8 concentration was increased over baseline. We have demonstrated a relationship between the sputum leukocyte count and MPO concentration, suggesting that these leukocytes are, as expected, predominantly neutrophils. Indeed, this was the only significant relationship demonstrated between inflammatory indices in sputum. One explanation for the absence of a relationship between sputum IL-8 concentration and leukocyte count would be that this cytokine is largely produced by airway epithelium. The general absence of relationships between sputum inflammatory markers at exacerbation of COPD highlights the complexity of using sputum to assess airway inflammation and the importance of selecting appropriate mediators to assay. These findings may also reflect the use of spontaneous sputum in the present study, with sputum induction being poorly tolerated in these patients with severe disease at the time of exacerbation. In contrast, individual mediators in serum and nasal wash were highly correlated, suggesting that in such samples the choice of marker assayed is less important.
This study is the first to report on upper airway involvement at exacerbation of COPD. We have recently reported that COPD in the stable state is associated with an up-regulated nasal IL-8 concentration in comparison to control subjects, the degree of which reflects that present in the sputum (8). The current study demonstrates that relationships between the upper and lower airway are also present at exacerbation, with increased nasal inflammation at exacerbation compared with stable disease, and therefore that exacerbation is associated with a pan-airway inflammatory response. Of note, the severity of upper airway symptoms at exacerbation was directly related to the nasal IL-6 and IL-8 concentrations. Relationships between the upper and lower airways have been extensively studied in asthma. Here, in response to a variety of clinical and pathologic evidence, the concept of a "united airways disease" has been proposed in which rhinitis and asthma are the upper and lower airway manifestations of the same disease process (28). Although the clinical relevance of such relationships remains controversial—in particular, whether treating the rhinitis of patients with asthma and rhinitis is able to improve asthma symptoms—the demonstration of pan-airway involvement at exacerbation of COPD is novel and suggests that the upper airway deserves further study in COPD.
We have reported on the microbiology of upper and lower airway samples at exacerbation of COPD. Our finding that 24% of exacerbations are associated with rhinovirus is similar to previous reports (29), as is the finding that rhinovirus is more commonly isolated in sputum than nasal wash. In experimental infections, rhinovirus titers in nasal samples peak at Day 2 and decline rapidly thereafter (30). We did not detect a difference in duration from exacerbation onset to sampling between those exacerbations in which rhinovirus was present in both nasal wash and sputum compared with sputum alone. It is therefore possible that the difference reflects lower titers of rhinovirus in nasal wash than sputum. Future studies using a quantitative approach would be useful to look at the dynamics of rhinoviral infection in the upper and lower airway. A total of 76% of exacerbations was associated with the presence of a sputum PPM, somewhat higher than previous reports, but an important finding given the association of a sputum PPM with greater lower airway and systemic inflammation. Overall, when a PPM was isolated in sputum, the same species was also present in 24% of the nasal wash samples. However, this masks differences in rates between individual species and the dynamics of upper and lower airway bacterial carriage also deserve further study. A further interpretation of these findings would be that upper airway sampling for microorganisms adds little to sputum sampling at the time of exacerbation.
Using the well characterized East London COPD cohort with daily recording of symptoms has allowed us to accurately diagnose the presence of exacerbations, and baseline preexacerbation clinical data was available on all 41 patients. Furthermore, samples were obtained before the initiation of any additional therapy, and this is the first study to examine samples from all three compartments at exacerbation of COPD. We have been able to compare these data with samples obtained in the stable state. Although guidelines for the standardization of sputum analysis are available and have been followed (31), no such recommendations for nasal wash are available. We have, however, previously reported on the reproducibility of this nasal wash technique for the assessment of both inflammatory cytokines and bacterial load (8). We have identified the most important bacterial and viral pathogens responsible for exacerbation of COPD. We did not, however, assess the presence of respiratory viruses other than rhinovirus. This cohort of patients is extensively influenza-vaccinated, and we have previously found few isolates of influenza during these events. The only other respiratory virus commonly isolated at exacerbation of COPD is respiratory syncytial virus, but respiratory syncytial virus is isolated as commonly in the stable state, and the significance of respiratory syncytial virus isolation at exacerbation is therefore unclear (29). Given that COPD is associated with predominantly neutrophilic inflammation, we chose to focus on such markers in nasal wash and sputum. We assayed IL-6 and CRP in serum because of the association between IL-6, fibrinogen, and cardiovascular morbidity, and the widespread availability of CRP testing respectively.
In conclusion, the degree of systemic inflammation at exacerbation of COPD is related to both the degree of lower airway inflammation and the presence of a sputum PPM. Furthermore, this is the first study to report upper airway involvement at exacerbation in this disease and therefore that exacerbation of COPD is associated with pan-airway inflammation. The results are of importance in understanding the pathophysiology of exacerbation and in the design of rational strategies to monitor and treat the considerable morbidity and mortality associated with this prevalent condition.
Acknowledgments
The authors thank Angela Whiley and Mark Wilks for the bacteriologic analysis and Linda Hammond for assistance with the rhinovirus polymerase chain reaction.
FOOTNOTES
Supported by St. Bartholomew's and the Royal London Charitable Foundation, and an unrestricted educational grant from GlaxoSmithKline.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.200505-704OC on September 22, 2005
Conflict of Interest Statement: J.R.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. W.R.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. T.M.A.W. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. G.C.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. J.A.W. has received honoraria for lectures at meetings or attendance at advisory boards from the following companies: GlaxoSmithKline (GSK), Boehringer Ingelheim, Astra Zeneca, Bayer, Roche, Vitalaire, and Aventis Pasteur. She has received research grants totaling approximately $600,000 from GSK for studies of COPD questionnaires, studies of COPD exacerbation time course, flow cytometric studies of the interaction of the upper and lower airway in COPD, study of time course of inflammatory markers at COPD exacerbation, analysis of long-term changes in airway and systemic inflammatory markers, clinical trial of anti-cough therapy in COPD, and clinical trial of Seretide in COPD. She has received a grant of approximately $450,000 for a study of tiotropium in COPD and $25,000 from Astra Zeneca for a health economic study.
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