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Inflammatory Profile of New Bacterial Strain Exacerbations of Chronic Obstructive Pulmonary Disease Sanjay Sethi1,2, Catherine Wrona3, Karen Eschberger2, Phyllis Lobbins3, Xueya Cai4, and Timothy F. Murphy2,3 1

Division of Pulmonary and Critical Care and Sleep Medicine, Department of Medicine, University at Buffalo, State University of New York; Veterans Affairs Western New York Healthcare System, Buffalo, New York; and 3Division of Infectious Diseases, Department of Microbiology, and 4Department of Biostatistics, University at Buffalo, State University of New York 2

Rationale: Whether the airway and systemic inflammatory profile in bacterial exacerbations of chronic obstructive pulmonary disease (COPD) is distinct from nonbacterial exacerbations is unclear. Previous studies have not used molecular typing of bacterial pathogens, which is required to accurately define bacterial infection in COPD. The relationship between clinical severity and course of exacerbation and inflammation is also not fully understood. Objectives: To determine if (1) systemic and airway inflammation is distinct in new bacterial strain exacerbations and (2) clinical severity and resolution of exacerbations is related to airway and systemic inflammation. Methods: In a prospective longitudinal cohort study in COPD, sputum and serum samples obtained before, at, and following exacerbations during a 2-year period were studied. Measurements and Main Results: Clinical information, molecular typing of bacterial pathogens, sputum IL-8, tumor necrosis factor (TNF)-a and neutrophil elastase, and serum C-reactive protein. From 46 patients, 177 exacerbations were grouped as new strain, preexisting strain, other pathogen, and pathogen negative. New strain exacerbations were associated with significantly greater increases from baseline in sputum TNF-a and neutrophil elastase, and in serum C-reactive protein compared with the other three groups. Increases in inflammatory markers were similar among the other three groups. Clinical resolution was accompanied by resolution of inflammation to preexacerbation levels, whereas persistent symptoms were paralleled by persistently elevated inflammation. Clinical exacerbation severity was significantly correlated with levels of all four markers. Conclusions: Neutrophilic airway inflammation and systemic inflammation are more intense with well-defined bacterial exacerbations than with nonbacterial exacerbations. Clinical course of exacerbation and inflammation are closely linked. Keywords: chronic obstructive pulmonary disease; exacerbation; inflammation

Inflammation in the lung is prevalent in all stages of chronic obstructive pulmonary disease (COPD) and is now regarded as a major contributor to disease pathogenesis (1, 2). Furthermore, an increase in airway inflammation above baseline has been demonstrated in exacerbations of COPD in several studies (3–7). Whether the etiology of an exacerbation determines the inflammatory profile seen in the airway is controversial (3, 5–8). In some studies, increased airway mediators and cells characteristic of neutrophilic inflammation have been described with

(Received in original form August 21, 2007; accepted in final form December 11, 2007) Correspondence and requests for reprints should be addressed to Sanjay Sethi, M.D., VA WNY Healthcare System (151), 3495 Bailey Avenue, Buffalo, NY 14215. E-mail: [email protected] This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Am J Respir Crit Care Med Vol 177. pp 491–497, 2008 Originally Published in Press as DOI: 10.1164/rccm.200708-1234OC on December 13, 2007 Internet address: www.atsjournals.org

AT A GLANCE COMMENTARY Scientific Knowledge on the Subject

Changes in airway inflammation with bacterial exacerbations of chronic obstructive pulmonary disease are not consistent among studies. This may be related to lack of molecular characterization of bacterial strains isolated at exacerbation and the timing of stable sampling. What This Study Adds to the Field

New bacterial strain exacerbations are associated with more intense airway and systemic inflammation than those with preexisting strains and nonbacterial episodes. Clinical severity and resolution of exacerbation and inflammation are closely linked.

bacterial exacerbations, whereas eosinophilic inflammation has been associated with viral exacerbations (3, 6, 7). Systemic inflammation is now increasingly recognized as a feature of COPD (9, 10). Increased serum C-reactive protein (CRP), fibrinogen, and IL-6 with exacerbation has been described (11, 12). However, whether systemic inflammation differs on the basis of etiology of exacerbations has not been systematically examined. Although much has been learned about inflammation in COPD exacerbations, several important questions remain in this area of research. The presence of bacteria in respiratory secretions at the time of exacerbation is not adequate to define the etiology as bacterial infection, as these patients are often chronically colonized in the airway with the same bacterial pathogens. Molecular characterization of the bacterial pathogens isolated is required to accurately define bacterial infection, because of the specific association with strains of bacteria that are new to the patient with development of an exacerbation and a specific immune response to the infecting strain of the pathogen (13, 14). To fully understand airway inflammation in exacerbation, it is necessary to perform molecular characterization of the isolated bacterial pathogens to accurately discriminate bacterial infection from preexisting colonization (3–7). Whether there is a difference in the systemic and airway inflammatory profile when new strains of bacteria are present as compared with preexisting strains is not known. There is wide variability in the baseline level of airway and systemic inflammation among patients with COPD. Therefore, to accurately elucidate changes in airway inflammation at the time of exacerbation, it is best to have baseline measurements from the same patient for comparison. Recent studies have used stable-state sampling for comparison and have contributed considerably to our knowledge in this field (3–5, 15, 16). In some of these studies, stable-state samples were obtained before the exacerbation and then the patients were prospectively

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followed until they had an exacerbation (4, 5, 15). One limitation of this approach is that the time interval between the two samples has been variable, and often the stable-state samples were obtained several months before the exacerbation (4, 5, 15). The progressive nature of COPD and changes in chronic treatment could potentially confound the results when stable samples are not obtained close to the onset of exacerbation. Alternatively, in other studies, the stable samples were obtained after resolution of exacerbation, in which case treatment effects could confound the measurements (3, 16). Another important question not fully answered is whether clinical resolution and resolution of inflammation in exacerbations are correlated. Available studies have been confined to purulent exacerbations or have only studied patients whose exacerbation resolved (3, 17). We have been conducting a prospective longitudinal study in a cohort of patients with COPD to understand the dynamics of bacterial infection in COPD and to evaluate immune and inflammatory responses to bacterial infection in this disease. The participants in this cohort study are seen on a monthly basis and at the time of suspected exacerbations in our study clinic. At each clinic visit, in addition to collecting clinical information, a spontaneously expectorated sputum sample as well as a serum sample are collected. Inflammatory markers from selected sputum supernatant and serum samples from this study were evaluated in this study to test the following specific hypotheses: (1) the increase in systemic and airway inflammation from baseline is greater in exacerbations associated with new strains of bacteria than in exacerbations associated with preexisting strains and nonbacterial exacerbations; (2) clinical resolution is related to decreased inflammation, and clinical failure is associated with a persistent increase in airway inflammation; (3) the intensity of airway and systemic inflammation is related to the clinical severity of an exacerbation. Whether any of the markers of airway and systemic inflammation that we measured could be clinically useful in distinguishing the etiology of exacerbations was also explored. Some of the results of these studies have been previously reported in the form of an abstract (18).

METHODS COPD Study Clinic Details of this study clinic have been described previously (13, 14, 19). The institutional review board of the Veterans Affairs Western New York Healthcare System (Buffalo, NY) approved the study protocol. All participants gave written, informed consent. Briefly, participants with smoking-associated chronic bronchitis or COPD are enrolled and followed on a monthly basis and also whenever they suspect they are experiencing an exacerbation. At each clinic visit, clinical information and sputum and serum samples are collected. Exacerbations were defined on the basis of symptoms as described earlier (14). Each exacerbation visit was designated as a new or a continuing exacerbation based on predetermined criteria as described previously. A cohort of 50 participants was initially enrolled in 1994, with additional participants enrolled as necessary to maintain the size of the cohort. For this study, selected clinic visits and corresponding sputum and serum samples obtained in 1999–2000 were analyzed.

Sample Selection Preexacerbation, exacerbation, and postexacerbation sputum and serum samples were selected for this study for the exacerbations experienced by the participants in the COPD study clinic during a 2-year period from January 1, 1999, through December 31, 2000. If an exacerbation sample was not available, the corresponding pre- and postexacerbation samples were also not included in this study. Preexacerbation samples were obtained before the onset of exacerbation in a clinically stable patient. Available samples closest to the onset of exacerbation were used. Postexacerbation samples were obtained when the patient was no longer experiencing symptoms of an exacerbation and no further treatment for

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the exacerbation was prescribed. If the patient had multiple visits for a continuing exacerbation, those were included in the analysis of the relationship between clinical resolution and inflammation.

Clinical Score For each clinic visit, a clinical score was calculated on the basis of symptoms and signs, and the range of possible clinical scores was from 10 to 30. Further details are provided in the online supplement.

Sputum and Serum Sample Processing Details have been described previously (13, 14, 19). Briefly, spontaneously expectorated morning sputum samples are homogenized with the addition of ditheothreitol (Sputolysin; Calbiochem, San Diego, CA) and an aliquot processed for quantitative bacteriology. Sputum supernatants are obtained by centrifugation and stored at 2808C. Serum was obtained from venous blood samples and stored at 2808C.

Strain Characterization and Designation Strains of nontypeable Haemophilus influenzae, Haemophilus haemolyticus, Streptococcus pneumoniae, Moraxella catarrhalis, and Pseudomonas aeruginosa were characterized by molecular typing as described earlier (14, 20). Strains of these pathogens not previously isolated from a participant were designated as new, whereas strains isolated previously were designated as preexisting strains. Strain characterization was not conducted for gram-negative bacilli, Staphylococcus aureus, and Haemophilus parainfluenzae.

Inflammatory Marker Measurement Sputum supernatant levels of IL-8, tumor necrosis factor (TNF)-a, and active neutrophil elastase (NE) were measured as described earlier (6). Serum CRP levels were measured by sandwich ELISA as described in the online supplement.

Statistical Analysis Data are presented as mean 6 SEM when normally distributed and as median 6 interquartile range when nonnormally distributed. Analyses were performed for paired data by comparing preexacerbation, with exacerbation, and postexacerbation samples. Changes in log-transformed CRP, IL-8, TNF-a, and NE were compared with paired t tests and analysis of variance, Tukey’s test was used to determine betweengroup differences to account for multiple comparisons. Nonnormally distributed data were analyzed with Wilcoxon signed rank and MannWhitney U tests.

RESULTS Number of Exacerbations and Samples

There were 1,033 clinic visits by 65 patients and 186 exacerbation visits by 50 patients in the 2-year time period. Of the exacerbation visits, 158 were initial visits and 28 were repeat visits for continuing exacerbations. Of these, sputum samples were not provided by the subjects in seven visits, and in an additional two visits sputum had been collected but was not available for this study. Therefore, 177 of the 186 (95.2%) exacerbation visit samples from 46 patients were included in this study, of which 150 were initial visits and 27 were repeat visits for a continuing exacerbation. Corresponding 148 preexacerbation and 133 postexacerbation samples were available and included in this study. All exacerbations were initially treated on an outpatient basis, and therefore would be considered as mild to moderate exacerbations. Subject Demographics

There were 65 subjects who had at least one COPD study clinic visit during the 2 years of the study. Of these, 46 subjects had at least one exacerbation and contributed samples to this study. The demographics of the subjects with COPD who contributed at least one sample to this study are shown in Table 1.

Sethi, Wrona, Eschberger, et al.: Inflammation in New Strain Exacerbation TABLE 1. DEMOGRAPHIC AND CLINICAL CHARACTERISTICS OF STUDY CLINIC SUBJECTS THAT CONTRIBUTED SAMPLES TO THIS STUDY Age, mean yr (range) Sex, n

66.1 (46–85) Male: 45 Female: 1 Whites: 42 African Americans: 4 11.9 (0–50) Ex-smokers: 31 Current smokers: 15 76.8 (10–150) 1.74 (0.56–4.07) 50.3 (17–99) 053 151 2 5 19 3 5 16 457

Race, n Mean yr since diagnosis (range) Smoking status on enrollment, n Mean smoking pack-years (range) Mean FEV1, L (range) Mean FEV1% predicted (range) GOLD stage (no. subjects)

Definition of abbreviation: GOLD 5 Global Initiative for Chronic Obstructive Lung Disease.

Designation of Exacerbation Etiology

Exacerbations were divided into four groups based on sputum culture results and strain characterization. New strain exacerbations had a new strain of H. influenzae, S. pneumoniae, M. catarrhalis, and P. aeruginosa isolated from sputum at the time of exacerbation. Preexisting strain exacerbations had a strain isolated of one of these four species that had been previously isolated from sputum in the same patient. Other pathogen exacerbations had S. aureus or gram-negative bacilli other than P. aeruginosa isolated from sputum. Pathogen-negative exacerbations had only normal flora, H. parainfluenzae, and/or H. haemolyticus isolated from sputum. Of the 150 exacerbations, 39 (26%) were new strain, 15 (10%) were preexisting strain, 30 (20%) were other pathogen, and 66 (44%) were pathogennegative exacerbations. Specific pathogens isolated are described in Table 2. Change in Inflammatory Markers with Onset and Etiology of Exacerbation

The measured levels of the inflammatory markers at preexacerbation, exacerbation, and postexacerbation visits are shown in Table E1 in the online supplement. Baseline inflammation varies among patients with COPD. Therefore, all analyses were performed for paired data by comparing preexacerbation, with exacerbation, and postexacerbation samples. To accurately measure the change in airway inflammation with onset of an exacerbation, changes in log-transformed values of the inflammatory markers with the onset of exacerbation was calculated by subtracting the preexacerbation level from the exacerbation level for each episode (n 5 148 pairs). Significant increases in IL-8 (1.38 ng/ml [1.20–1.58 ng/ml]), geometric mean (95%

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confidence interval [CI], P , 0.0001), TNF-a (3.72 ng/ml [2.59– 3.76 ng/ml], P , 0.0001), and NE (6.36 nM [3.51–11.5 nM], P , 0.0001) were seen with the onset of exacerbation, as well as in serum CRP (2.12 mg/L [1.7–2.65 mg/L], P , 0.0001). New strain exacerbations had the largest increase in airway inflammation with significantly greater changes in TNF-a and NE when compared with pathogen-negative and other pathogen exacerbations (Figure 1). By contrast, the change in sputum IL-8 showed no differences among the four groups of exacerbations. Increase in systemic inflammation (i.e., serum CRP) was also significantly higher with new strain exacerbations in comparison to each of the other three groups of exacerbations. Changes in sputum and serum inflammatory markers did not differ among preexisting strain, other strain, and pathogen-negative exacerbations. Change in Inflammatory Markers with Resolution of Exacerbations

Inflammatory marker changes with resolution of exacerbation were calculated by subtracting the postexacerbation level from the exacerbation level for each marker (n 5 133 pairs). Both airway and systemic inflammation decreased with resolution of exacerbation. Significant decreases in IL-8 (1.30 ng/ml [1.12– 1.51 ng/ml]), geometric mean (95% CI, P 5 0.0009), TNF-a (3.37 ng/ml [2.27–5.02 ng/ml], P , 0.0001), and NE (5.65 nM [3.13–10.2 nM], P , 0.0001) were seen with the resolution of exacerbation, as well as in serum CRP (1.73 mg/L [1.35–2.21 mg/L], P , 0.0001). Changes between pre- and postexacerbation levels were compared by subtracting the postexacerbation level from the preexacerbation levels. Postexacerbation levels of sputum IL-8, TNF-a, and NE, and serum CRP were no different from preexacerbation levels (data not shown). In 27 instances, patients experienced continuing exacerbations where sputum and/or serum samples had been collected. Sputum IL-8, TNF-a, and NE did not change significantly in these 27 visits when compared with the index exacerbation visits. Changes in the inflammatory markers on the nonresolving visit when compared with the index exacerbation visit were as follows: IL-8 (1.03 ng/ml [0.76–1.38 ng/ml]), geometric mean (95% CI, P 5 0.86), TNF-a (2.10 ng/ml [0.84–5.24 ng/ml], P 5 0.12), and NE (0.68 nM [0.14–3.34 nM], P 5 0.63). Change in serum CRP from the index exacerbation visit was also not significant in the 17 serum samples obtained in these continuous exacerbation visits (1.1 mg/L [0.69–1.75 mg/L], P 5 0.70). These results indicate that persistent symptoms of exacerbation were paralleled by persistent elevation in inflammatory markers. Correlations between Clinical Severity and Inflammatory Markers

Clinical severity of the exacerbation was assessed by measuring the clinical score, a combination of symptoms and signs. This clinical score at exacerbation was significantly related with levels of sputum TNF-a, sputum NE, and serum CRP (Pearson

TABLE 2. POTENTIAL BACTERIAL PATHOGENS ISOLATED FROM SPUTUM New Strain Exacerbations (n 5 39) Nontypeable Haemophilus influenzae 5 20 Streptococcus pneumoniae 5 5 Moraxella catarrhalis 5 12 Pseudomonas aeruginosa 5 5

Preexisting Strain Exacerbations (n 5 15)

Other Pathogen Exacerbations (n 5 30)

Pathogen-negative Exacerbations (n 5 66)

Nontypeable Haemophilus influenzae 5 4 Streptococcus pneumoniae 5 2 Moraxella catarrhalis 5 6 Pseudomonas aeruginosa 5 7

Enterobacter spp. 5 12 Klebsiella pneumoniae 5 7 Serratia spp. 5 4 Stenotrophomonas maltophila 5 3 Escherichia coli 5 2 Staphylococcus aureus 5 5 Others 5 5

Haemophilus hemolyticus 5 2 Haemophilus parainfluenzae 5 54 Normal flora 5 12

The number of pathogens in each group exceeds the number of exacerbations because of simultaneous multiple pathogen isolation from some sputum samples.

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Figure 1. (A–D) Changes in inflammatory markers from baseline in a paired analysis by patient and etiology. Geometric means 6 1 SEM of logtransformed values are shown. The P values are by analysis of variance. Significant differences between groups are depicted by arrows. CRP 5 C-reactive protein; IL-8 5 interleukin-8; NE 5 neutrophil elastase; TNF-a 5 tumor necrosis factor-a.

correlation coefficients of 0.16 [P 5 0.05], 0.23 [P 5 0.005], and 0.27 [P 5 0.002], respectively). Sputum IL-8 did not demonstrate this relationship (Pearson correlation coefficient of 0.08, P 5 0.36). A similar analysis using the change in each of these marker levels from the preexacerbation baseline with clinical score showed a stronger relationship with all four markers demonstrating a significant association with the clinical score at exacerbation (Figure 2). Changes in sputum NE and serum CRP had stronger correlations with the clinical severity of the exacerbation than the other two inflammatory markers. Consistent with the observation that inflammatory changes were greater with new strain exacerbations, these exacerbations were also associated with significantly higher clinical scores (new strain [group 1], 18.95 6 0.56, vs. no new strain [groups 2–4], 17.82 6 0.27; mean 6 SEM, P 5 0.04). Predictive Value of Biomarkers

A biomarker that could distinguish bacterial from nonbacterial exacerbations could be clinically extremely useful in tailoring antibiotic treatment of exacerbations. To determine whether inflammatory markers singly or in combination could be used

to distinguish new strain exacerbations from the other three groups of exacerbations, we calculated area under the receiver operating characteristic (ROC) curves for each of these inflammatory markers (Table 3). As a single marker, sputum TNF-a had the largest area under the ROC of 0.79. Combinations of inflammatory markers performed better than single markers, with the largest area under ROC of 0.85 achieved with the combination of sputum TNF-a, sputum NE, and serum CRP (Figure 3).

DISCUSSION Several studies in the past few years have clearly established that exacerbations of COPD are inflammatory events, and are accompanied by both airway and systemic inflammation (3–7). The present study adds to this body of literature in several ways. In this study, exacerbations associated with acquisition of new strains of bacteria, specifically H. influenzae, S. pneumoniae, M. catarrhalis, and P. aeruginosa, were clearly associated with a more intense neutrophilic inflammatory response in the airway, as well as more intense systemic inflammation, compared with exacerbations not associated with such strain acquisition. This

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Figure 3. Receiver operating characteristic curve for distinguishing new strain exacerbations with sputum tumor necrosis factor-a, sputum neutrophil elastase, and serum C-reactive protein included in the prediction model. AUC 5 area under the receiver operating characteristic curve.

Figure 2. Correlation between changes from baseline in inflammatory markers (log-transformed values) and clinical severity of exacerbation measured as clinical score. Pearson’s correlation coefficient r and the corresponding P value are shown. CRP 5 C-reactive protein; IL-8 5 interleukin-8; NE 5 neutrophil elastase; TNF-a 5 tumor necrosis factor-a.

adds to evidence gathered from bronchoscopic, molecular epidemiology, and immune response studies that these bacteria indeed cause a substantial proportion of exacerbations of COPD (13, 14, 19, 21). In contrast, the fact that preexisting strain exacerbations had an inflammatory profile similar to pathogen-negative exacerbations suggests that, in these exacer-

bations, the bacteria isolated are indeed ‘‘innocent bystanders’’ and likely not causative. A variety of other gram-negative bacilli and gram-positive bacteria in addition to the pathogens discussed above can be isolated from the sputum of patients with exacerbations of COPD and were grouped as ‘‘other bacteria’’ in this study. Neutrophilic airway inflammation and systemic inflammation in this group was no different from pathogen-negative exacerbations in this study. This would suggest a nonpathogenic role of these bacteria. One limitation is the lack of molecular typing to distinguish new acquisitions from preexisting strains among these species. Therefore, it is still possible that new strains of these bacteria indeed cause exacerbations, a possibility that needs further study. Previous studies have had contradictory observations with regard to whether exacerbations associated with bacterial pathogen isolation are any different from nonbacterial exacerbations (3, 5–8). Papi and colleagues demonstrated that patients with exacerbations uniformly have neutrophilic inflammation; however, there was no difference between bacterial (positive sputum culture) and nonbacterial (negative sputum culture) exacerbations (3). We did not measure sputum neutrophil counts; however, in contrast to their results, our study shows a clear difference in markers of neutrophilic inflammation between bacterial (new strain, group 1) and nonbacterial (no new strain, groups 2–4) exacerbations. One likely reason for this different result is that our study distinguished colonizing preexisting strains and infecting new strains by molecular typing. In fact, as discussed above, the inflammatory profile of exacerbations with preexisting strains isolated from sputum was no different from pathogen-negative exacerbations, emphasizing the importance of molecular characterization of these strains. Another likely reason for variant results in the two studies is the difference in the timing of the baseline visit. In Papi and colleagues’ study, the baseline visit was after rather than before the exacerbation, as in this study. Treatment of exacerbation with potent antibiotics and corticosteroids could make long-lasting differences in airway inflammation, especially if such treatment leads to bacterial eradication, thereby confounding the results (7, 17, 22). Of the inflammatory markers measured, sputum IL-8 did not distinguish new strain exacerbations from the others and had the weakest correlation with the severity of the exacerbation. This is likely related to the fact that other potential causes of exacerbations, including rhinovirus, particulate airborne pollution, and cigarette smoke, are all capable of inducing IL-8 from respiratory epithelium (23–25).

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TABLE 3. RECEIVER OPERATING CURVE ANALYSIS FOR NEW STRAIN EXACERBATIONS AND INFLAMMATORY MARKERS Inflammatory Marker Sputum IL-8 ng/ml Sputum TNF-a ng/ml Sputum NE nM Serum CRP mg/L Sputum TNF-a, sputum NE, serum CRP* Sputum TNF-a, sputum NE, serum CRP†

AUC for Change from Baseline

AUC for Absolute Value

Cutoff Level

Sensitivity (%)

Specificity (%)

Positive Predictive Value (%)

Negative Predictive Value (%)

0.58 0.70 0.74 0.71 NA NA

0.68 0.79 0.76 0.71 0.85 0.85

1.39 0.32 0.76 2.37 NA TNF 5 0.32 NE 5 0.76 CRP 5 2.37

66.7 71.8 71.1 61.8 72.7 84.8

65.8 70.3 68.2 68.3 85.0 71.0

40.6 45.9 43.5 39.6 61.5 49.1

84.9 87.6 87.2 84.1 90.4 93.4

Definition of abbreviations: AUC 5 area under the receiver operating characteristic curve; CRP 5 C-reactive protein; NE 5 neutrophil elastase; TNF 5 tumor necrosis factor. The cutoff level, sensitivity, specificity, and positive and negative predictive values for distinguishing new strain exacerbations are for the absolute values of each marker. * Based on the best combination of TNF-a, NE, and CRP according to the receiver operating characteristic curve. † Based on the best cut-point of TNF-a, NE, and CRP in the univariate analysis.

Gompertz and coworkers have shown that, when purulent sputum exacerbations are treated with antibiotics, resolution of exacerbations is associated with progressive reduction of neutrophilic airway inflammation (7). This study adds to these observations by demonstrating that the reverse phenomenon is also seen: nonresolution of symptoms is associated with nonresolution of airway inflammation. These observations suggest that persistently elevated inflammation drives the symptoms of an exacerbation and could potentially be used as a measure to objectively assess response to treatment. Severity of exacerbations of COPD has been measured in several different ways. In this study, we used a composite clinical score based on symptoms and signs to determine severity, and saw that acute changes in this score were significantly correlated with acute changes in airway and systemic inflammation. This quantitative link between inflammation and clinical manifestations strengthens the link between airway inflammation and the occurrence of an exacerbation. Several recent studies have strengthened the link between airway neutrophilic inflammation and the pathogenesis of COPD. Sputum neutrophilia has been linked to peripheral airway dysfunction; bronchoalveolar lavage NE levels have been related to subclinical emphysema and peripheral airway dysfunction; and bronchial wall neutrophil counts are related to peripheral airway dysfunction (26–29). In contrast to older data, new studies have demonstrated a link between frequency of exacerbations and progression of airflow obstruction in COPD (30, 31). However, the etiology of exacerbations was not determined in these studies. One can speculate that the accentuation of neutrophilic inflammation associated with exacerbation is the mechanism of progression of lung damage and consequent airflow obstruction seen with exacerbations. Our study would then suggest that all exacerbations may not be the same with regard to loss of lung function. Bacterial exacerbations, associated with new strain acquisition, with significantly greater amounts of NE released in the airways, may cause more lung damage and contribute to a greater extent to progressive airflow obstruction in COPD. Studies with careful delineation of the etiology of exacerbation and accurate measurement of lung function in a large number of subjects are required to test this hypothesis. COPD is a systemic disease and systemic inflammation is a feature of stable COPD and during exacerbations (11, 12, 32). We extend those observations with clear evidence that the extent of systemic inflammation is much greater in carefully defined bacterial (new strain, group 1) exacerbations, as compared with nonbacterial (no new strain, groups 2–4) exacerbations. The systemic consequences of exacerbations that are linked to systemic inflammation may therefore not be the same for all exacerbations;

indeed, they may depend on the etiology of an exacerbation. These data suggest that bacterial exacerbations may cause greater systemic harm than nonbacterial exacerbations, and that this harm may be perpetuated by inadequate treatment of these exacerbations. Indeed, persistently elevated levels of CRP 14 days after initiation of treatment of an exacerbation have been described as predictive of early relapse (33). Limitations of this study include absence of cell counts, study of viral and atypical bacterial pathogens, and determination of eosinophilic and other cellular inflammation (3, 16, 34, 35). Viral infection likely contributed to a significant proportion of exacerbations among the four groups. Whether these exacerbations have an inflammatory profile distinct from noninfectious exacerbations cannot be discerned from this study. Future studies should attempt to address these limitations to provide an even more complete understanding of infection and inflammation in COPD exacerbations. In summary, this study has demonstrated that neutrophilic airway inflammation and systemic inflammation are more intense with well-defined bacterial exacerbations than nonbacterial exacerbations. On the other hand, exacerbations associated with preexisting strains are likely to be nonbacterial. In addition, clinical manifestations of the onset and resolution of exacerbation and inflammation are closely linked. Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

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