Published ahead of print on December 13, 2007, doi:10.1164/rccm.200708-1234OC
© 2008 American Thoracic Society doi: 10.1164/rccm.200708-1234OC
Inflammatory Profile of New Bacterial Strain Exacerbations of Chronic Obstructive Pulmonary Disease1 Division of Pulmonary and Critical Care and Sleep Medicine, Department of Medicine, University at Buffalo, State University of New York; 2 Veterans Affairs Western New York Healthcare System, Buffalo, New York; and 3 Division of Infectious Diseases, Department of Microbiology, and 4 Department of Biostatistics, University at Buffalo, State University of New York 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: ssethi{at}buffalo.edu
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)- 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.
Key Words: 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 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 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).
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
Clinical Score
Sputum and Serum Sample Processing
Strain Characterization and Designation
Inflammatory Marker Measurement
Statistical Analysis
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
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 pathogen-negative 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 = 148 pairs). Significant increases in IL-8 (1.38 ng/ml [1.20–1.58 ng/ml]), geometric mean (95% confidence interval [CI], P < 0.0001), TNF-  (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-
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 = 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 = 0.0009), TNF- (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-, 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-
Correlations between Clinical Severity and Inflammatory Markers
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- 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-, sputum NE, and serum CRP (Figure 3).
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 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 exacerbations, 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). 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.
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.200708-1234OC on December 13, 2007 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. Received in original form August 21, 2007; accepted in final form December 11, 2007
This article has been cited by other articles:
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TOP
ABSTRACT
AT A GLANCE COMMENTARY
