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Departments of Pulmonology and Medical Decision Making, Leiden University Medical Center, Leiden, The Netherlands
Department of Pathology, Sao Paulo University Medical School, Sao Paulo, Brazil
ABSTRACT
Rationale: Patients with asthma have an accelerated decline in lung function, which can lead to irreversible airway obstruction. It is generally assumed that this is related to specific aspects of airway inflammation and/or remodeling.
Objective: We investigated the prognostic significance of bronchial eosinophil and CD8+ cell counts and subepithelial reticular layer thickness for the subsequent decline in lung function in patients with asthma after 7.5 years of follow-up.
Methods: In a prospective study, pre- and post-bronchodilator lung function (FEV1) was measured at baseline, and after 2 years and 7.5 years in 32 patients with asthma. Annual decline in lung function after 7.5 years of follow-up was related to type and severity of airway inflammation and remodeling in bronchial biopsies, which were taken at baseline and at Year 2.
Results: Annual decline in post-bronchodilator FEV1 (mean [SD], 46.6 [53.4] ml/year) was significantly larger than the decline in prebronchodilator FEV1 (mean [SD], 27.5 [62.5] ml/year), indicating loss in reversibility. Although annual fall in post-bronchodilator FEV1 was not related to thickness of the reticular layer or to eosinophil counts in bronchial biopsies, there was a significant correlation with CD8+ T cells (r = eC0.39, p = 0.032). Analyzing the biopsies taken at Year 2, the significant association between annual fall in post-bronchodilator FEV1 and CD8 cells could independently be confirmed (r = eC0.39, p = 0.036).
Conclusion: The outcome of asthma, as determined by the annual decline in FEV1, can be predicted by the bronchial CD8+ cell infiltrate. This suggests that the inflammatory phenotype in asthma has prognostic relevance, which may require phenotype-specific therapeutic strategies.
Key Words: asthma CD8 cells inflammation lung function decline prognosis
Asthma is a chronic inflammatory disease that is characterized by variable airway obstruction to various inhaled stimuli (1). Although this is largely reversible in most patients, some individuals with asthma develop persistent, nonreversible airway obstruction despite adequate treatment (2). Longitudinal studies have shown that adult patients with asthma have an accelerated decline in lung function (FEV1) as compared with control subjects (3eC5). However, the rate of decline demonstrates large variability between patients, which seems to be associated with disease duration, baseline lung function, and airway responsiveness (6, 7). Eventually, the lung function decline may progress to irreversible airway obstruction in a subgroup of patients with asthma (8).
The current working hypothesis is that chronic inflammation promotes restructuring of the airways, which in turn results in accelerated decline in lung function in some but not all patients with asthma. Airway inflammation in asthma is characterized by infiltration of lymphocytes and eosinophils in the bronchial epithelium and lamina propria (9) and of mast cells in the smooth muscle layer (10). Because of the release of growth factors and other mediators, the infiltrate is believed to induce structural changes in the bronchial wall, often referred to as "tissue remodeling" (11). Because this process begins early in the development of asthma, remodeling may occur in parallel or could even be required for the development of persistent inflammation (12). The features of airway remodeling in asthma include the following: thickening of the subepithelial reticular layer; changes of the interstitial matrix composition; increases in blood vessel area, airway smooth muscle, goblet cells in the surface epithelium; and number of mucous glands (11).
The prognostic significance of airway inflammation and remodeling for the decline in lung function is still unclear. In patients with chronic obstructive pulmonary disease, fixed airway obstruction is often found to be associated with bronchial CD8+ T-cell infiltration (13, 14). Cross-sectional studies in severe asthma have demonstrated that sputum and tissue eosinophil counts are associated with a lower lung function (8, 15). Furthermore, in some (16, 17), but not all, studies (15), the thickness of the subepithelial reticular layer was inversely associated with the level of lung function in asthma. However, it remains questionable whether these cross-sectional associations hold after longitudinal follow-up.
We postulated that the type and severity of inflammation or remodeling in bronchial biopsies are predictive of the subsequent annual decline in lung function in patients with asthma. For this reason, we performed a prospective follow-up study in a previously reported group of patients with asthma (Asthma Management Project University Leiden cohort) (18) who underwent repeated bronchoscopies and extensive clinical measurements at baseline. We aimed to investigate the relationship of bronchial eosinophil and CD8+ cell counts and the thickness of the subepithelial layer as measured at baseline with the subsequent annual decline in lung function after 7.5 years of follow-up.
Some of the results of this study have been previously reported in the form of an abstract (19).
METHODS
See the online supplement for further detail on METHODS.
Subjects
Seventy-five atopic patients with mild to moderate persistent asthma participated in the study (18). Forty-five patients underwent a successful bronchoscopy at entry and 37 patients at t (time) = 2 years. At inclusion, all patients (age, 18eC50 years) were non- or ex-smokers (< 5 pack-years); all had symptoms of episodic chest tightness and wheezing, whereas 77% of patients were using regular inhaled steroids. Prebronchodilator FEV1 was greater than 50% of predicted and more than 1.5 L, whereas post-bronchodilator was within the normal range (> 80% predicted) (20). All patients were hyperresponsive to methacholine (provocative concentration causing 20% fall in FEV1 [PC20] < 8 mg/ml).
The Medical Ethics Committee of the Leiden University Medical Center approved the study, and all participants gave written, informed consent.
Design
In a prospective study design, pre- and post-bronchodilator FEV1 and PC20 were measured at baseline, and at Years 2 and 7.5. Bronchoscopies were performed at baseline and Year 2.
During the first 2 years, patients were treated according to standardized guidelines, and treatment was adjusted by a chest physician every 3 months (18). To make this study representative for daily practice, the each patient's physician was instructed to adjust treatment according to Dutch Global Initiative for Asthma (GINA)eCderived guidelines between 2 and 7.5 years of follow-up.
Spirometry and Airway Responsiveness
Spirometry was performed according to the same procedures throughout the study (18). Patients withheld use of short-acting 2-agonists for 8 hours and long-acting 2-agonists for at least 24 hours before the measurements. Post-bronchodilator FEV1 was measured 15 minutes after inhalation of 400 e salbutamol (20). Airway hyperresponsiveness was determined using a methacholine challenge and was expressed as PC20.
Bronchoscopy and Immunohistochemistry
At baseline and after 2 years, five bronchial biopsies were taken for electron and light microscopy from right lower lobe subsegments, the middle lobe, and the main carina using a pair of cup forceps (Olympus FB-21C; Olympus Corp., Tokyo, Japan).
Two biopsies were fixed immediately in Trump's fixative, and ultra-thin sections were processed for electron microscopy. The thickness of the subepithelial reticular basement membrane was determined by measuring area divided by length on electron microscopy pictures in 2- to 5-welleCoriented electron micrographs (x5,700, 35 x 42 e), using computerized analysis (18).
Three biopsies were immediately embedded in ornithyl carbamyltransferase medium and snap-frozen in isopentane. Immunohistochemistry was performed on 6-e cryostat sections. Sections were stained with monoclonal antibodies against EG2 (eosinophils) (Pharmacia, Uppsala, Sweden), and CD8+ cells (Becton Dickinson, Mountain View, CA).
A validated method using computerized analysis was applied to examine the coded biopsy specimens (21). Two areas were selected and the number of positively stained cells was determined in the lamina propria. Values were expressed as cells/0.1 mm2.
Detailed biopsy methods and cell number data, including AA1 (mast cells), CD3, and CD4+ cells, have been previously published (18).
Analysis
Post-bronchodilator FEV1 was applied in the analysis to minimize the contribution of varying degrees of smooth muscle contraction to the level of airway obstruction. The decline in post-bronchodilator FEV1 was determined between baseline and t = 7.5 years (FEV1 at 7.5 years eC FEV1 at baseline/7.5) and between t = 2 and t = 7.5 years (FEV1 at 7.5 years eC FEV1 at 2 years/5.5), and was expressed as annual decline in milliliters/years. The declines in pre- and post-bronchodilator FEV1 were compared using a paired t test. Linear regression analysis was used to investigate the association between inflammation (EG2 and CD8+ cells and reticular layer thickness) in bronchial biopsies and annual decline in post-bronchodilator FEV1 during follow-up.
RESULTS
Patient Characteristics
Thirty-two of the 45 patients who underwent the bronchoscopy at baseline participated at follow-up after 7.5 years (71% response rate; Figure 1). The participating patients were not different from the nonparticipants with respect to disease severity, spirometry, reticular layer thickness, and EG2 and CD8+ cells (p > 0.2). In 30 of these 32 patients, biopsies were also taken at Year 2.
The total follow-up period was a mean of 7.6 (SD, 0.6) years. At all three time points, approximately 70% of the patients were using inhaled steroids (Table 1). None of the patients were using long-acting 2-agonists at t = 0 and t = 2 years, compared with six patients at t = 7.5 years. Seven patients stayed under regular care of a chest physician, whereas 23 patients were treated by a general practitioner. Only two patients stopped using any asthma medication and were free of symptoms. During the follow-up period, one in five received treatment with one or more courses of oral corticosteroids. Two patients had become current smokers after 7.5 years, whereas none smoked during the first 2 years. PC20 methacholine was less than 8 mg/ml in all patients at baseline, and in 28 of the 32 patients at t = 7.5 (range, 0.02eC16.3 mg/ml; Table 1).
Lung Function Decline
The mean pre- and post-bronchodilator FEV1 in % predicted stayed within the normal range at all visits, with considerable scatter (Table 1). The annual decline in prebronchodilator FEV1 during follow-up was a mean of 27.5 (SD, 62.5) ml/year, whereas the annual drop in post-bronchodilator FEV1 was 46.6 (SD, 53.4) ml/year (Figure 2). The variability in decline in post-bronchodilator FEV1 between individual patients was large, ranging from an annual increase of 39 ml/year to an annual fall of 149 ml/year. The decline in post-bronchodilator FEV1 was significantly larger than in prebronchodilator FEV1 (p = 0.022), indicating loss in reversibility (Figure 2).
Prognostic Significance of Airway Inflammation
The annual decline in post-bronchodilator FEV1 during the follow-up period was not related to thickness of the bronchial subepithelial reticular layer at t = 0 (r = eC0.02, p = 0.92; Figure 3). In addition, the fall in post-bronchodilator FEV1 during follow-up showed no correlation with eosinophils at baseline (r = 0.02, p = 0.90).
On the other hand, the annual change in post-bronchodilator FEV1 during the follow-up period of 7.5 years was significantly and inversely correlated with the bronchial CD8+ cells at t = 0 (r = eC0.39, p = 0.032). The slope of the linear regression analysis showed that for each doubling in CD8+ cells, post-bronchodilator FEV1 declined by an additional 13.8 ml/year.
When repeating the analysis using the bronchial biopsies taken at 2 years, these findings were entirely confirmed. There was a consistent, significant correlation of annual fall in post-bronchodilator FEV1 with the number of CD8+ cells at t = 2 years (r = eC0.39, p = 0.036), but not with bronchial eosinophils or reticular layer thickness (Figure 3).
All other cell types (AA1, CD3, and CD4) demonstrated no significant associations with the annual change in FEV1 (r < eC0.20, p > 0.28).
DISCUSSION
The results of this study show that the number of CD8+ cells in bronchial biopsies in patients with asthma is associated with disease outcome, as determined by loss of lung function. Other markers of inflammation or remodeling were not related to the decline in lung function during follow-up. Furthermore, the loss in post-bronchodilator FEV1 was significantly larger than the decline in prebronchodilator FEV1. These findings indicate that CD8 cells can be predictive of disease outcome in asthma and therefore suggest that targeting specific elements of inflammation may be required when aiming to prevent the accelerated decline in lung function in patients with asthma.
To our knowledge, this is the first longitudinal study showing the prognostic significance of type and severity of inflammation on the outcome of asthma. A cross-sectional relationship between CD8+ cells and the outcome of asthma has been observed in patients with fatal asthma (22). Recently, increased cytokine production of sputum CD8+ cells has been shown in patients with asthma that was related with disease severity (23). Interestingly, the association between lung function and CD8 cells has also been demonstrated in other diseases, not only by cross-sectional analysis in patients with chronic obstructive pulmonary disease (13) but also regarding decline in lung function in patients with systemic sclerosis (24). This shows that our longitudinal findings in asthma are in line with those in other inflammatory lung disorders.
The magnitude of the annual decline in lung function is in keeping with other longitudinal studies in patients with asthma and is higher as compared with the figures previously published for normal subjects (normal subjects, 22 ml/year, and patients with asthma, 38 ml/year) (5). Our results extend previous findings by demonstrating that the decline in post-bronchodilator FEV1 is larger than the decline in prebronchodilator FEV1. This puts emphasis on measuring post-bronchodilator FEV1, as a ceiling of lung function, in prospective studies in asthma.
The present study design may have potential limitations. During the follow-up period of 7.5 years, the patients were treated by their own physician as opposed to controlled, standardized therapy. This may have introduced variability in asthma control, because some patients were seeing a chest physician regularly (22% of patients), whereas others had not been visiting their doctor for asthma symptoms at all (6% of patients). We consider this strategy to be representative of daily practice. Before the baseline bronchoscopy, the patients were also treated by their own physician, or were newly diagnosed (23% of patients). Moreover, any variability in therapy may have led to a broader disease outcome, which is likely to be represented by the large range in annual decline in FEV1. For that reason, we chose common asthma management as opposed to protocolized therapy during follow-up of this cohort.
It is unlikely that the present association between decline in FEV1 and CD8+ cells is due to chance. This was a consistent finding when using bronchial biopsies of two separate bronchoscopies 2 years apart. The first and second bronchoscopy differed in that the second biopsy was taken after 2 years of optimal treatment according to the GINA guidelines or with management additionally based on airway hyperresponsiveness (18). This suggests that treatment level is not affecting the association between airway inflammation and lung function decline in asthma.
How can CD8 cells contribute to the accelerated, irreversible airway obstruction in asthma In vitro studies have characterized CD8+ cells with regard to their cytokine production (Tc1 vs. Tc2) and populations (effector vs. memory) (25). Interestingly, a subset of an antigen-specific, "nonlymphoid," memory CD8 T-cell population, which can be isolated from several organs, including the lungs, demonstrates a high lytic activity and proliferates rapidly (26). Various antigens, like allergens and viruses, can rapidly activate specific effector/memory T cells (27). In mice models, CD8 cells are required for the development of airway hyperresponsiveness after allergic sensitization (28), leading to increased inflammation (29). During respiratory virus infections, CD8 cells appear to be essential for the influx of eosinophils into the lung and the development of airway hyperresponsiveness in mouse models (30). Indeed, we have recently demonstrated that rhinovirus infection in subjects with asthma is associated with accumulation of CD8 cells (31). Interestingly, antigen-specific CD8 cells can persist in the lung for several months (32) and may also activate resident cells, such as epithelial cells (33). Therefore, CD8 cells can induce potential conditions that are required for changes in airway structure, which eventually may lead to changes in airway structure. However, we cannot exclude the possibility that the association of CD8 cells with lung function decline is just an epiphenomenon and a marker of a complex immunopathologic pathway.
Eosinophils were not predictive for the decline in lung function in our study. Increased numbers of sputum and tissue eosinophils have been associated with persistent airway obstruction in patients with severe asthma (8, 15). However, these conclusions were derived from cross-sectional data. Interestingly, it has been shown that elevated sputum eosinophil numbers may predict the short-term worsening of asthma as reflected by exacerbations (34). This suggests that the inflammatory profile may have distinct effects on short- and long-term disease outcome.
Remarkably, the thickness of the subepithelial reticular layer was also not related to lung function decline. This probably illustrates that restructuring of the airways as measured in large airway biopsies is not sufficient to represent other aspects of (small) airways remodeling (35). When sampling the latter in patients with chronic obstructive pulmonary disease, Hogg and coworkers (36) recently showed an association between airway structure and lung function level. However, comparable data in asthma will not be readily available.
Our findings can have implications for clinical management and drug development. First, the consistent association between FEV1 decline and CD8 cells even after 2 years of optimal standardized therapy in our study suggests that the current treatment strategies for asthma may not be effective in preventing or reversing the accelerated fall in lung function in patients with asthma. Second, even though the CD8 cell may just be a marker of another causative mechanism, the possibility of manipulating the presence and/or phenotype of CD8 cells should be considered. Glucocorticoids are able to induce a CD8-cell phenotype that produces high levels of interleukin 10 and reduced levels of interleukins 4 and 5 (37). However, the effect of glucocorticoid modulation of CD8-cell cytokine production is much smaller as compared with CD4 cells (37). Therefore, the development of new interventions specifically targeting CD8+ T cells may need to be explored when aiming to prevent the persistent airway obstruction in asthma.
In conclusion, we have shown that outcome of asthma, as determined by the annual decline in FEV1, can be predicted by bronchial CD8-cell infiltrate. CD8+ cells may have, as previously suggested in patients with chronic obstructive pulmonary disease, a significant role in the clinical course of asthma. We could speculate that this requires phenotype-specific therapeutic strategies to prevent the accelerated decline of lung function in asthma.
Acknowledgments
Departments of Medical Decision Making (J. K. Sont), Pulmonology (E. H. Bel, C. E. Evertse, K. F. Rabe, E. L. J. van Rensen, P. J. Sterk, L. N. A. Willems), Pathology (J. H. J. M. van Krieken), and Hematology (J. C. Kluin-Nelemans), Leiden University Medical Center, Leiden; St. Antoniushove Hospital, Leidschendam (C. R. Apap); Langeland Hospital, Zoetermeer (A. C. Roldaan, K. W. van Kralingen); and Diaconessenhuis Hospital, Leiden, The Netherlands (H. C. J. van Klink).
Supported, in part, by the Netherlands Asthma Foundation.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
AAPUL Study Group members are listed at the end of the article.
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