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ELEGI, Colt Research Laboratories; and MRC/UoE Centre for Inflammation Research, The Queen's Medical Research Centre, University of Edinburgh, Edinburgh, United Kingdom
Correspondence and requests for reprints should be addressed to William MacNee, M.D., MRC/UoE Centre for Inflammation Research, The Queen's Medical Research Centre, University of Edinburgh, Little France Crescent, Edinburgh, UK. E-mail: w.macnee{at}ed.ac.uk
I found it both challenging and rewarding to summarize the 48th Aspen Lung Conference on the Pathobiology of Chronic Obstructive Pulmonary Disease (COPD). I would like to thank Norbert Voelkel, Tom Petty, and William Vandivier for giving me the honor of being the conference summarizer. The last Aspen conference on COPD took place in 2000. The conference summarizer on that occasion, Robert Senior, indicated that, by the next time we met on this topic, we would know "much more about the mechanisms of COPD . . . , but not everything." Well, we certainly know much more about the pathobiology of COPD as judged by the amount of new data presented at this Aspen Lung Conference, but we are still far away from knowing everything. Much has changed in COPD in the recent past; not least has been the recognition that what was once thought to be a "completely irreversible," untreatable condition does have treatments that can improve symptoms and health status, reduce exacerbations, and, in some cases, improve mortality.
The challenge from Norbert Voelkel in his introduction to the conference was to "answer the old questions and the new hard ones" related to the pathobiology of COPD, with a view to new treatment strategies for this condition.
COPD PHENOTYPE
Appropriately, the opening lecture in the conference from Bart Celli was on the topic of COPD phenotypes, a fundamental and important issue, which was mentioned in many of the presentations at the conference. We now recognize that COPD is a complex multicomponent disease in which chronic airflow limitation, although a characteristic feature, is only one aspect. Improvement in the FEV1 or reduction in the decline in FEV1, which so often in the past has been the critical endpoint for intervention, is certainly not the only outcome worth targeting for therapeutic intervention in COPD.
Measurements that relate to overinflation of the lungs, such as the inspiratory capacity (IC), have been increasingly shown to be important in relation to exercise capacity and symptoms in patients with COPD. Indeed, changes in IC after an intervention correlate better with changes in exercise endurance time than do changes in the FEV1 (1). Recent evidence has also shown a relationship between the IC/TLC ratio and survival in patients with COPD (2). Thus, the presence of static hyperinflation, and the dynamic hyperinflation that occurs on exercise, may represent a phenotype of COPD that predicts outcome in terms of dyspnea, exercise, and mortality, and is therefore is a target for therapy.
In addition, other phenotypic features of COPD to the FEV1, such as body mass index and measures of breathlessness and exercise tolerance (6-minute walking distance, Medical Research Council [MRC] dyspnea scale, body-mass index, airflow obstruction, dyspnea, and exercise capacity [BODE] index), enhance the prediction of mortality, in comparison with the FEV1 alone (3). Studies are needed to define different COPD phenotypes and their relation to relevant outcome measurements in COPD and the response to treatment.
Whether biomarkers are useful in phenotyping patients with COPD has yet to be determined. Data were presented at this meeting on biomarkers in serum assessed by proteomics, showing relationships with clinical parameters such as 6-minute walking distance, transfer factor for carbon monoxide, and the BODE index. Other studies, which used microarray and serial analysis of gene expression to assess patterns of gene expression, demonstrated differences in gene expression in lung tissue between at-risk patients and patients with COPD. The biological significance of some of the differences in gene expression was then validated in in vitro studies. An example was the difference in the expression of the gene encoding the transcription factors (early growth response gene [Egr-1], Fos) between smokers at risk and patients with COPD. In vitro studies indicate the biological relevance of this finding by demonstrating that the induction of matrix metalloproteinase (MMP)-2 in lung fibroblasts by cigarette smoke extract is Egr-1 dependent. These findings have relevance to the excessive matrix remodeling that occurs in the small airways of cigarette smokers. Studies were also presented that used cDNA, reverse transcriptase–polymerase chain reaction, and laser capture techniques to assess antioxidant enzyme and cytokine expression in the terminal bronchioles in resected lung tissue and showed a decrease in catalase expression in COPD, in association with up-regulation of inflammatory cytokines, such as interleukin (IL)-8 and monocyte chemotactic peptide (MCP)-1. The decrease in this important antioxidant enzyme in the lungs of patients with COPD would enhance the injurious and proinflammatory effects of oxidative stress.
GENETICS AND COPD
The "holy grail" in COPD is determination of the susceptibility factors that lead to only a proportion of smokers developing clinically significant COPD. Professor Bill Cookson provided an insight into the quest to find susceptibility genes for a complex genetic disease such as COPD. Studies using techniques such as candidate genes, positional cloning, and linkage disequilibrium mapping have all produced somewhat conflicting results. It may be more relevant to relate genetic susceptibility to the different phenotypes of COPD. The search for asthma genes that may be relevant to COPD has come up with some interesting associations with COPD. For example, the PHF11 gene, which has a role in histone acetylation/deacetylation, is associated with the emphysema phenotype in a COPD population and with body mass index. Professor Cookson emphasized the importance of assessing differences in gene expression in the epithelium between patients with COPD and smokers. An approach that he has taken is to study gene expression profiling during differentiation of airway epithelium, which may identify relevant genes that may have importance in COPD. There is clearly still a lot to do in COPD genetics!
Microarray techniques have also been used to identify and prioritize susceptibility genes in populations of patients with COPD. These studies have identified a number of biologically relevant candidate genes associated with increased susceptibility to COPD, including SERPINE2.
The National Emphysema Trial database has been used to define different COPD phenotypes and several polymorphisms, particularly in the epoxide hydrolase gene, which has a protective function against xenobiotics, and in the transforming growth factor ß1 (TGF-ß1)_gene, which may be involved in airway remodeling. These have been shown to be related with measures of COPD severity other than the FEV1, such as breathlessness and exercise capacity. Mice with different genetic backgrounds were used in one study to show that genetic influences regulate the effect of or responses to TGF-ß1 in the lungs and that these genetic controls may regulate the degree to which TGF-ß induces tissue remodeling responses results in emphysema and/or fibrosis in the lung. Polymorphisms of the human MMP-1 promoter also result in enhanced responsiveness of cells to cigarette smoke and may predispose to enhanced MMP-1 expression in the lungs and contribute to the development of COPD.
Studies in genetically manipulated (CD-1, emphysema-resistant) mice in which the NFR2 gene (important in signaling pathways for antioxidant enzymes) was disrupted showed enhanced cigarette smoke–induced airspace enlargement, associated with enhanced inflammation, apoptosis, and oxidative stress. Furthermore, other studies presented at the conference show that transgenic mice that overexpress copper-zinc superoxide dismutase (SOD) have decreased inflammation and oxidative stress in the lungs and developed less emphysema when exposed to cigarette smoke compared with wild-type mice. These studies lend further support to the role of oxidative stress in the pathogenesis of emphysema.
COPD IS A SYSTEMIC DISEASE
It is increasingly recognized that, although primarily a disease of the lungs, COPD has important systemic consequences (4, 5). The systemic effects of COPD are now well established. They range from systemic inflammation and oxidative stress, skeletal muscle dysfunction, and abnormal nutrition and metabolism, to endothelial dysfunction and effects on other organs. An interesting new concept, which was discussed at the conference, was the hypothesis that there may be individuals who have an enhanced systemic inflammatory response, an "inflammatory phenotype," which leads to susceptibility to COPD. Related to this, Professor Alvar Agusti introduced the concept that systemic inflammation may be both a cause and/or a consequence of COPD. It is clear that the influx of inflammatory cells into the lungs is derived from circulating blood cells and there is clear evidence of a systemic effect of cigarette smoke on circulating leukocytes, as shown by the increase in circulating neutrophil counts that occurs in smokers, the release of leukocytes from the bone marrow, and the acute sequestration of neutrophils in the pulmonary circulation that occurs during smoking. The intriguing possibility, therefore, is that patients with COPD may be prone to an enhanced systemic inflammatory response to cigarette smoke or infection, which results in abnormal inflammatory responses in the lungs.
One systemic manifestation of COPD that is gaining increasing prominence is the relationship between COPD and coronary heart disease. Recent studies have shown the interaction between systemic inflammatory markers, such as C-reactive protein, relevant to the pathogenesis of coronary artery disease, and COPD enhances cardiovascular risk (6). Interesting studies, which require further confirmation, also suggest that inhaled corticosteroids can reduce systemic inflammatory markers (7), and there are some data, albeit retrospective, that suggest that inhaled corticosteroids may in fact reduce the risk of death from myocardial infarction in patients with COPD (8). This has implications for the development of novel therapeutic interventions, which may both decrease the inflammation in the lungs in COPD and reduce systemic inflammation/oxidative stress, and thereby decrease the enhanced cardiovascular risk in COPD. Treatments such as statins and angiotensin-converting enzyme inhibitors should be considered in this regard. In addition, there is preliminary evidence that antioxidant therapy, principally N-acetylcysteine, given systemically can reduce markers of oxidative stress in blood and presumably also in muscle, and enhance muscle performance (9).
AN AUTOIMMUNE HYPOTHESIS OF COPD
The most recent definitions of COPD have included statements that COPD results from an enhanced or abnormal inflammatory response in the lungs to inhaling particles and gases, usually cigarette smoke (4, 10). This "enhanced inflammation" hypothesis has been the subject of much research. It is clear that all smokers develop inflammation in their lungs. This hypothesis suggests that patients who develop significant COPD have an enhanced inflammatory response. The trigger for such an enhanced inflammatory response is still debated. It has been suggested that lower respiratory tract infection, bacterial or viral, may enhance an adaptive immune response in the lungs (11). More recently, it has been suggested that an autoimmune component may enhance airspace inflammation (12). The question arises: What are the possible antigens in COPD and what evidence is there for autoimmunity in COPD?
Data presented at the conference showed evidence of oligoclonal CD4+ T cells in the lungs of patients with severe emphysema who underwent lung transplantation. These data suggest a response to an antigen may have a role in the persistent lung inflammation that characterizes severe COPD.
Studies in animal models also suggested that autoimmune responses may result in the development of emphysema. In a novel animal model, rats injected with human umbilical vein endothelial cells (HUVECs) or with pulmonary artery smooth muscle cells developed antibodies to these cells. The animals that developed antibodies to HUVECs also developed airspace enlargement. In this animal model, similar events occurred as had been noted in previous models of apoptosis-mediated emphysema, produced by down-regulation of the VEGF receptor, such that HUVEC-immunized animals developed endothelial cell death, significant up-regulation of MMPs, and accumulation of CD4+ T cells in the lungs. Adoptive transfer of pathogenic, spleen-derived CD4+ cell populations into naive immunocompetent animals also resulted in emphysema (13). These studies show that humeral and CD4-dependent mechanisms are sufficient to trigger the development of airspace enlargement in these animal models. Intriguingly, data were also presented that intraperitoneal injection of cigarette smoke condensate in rats produced airspace enlargement after only 5 weeks, suggesting an autoimmune response to cigarette smoke! Other studies presented at the meeting demonstrated the presence of activated peripheral blood CD4+ T cells in patients with COPD and that this correlated with the degree of airway obstruction. All of these studies support a role of autoimmunity in the development of enhanced inflammation and in at least the emphysematous phenotype of COPD.
BRONCHIOLITIS AND AIRWAY REMODELING
Professor Jim Hogg presented his fascinating studies of resected lung tissue at the conference. His most recent data showed that increased small airway occlusion occurs with increased severity of the disease, and intriguingly, preliminary data suggest that airway occlusion with mucus and cells may affect survival. This fits with some epidemiologic studies which show that, with increasing airway obstruction, the presence of chronic hypermucus secretion influences survival, which is not the case in the early stage of the disease (14). It is likely that the peripheral airway mucus plugging contributes to the overinflation that characterizes COPD. Because both IC/TLC and small airway occlusion have been shown in independent studies to relate to survival, it is interesting to speculate that these two are related and that IC/TLC may well be the phenotypic characteristic of mucus plugging in the peripheral airways in the lungs of subjects with COPD.
Professor Hogg developed a hypothesis for the mechanism of the airway remodeling that occurs in the small airways and accounts for the increased airway resistance. He invoked a hypothesis involving injury to the epithelium; up-regulation of IL-1ß/IL-1ß receptor, resulting in up-regulation of p38 mitogen activated protein (MAP) kinase; mRNA stabilization for the platelet-derived growth factor receptor (PDGFR)
gene because of the up-regulation of IL-13; signal transducer and activator of transcription (STAT)6 signaling; and PDGFR expression leading to cell proliferation. In addition, TGFß has a role in decreasing PDGFR growth inhibition and in increasing collagen synthesis. Using a combination of immunostaining, protein expression, and laser capture microdissection of small airways to assess gene expression by real-time polymerase chain reaction, Professor Hogg showed that all of the players in the hypothesis are present in the airways and that there is a relationship between gene expression for IL-1ß and tumor necrosis factor (TNF)- and measures of airflow obstruction in COPD. He has shown a clear increase of lymphoid follicles in the airways in patients with GOLD (Global Initiative for Chronic Obstructive Lung Disease) stages III and IV COPD, and he hypothesized that this may be related to the development of lower respiratory tract infections at this stage of the disease. He presented some interesting preliminary data which suggest that corticosteroid treatment may cause a reduction in airway follicles and the adaptive immune response in the later stages of the disease.
Related to the mechanism of the bronchiolitis and airway remodeling in COPD, data presented by Dr. Andrew Churg using cigarette smoke–exposed tracheal explants showed that increased TGF-ß through SMAD signaling pathways increased connective tissue growth factor (CTGF), leading to increased procollagen gene expression, a process that would drive fibrosis. His data also showed that cigarette smoke can directly induce the release of pre-formed TGF-ß by oxidizing the latency-associated peptide. Thus, remodeling may reflect a direct induction of growth factors without the need to invoke an inflammatory response.
MOLECULAR AND CELLULAR MECHANISMS OF ALVEOLAR DESTRUCTION IN EMPHYSEMA
In a series of elegant studies of knockout transgenic animal models, Dr. Jack Elias elucidated the role of Tc1 and Tc2 (IL-13/IL-4) pathways in emphysema and mucus hypersecretion. He stressed that there may be both inflammatory and destructive pathways in the pathogenesis of emphysema and that there may be genetic modifiers that influence these pathways in addition to the presence of cytokine modifiers such as VEGF. The relevance of these animal models needs to be applied to human disease.
Professor David Lomas provided us with an insight into the mechanism of PiZZ1-antitrypsin (PiZZ-AT) deficiency and presented elegant work showing the formation of 1-antitrypsin (1-AT) polymers in vivo and the molecular structural changes that occur to produce these polymers. This will lead to the development of therapeutic strategies to prevent (1-AT) polymerization, either using chemical chaperones or blocking surface cavities by applying a cavity-filling molecule to attenuate polymerization. He also presented some fascinating data on another of the serpinopathies, showing the presence of neuroserpin polymers in the brain and their association with a new form of dementia. He also gave us an insight into how intralung polymers might not only result in inactivation of 1-AT but may themselves be proinflammatory, enhancing the development of emphysema.
Other studies showed that polymeric forms of 1-AT were only present in PiZZ-AT emphysematous lungs and that the oxidized form was only found in PiMM-AT emphysematous lungs, the latter in association with a significant increase in the amount of collagen. These data suggested that conversion of native 1-AT into oxidized form not only depletes the lungs of antiprotease protection but may also contribute to tissue repair.
Two state-of-the-art presentations at the conference demonstrated the role of apoptosis in the development of emphysema. Dr. Rubin Tuder gave a fascinating talk suggesting that emphysema may well be an accelerated aging process. He also showed the evidence that linked VEGF receptor blockade, the development of apoptosis and hence emphysema, and a role for oxidative stress in this process. Studies in which endothelial cells were targeted with lung homing/killing chimeric peptides resulted in the development of alveolar wall cell apoptosis and airspace enlargement indicative of emphysema, and showed that oxidative stress played a critical role in this process. He suggested that oxidative stress may be the link between protease/antiprotease imbalance and apoptosis in COPD and that it may be an amplification factor in the process of lung destruction. Further studies from his group showed a role for ceramide in pulmonary cell apoptosis in emphysema and an important role for sphingolipid homeostasis in the maintenance of alveolar cells. Other lipid pathways could also be involved in this process, suggesting that these may be novel therapeutic targets in this condition.
In a further paper, the role of peroxisome proliferators-activated receptor (PPAR)
as an endogenous regulator of normal lung maturation and maintenance was proposed. PPAR may be an important area of study because it is a nuclear hormone receptor that regulates gene expression, cellular differentiation, and inflammation. PPAR knockout mice develop airspace enlargement during development and smoke exposure in these mice produces enhanced emphysema compared with smoke exposure in wild-type mice. These studies suggest a potential role for PPARs in COPD.
Following a similar theme, Peter Henson provided us with a state-of-the-art review on phagocytosis and clearance of apoptotic cells in COPD. His hypothesis was that the presence of apoptosis in lung tissue or animal models of emphysema may be a result of one of two events, either massive apoptosis or failure of clearance of apoptotic cells. He suggested that there may be an abnormal response to apoptotic cells in the lungs of emphysematous subjects and in addition there may be inflammatory responses induced by postapoptotic cell necrosis. He proposed that emphysema may result from a defect in the normal cell replacement and clearance responses to apoptosis and indicated that this apoptotic process was significantly increased with age and is oxidant driven. Studies were also presented that statins enhance the clearance of apoptotic cells and may therefore have a role as a therapeutic intervention in this condition.
LUNG IMAGING
One of the most important developments in COPD research over the last 10 years has been the development of lung imaging, particularly imaging of emphysema. Eric Hoffman presented a state-of-the-art review on computed tomography (CT) scanning techniques and the ability of CT to determine lung dimensions and ventilation perfusion within the lungs. Presentations at the conference also highlighted the use of CT scanning to define distinct emphysematous and nonemphysematous phenotypes of COPD and showed that these phenotypes have different relationships to cigarette smoking. Other studies showed the relationship between pathologic characteristics of peripheral airways and outcome after lung volume reduction surgery in severe emphysema. In these studies, peripheral airway luminal content and not airway wall thickness was associated with poor survival and greater decline in exercise tolerance and dyspnea in patients with COPD.
COPD INFLAMMATION AND LUNG CANCER
Jerome Brody presented data that showed the interrelationships and differences between inflammation in COPD and lung cancer. Both conditions are associated with smoking, oxidative stress, and inflammation. He showed data of the gene expression profiling in COPD biopsy specimens and cells from patients with COPD that demonstrated the hallmarks of cancer and of COPD and the differences between these two conditions. In cancer, the hallmarks involve evading apoptosis, self-sustaining growth, tissue invasion, sustained angiogenesis, and limitless replication. In COPD, increased apoptosis, matrix aggregation, ineffective tissue repair, unique immune responses, and very little angiogenesis are characteristic.
The use of gene expression profiling to determine the epithelial field of injury in smokers, the so-called airway transcriptone, produced some interesting results showing increased expression in smokers of genes that have a protective role, such as antioxidant genes. Some smokers clearly do not react to smoke by up-regulating the genes for these protective mechanisms and may therefore be susceptible to either COPD or lung cancer. Interestingly, some of the changes in the expression of these genes do not return to normal after smoking cessation. The influence of epigenetic factors that either activate or silence genes, such as histone acetylation/deacetylation and methylation, may be important in this context. Dr. Brody put forward the proposal that the ability of an individual to maintain a stable genome may be a characteristic of cancer but not COPD, whereas in COPD, a specific immune response occurs.
CONCLUSIONS
The increasing complexity of the pathogenesis of COPD was emphasized at this meeting. There were some important new hypotheses on the pathobiology of COPD presented and discussed. The role of inflammation in both the systemic effects of COPD and in the destructive and remodeling processes that occur in COPD still remains to be fully elucidated. Whether inflammation is a consequence of tissue destruction or an integral part of processes such as protease/antiprotease, apoptosis and oxidative stress, which interact to produce COPD, is still debated. Whether a systemic inflammatory phenotype has a role in COPD or amplification of the inflammation occurs in certain smokers by genetic, infection, autoimmunity, oxidative stress, or epigenetic factors still needs to be worked out. It is important that we translate the basic mechanisms described in in vitro and in animal model studies into the human disease, which was exemplified by some of the studies at this meeting. We may be near to testing some of these theories in proof-of-concept studies with therapeutic interventions.
FOOTNOTES
Conflict of Interest Statement: W.M. has been reimbursed for travel by GlaxoSmithKline, Zambon, AstraZeneca, Boehringer Ingelheim, Pfizer, and Micromet for attending conferences. He has received honoraria from GlaxoSmithKline, AstraZeneca, Zambon, and Pfizer for participating as a speaker in scientific meetings. He served on advisory boards for Pfizer, Almirall, Amgen, Bayer, and Micromet, and serves on an expert panel for GlaxoSmithKline. He serves as a consultant for Pfizer and GlaxoSmithKline. Research grants to support work carried out in his laboratory come from Pfizer and Umlever.
(Received in original form March 20, 2006; accepted in final form March 22, 2006)
REFERENCES
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