Smoking History Effect on Peripheral Lung Inflammation and Gene Transcription in Chronic Obstructive Pulmonary Disease

In this issue of AJRCCM (pp. 41-50), Szulakowski and coworkers (1) utilized peripheral lung parenchyma from age-matched nonsmokers, current smokers with normal lung function, and current and ex-smokers with Global Initiative for Chronic Lung Disease (GOLD) stages I to III chronic obstructive pulmonary disease (COPD) to explore the effects of current/past cigarette smoke exposure on proinflammatory cytokines, oxidative stress markers, histone acetylation/deacetylation balance, and the activation of the transcriptional regulatory proteins nuclear factor-?B (NF-?B) and activator protein-1 (AP-1). The article focuses our attention on the relationship between smoking history and the modulation of inflammation and gene transcription in COPD peripheral lung. This topic is certainly relevant and needs to be adequately addressed in future studies of the molecular pathology of COPD.

Some crucial aspects of this study and some inconsistencies with previously published studies deserve discussion. The major limitation, recognized by the authors, is the absence of a control group of ex-smokers with normal lung function. These investigations were conducted in humans, and looked at the molecular pathways modulated by tobacco exposure. In such human studies it is important to obtain biochemical verification of tobacco use and cessation (2). This is particularly relevant here, where many correlations between time since last cigarette smoked and inflammatory markers are evaluated (e.g., histone H4/actin ratio in Figure 8B and 8-isoprostane levels in Figure 12B). Indeed, previous studies have shown that regular cigarette smokers are highly heterogeneous (3). Of note, in a controlled clinical trial of nicotine replacement therapy, 23% of participants (presumably fairly motivated to quit) who self-reported 7 days of smoking abstinence had biochemical verification of tobacco use (4). Also, it is a common experience for respiratory clinicians to see a declared "ex-smoker" light up a cigarette just outside the outpatient clinic at the end of a follow-up visit. Acknowledging the complexity of cigarette smoking and classifying smokers into more specific subgroups based on their smoking behavior will enable future studies to better evaluate the effects of the amount of tobacco exposure on COPD pathogenesis. In addition, although the presence or absence of chronic bronchitis influences the type and degree of inflammation in COPD peripheral lung (5), this issue was not investigated in the current study. Another interesting characteristic of the study population (Table 1) is the presence of many females in all groups of smokers except in current smokers with COPD. This may reflect different smoking habits in Scotland, compared with other areas of the world, such as Italy, where there is still an overwhelming prevalence of male smokers (6). Because it is still unclear whether women are more susceptible to the proinflammatory effects of cigarette smoke than are men, this topic deserves future controlled studies.

Because nuclear and cytosolic proteins were isolated from blocks of peripheral lung, there are potential methodologic issues with some of the molecular data presented in this study (e.g., histone acetylation/deacetylation balance as well as activation of NF-?B and AP-1). In the online supplement (Figure E1), the authors confirm the presence of nuclear contamination in their peripheral lung cytoplasmic extracts in at least 15% of all their samples, but do not present any evidence about the possible contamination of nuclear extracts with cytoplasmic proteins. This reflects the methodologic limitations that we currently face in the absence of standardized techniques to extract nuclear proteins from complex tissues, such as peripheral lung. NF-?B is a major family of transcription factors that primarily resides in the cytoplasm, and is held in an inactive state by an inhibitor protein, I?Ba. Phosphorylation of I?Ba results in the release of active NF-?B, enabling it to translocate to the nucleus where it binds to target DNA elements and up-regulates the transcription of many genes involved in immune and inflammatory responses potentially relevant to the pathogenesis of COPD (7). In the current study, NF-?B and AP-1 activation has only been investigated using electrophoretic mobility shift assay (EMSA), using nuclear extracts from peripheral whole lung (a complex mixture of structural and inflammatory cells). Previous studies have clearly shown that, in the COPD lung, the NF-?B pathway is activated only in selected cell populations, such as alveolar macrophages and epithelial cells (7, 8). The present study would have benefited from immunohistochemical analysis to show if there is a differential activation of NF-?B in specific pulmonary cell populations.

AP-1 is a collection of related transcription factors and, like NF-?B, regulates many of the inflammatory and immune genes that are potentially relevant to the COPD pathogenesis (9). Many of these genes require the simultaneous activation of both transcription factors that work together cooperatively (9). This study is the first investigation of AP-1 activation in COPD peripheral lung. The authors have been unable to find any significant difference in AP-1 binding to DNA among the groups of subjects examined. This is in keeping with the results of a previous study performed on bronchial biopsies of patients with chronic bronchitis, including some with COPD (10). Again, the method used cannot rule out possible activation of AP-1 family members in selected cell types. It has become clear that attempts at global analysis of transcription factor activity using EMSA, or similar techniques, in tissues where multiple cell types are involved may give unrepresentative results. The combination of EMSA with cellular localization is important as are attempts to relate these changes to the transcription of individual genes using chromatin immunoprecipitation assays.

As with clinical controlled trials, the evidence-based medicine approach applied to the molecular biology of the lung dictates that many well-designed, carefully conducted clinical studies must be performed before new evidence can become a grade A recommendation (11). In the last decade, the scientific community has just started to use progressively more sophisticated methodologies to unravel the complex network of interactions among cigarette smoking, lower airway inflammation, gene transcription, and development of COPD (12-15). To accelerate research in this field, substantial investments are required at all levels, including the public and private sectors, with the ambitious aim, paraphrasing the recent American Thoracic Society/ European Respiratory Society guidelines (16), of making COPD a preventable and fully treatable disease.

Conflict of Interest Statement: Neither author has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.