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Published online before print May 15, 2003, 10.1148/radiol.2281020187
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(Radiology 2003;228:85-94.)
© RSNA, 2003

Thoracic Imaging

Structure and Function of Small Airways in Smokers: Relationship between Air Trapping at CT and Airway Inflammation1

Patrick Berger, MD, PhD, François Laurent, MD, Hugues Begueret, MD, Vincent Perot, MD, Rozen Rouiller, MD, Chantal Raherison, MD, Mathieu Molimard, MD, PhD, Roger Marthan, MD, PhD and J. Manuel Tunon-de-Lara, MD, PhD

1 From the Laboratoire de Physiologie Cellulaire Respiratoire (INSERM E-9937), Université Victor Ségalen Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France; and Service d’Imagerie Médicale and Service des Maladies Respiratoires, Hôpital Haut-Lévêque, CHU de Bordeaux, France. Received March 7, 2002; revision requested May 22; final revision received October 30; accepted December 10. Supported by grants from Programme Hospitalier de Recherche Clinique (PHRC) 1997 and Institut Pneumologique d’Aquitaine. Address correspondence to J.M.T.d.L. (e-mail: manuel.tunondelara@u-bordeaux2.fr).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 APPENDIX
 REFERENCES
 
PURPOSE: To use quantitative computed tomography (CT) to compare lung attenuation with both inflammatory infiltration and in vitro reactivity of peripheral airways in smokers scheduled to undergo lung resection for localized pulmonary lesions.

MATERIALS AND METHODS: Attenuation was measured in nine ex-smokers, 13 current smokers, and eight nonsmoking control subjects by using CT with respiratory gating and a contour-tracing algorithm. After lung resection in smokers, peripheral bronchi were dissected and studied in terms of both inflammation (by using immunohistochemistry to examine glycolmethacrylate-embedded specimens) and mechanical activity (by using an isolated organ bath system). Comparisons between groups were made by using analysis of variance and subsequent unpaired t tests. Correlations were evaluated by using the Pearson coefficient and stepwise multiple regression analysis.

RESULTS: The difference between inspiratory and expiratory attenuation was significantly higher in control subjects (-128 HU ± 11 [SD]) than in ex-smokers (-77 HU ± 10; P = .004) or current smokers (-67 HU ± 11; P = .001). Cells infiltrating the smooth muscle increased with the decrease in expiratory attenuation (r = -0.46; P = .03) and the increase in inspiratory versus expiratory attenuation (r = 0.66; P = .001). Mast cell and neutrophil infiltration of smooth muscle was the most important factor in this relationship. Cellular infiltration of the smooth muscle increased with the decrease of in vitro relaxation response to salbutamol.

CONCLUSION: In smokers, air trapping is correlated with inflammatory infiltration of the smooth muscle layer of small airways.

© RSNA, 2003

Index terms: Bronchi, abnormalities, 60.2191, 60.793 • Bronchi, anatomy • Computed tomography (CT), thin-section, 60.12118 • Emphysema, 60.751 • Lung, air trapping • Lung, CT, 60.12115, 60.12118


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
 APPENDIX
 REFERENCES
 
Small airways have long been recognized to play an important role in the airway obstruction observed in chronic obstructive pulmonary disease (COPD) (1) and, more recently, have been implicated in the pathophysiology of asthma (2,3). In such bronchial diseases, studies of autopsy material, lung resection specimens, and fiberoptic bronchoscopy biopsy specimens have indicated an infiltration of both proximal and peripheral airways by inflammatory cells (4,5). Since the analysis of small airways, defined as airways smaller than 2 mm in diameter, requires large samples of lung tissue, most data have been obtained in patients who are dying of asthma or undergoing lung resection for bronchial carcinoma (3,5–7). In COPD, peripheral airways are infiltrated by neutrophils, macrophages, and T lymphocytes, and the number of CD8+ T lymphocytes seems to correlate with symptoms and obstruction (8). In addition, it has been demonstrated that smokers develop a similar bronchial inflammatory process, even in the absence of symptoms or significant obstruction (9). Peripheral airways are also involved in the pathophysiology of respiratory bronchiolitis, a common consequence of heavy smoking (10).

Recent advances in imaging techniques have not yet allowed direct radiographic assessment of luminal caliber and wall thickness of small airways. However, thin-section computed tomography (CT) provides an indirect means of evaluating changes in peripheral airways caliber by measuring changes in regional air trapping (11,12). Air trapping reflects the retention of excess gas in all or part of the lung at any stage of respiration and is detected, preferentially, at expiratory CT as a pattern of areas with abnormally low attenuation. Such an appearance is related to peripheral airway obstruction and has been reported in a variety of lung diseases, including emphysema, bronchiectasis, bronchiolitis obliterans, and, more recently, asthma (13–16). In one study (17), air trapping was also observed in asymptomatic smokers with a history of smoking more than 10 pack-years. Air trapping is generally evaluated by using a scoring system based on the detection of trapped lobules (18). It has been quantified more recently by analyzing lung attenuation curves that indicate the distribution of lung attenuation in regions of interest within the lung. Such a method has been used by Goldin and co-workers (12) to assess peripheral airway hyperreactivity in patients with asthma. In COPD, CT has been used to evaluate lung attenuation in emphysematous lesions. However, few studies have been conducted on peripheral air trapping with this disease, and lung attenuation has not been evaluated quantitatively (19). In addition, to the best of our knowledge, the relationship between the inflammation of small airways and air trapping has not yet been studied.

Thus, the purpose of our study was to use quantitative CT to compare lung attenuation with both inflammatory infiltration and in vitro reactivity of peripheral airways in smokers scheduled to undergo lung resection for localized pulmonary lesions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 APPENDIX
 REFERENCES
 
Participants
Twenty-two patients with a history of cigarette smoking who were scheduled to undergo resection of an isolated pulmonary nodule gave their written informed consent to participate in the study after the nature of the procedure had been fully explained. The study involved acquisition of inspiratory and expiratory CT sections and use of lung specimens obtained routinely following surgical resection. The study received the approval of the local ethics committee (Comité Consultatif pour la Protection des Personnes en Recherche Biomédicale). Lung function tests were performed systematically before surgery, including plethysmography, CO transfer, and arterial gas analysis. Patients were neither atopic (they had negative skin test results for common aeroallergens) nor asthmatic (on the basis of clinical history and lung function). They did not receive oral or inhaled corticosteroids within 4 weeks prior to the study. Also, they did not experience bronchial symptoms or any respiratory infection within 2 months prior to the study. None of them had emphysema as detected with thin-section CT. Nine patients were ex-smokers—that is, they did not smoke cigarettes for a period of 3 years before entering the study. Thirteen patients were current smokers with a history of cigarette smoking from 10 to 60 pack-years. Eight healthy volunteers were also included as a nonsmoking control group. The control group underwent only lung function tests and CT performed below the level of the carina (including acquisition in the lower and middle lobe).

CT Assessment
CT was performed for quantitative assessment of lung attenuation the day before lung surgery.

CT scanning technique.—CT scans (Somatom Plus 4S; Siemens, Erlangen, Germany) were obtained with the following parameters: 1-mm section thickness, 120 kV, 165 mA, 0.75-second rotation time, pitch of 2, with 2–3 cm spiral acquisitions. Spiral acquisitions were performed at two breath hold levels that corresponded to residual volume and total lung capacity. Images were acquired in single-volume data sets of 7.5–11.0 seconds through each anatomic region of interest, which were chosen according to distance from the nodule. The volume of acquisition was chosen in the lobe to be resected. Images were reconstructed in thin-section mode at 1-mm intervals. Two sets of 20–30 images were obtained at end inspiration and end expiration for each participant. All scans were reviewed to select the five sections with the closest anatomic match between inspiration and expiration on the basis of visual bronchial and vascular anatomy. These sections were then used for visual inspection and image evaluation.

Respiratory gating.—All images were acquired with spirometric gating (20). The gating device was used to trigger scanning at a selected level of respiration and to interrupt air flow during scanning. The level of inspiration was defined by means of a small hand-held transducer (Micro Medical Instruments, Rochester, England) through which the individual was asked to breathe with his or her nose occluded. A microcomputer that was connected to the transducer determined vital capacity and generated trigger signals at a user-selected level of inspiration. These levels could be chosen as a percentage of vital capacity. As the trigger signal is generated and sent to the CT scanner, air flow is interrupted mechanically by closing a valve attached to the transducer. Therefore, the momentary status is kept constant for the duration of CT scanning. All participants were alert and cooperative and had prepared for the examination by practicing the breathing maneuver before the CT study. When the individual was positioned in the scanner, a spirometric measurement of vital capacity was obtained. Then, acquisitions were performed at 90% of vital capacity for end inspiration and 20% of vital capacity for end expiration. All participants completed the study within 10 minutes.

Image evaluation.—A semiautomatic program was used to evaluate the CT scans as described previously (21). This program is based on a contour-tracing algorithm that automatically isolates the left and right lungs on each CT scan, determines histograms of attenuation values in Hounsfield units, and calculates the mean lung attenuation with SDs. The selection of inspiratory images was performed in consensus by two observers (F.L., R.R.). Once the inspiratory images had been selected, choosing the matched expiratory images and performing segmentation of the resected lobe were conducted independently by two observers (F.L., R.R.), so that each observer evaluated all scans and selected the five anatomic levels to best match the inspiratory and expiratory images. Two independent observers (F.L., V.P.) analyzed the images and looked for emphysema, ground-glass opacities, and micronodules.

Tissue Preparation
Human lung tissue was obtained at thoracotomy as described previously (22). Briefly, lung specimens were immediately transferred to the laboratory in ice-cold oxygenated Krebs-Henseleit solution (composition: 118.4 mmol/L NaCl, 4.7 mmol/L KCl, 2.5 mmol/L CaCl2.2H2O, 1.2 mmol/L MgSO4.7H2O, 1.2 mmol/L KH2PO4, 25.0 mmol/L NaHCO3, and 11.1 mmol/L D-glucose). From a macroscopically tumor-free part of each specimen, segments of human peripheral bronchus (1–2 mm in internal diameter) were carefully dissected. After removal of adhering fat and connective tissue, bronchi were cut into rings and prepared for mechanical recording and immunohistochemical processing. Surrounding parenchyma was embedded in paraffin and stained with hematoxylin-eosin stain.

Sample Processing and Immunohistochemistry Examination
Bronchial tissues were embedded in glycolmethacrylate for immunostaining, as described previously (22,23). Bronchial rings were cut into small fragments of 2 x 2 x 2 mm and placed into ice-cooled acetone containing protease inhibitor. Embedding resin was prepared with benzoyl peroxide, glycolmethacrylate monomers, and N,N-dimethylaniline and polymerized overnight at 4°C. The glycolmethacrylate 2-µm sections were incubated overnight with mouse monoclonal antibodies, including antihuman mast cell tryptase AA1 (24), antihuman mast cell chymase (Chemicon, Souffelweyersheim, France), antihuman neutrophil elastase (Dako, Trappes, France), antihuman T lymphocytes CD3 (Dako), antihuman monocytes CD68 (Dako), and antihuman activated eosinophils EG2 (Pharmacia, Saint-Quentin-en-Yvelines, France). Control sections were treated similarly, with an irrelevant primary monoclonal antibody. Amino-ethyl carbamazole (Sigma) in acetate buffer (pH 5.2) and hydrogen peroxide were used as substrate to develop a peroxide-dependent red color reaction at 37°C. The sections were rinsed and counterstained with Mayer’s hematoxylin.

Tissue Analysis
Light microscopy was performed by using an Optiphot microscope (Nikon, Tokyo, Japan). Inflammation of peripheral airways was quantified by using an automated method of immunostaining analysis. Cells staining positively with each monoclonal antibody were counted in the specimen, excluding alveoli and cartilage, by using the computerized cell recognition system Quancoul (Quant’Image, Bordeaux, France), as described previously (7). Two independent observers (P.B., R.R.) delineated the area of the section by using an interactive video display system (original magnification, x100), and the total area examined was calculated by using Quancoul software. Cell counts were expressed as number of cells per square millimeter in each histologic layer.

Surrounding parenchyma was analyzed to detect histologic lesions of emphysema. For this purpose, one observer (H.B.) specifically evaluated alveolar wall integrity and classified parenchymal alteration in a blinded fashion as follows: grade 0 = no emphysema, grade 1 = several alveolar septa show emphysematous dystrophies (ie, loss of alveolar walls), and grade 2 = most alveolar septa show emphysematous dystrophies. We also evaluated the presence of respiratory bronchiolitis (ie, respiratory bronchioles, alveolar ducts, and peribronchiolar alveolar spaces containing clusters of dusty brown macrophages accompanied by a mild peribronchiolar fibrosis and a patchy submucosal and peribronchiolar chronic infiltrate) or desquamative interstitial pneumonia (ie, diffuse involvement of lung by numerous macrophage accumulations within most of the distal air spaces).

Mechanical Recording
To evaluate the ability of peripheral airways to respond to bronchodilators, we assessed in vitro bronchial responsiveness by using an organ bath system (EMKA Technologies, Paris, France) as described previously (25). Briefly, resting tensions were adjusted to 1.5 g in airways, a passive load that stretches this type of preparation to optimal length. The relaxation response to salbutamol from 10-8 to 10-4 mol/L (Sigma) was studied in at least two rings per subject (n = 17). The maximal relaxation was assessed in response to 10-3 mol/L bamifylline (Rhône-Poulenc Rorer, Neuilly sur Seine, France). A cumulative concentration-response curve for salbutamol was then constructed by two observers (P.B., M.M.). Results were expressed as the geometric mean of IC50, the concentration of salbutamol that produced half of the maximal relaxation (25) and the plateau of the cumulative concentration-response curve in percentage of the maximal relaxation response to bamifylline.

Statistical Analysis
Clinical and functional characteristics were compared by using unpaired t tests. CT parameters between resected and contralateral lobes or between groups of participants were compared by using analysis of variance. Since cell counts were not distributed normally, results from immunohistologic studies were log transformed to obtain more normal variables. Unpaired t tests were used to compare the number of cells of each different phenotype (in log-transformed units) according to the smoking status of participants. Pearson correlation coefficients were used to examine the relationships between tobacco smoking and attenuation parameters and between in vitro bronchial responsiveness and log-transformed inflammatory cell counts. Stepwise multiple regression analysis was performed to further analyze the relative contribution of each inflammatory cell phenotype to the relationship between inflammation and expiratory lung attenuation (Appendix). Results were considered significant when P < .05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 APPENDIX
 REFERENCES
 
Participant Characteristics, Tissue Analysis, and CT
Parameters Clinical and functional characteristics in healthy volunteers and in patients assessed prior to surgery are reported in Table 1. There was no difference between groups in terms of height or weight. Healthy volunteers were younger than patients (P < .001), however, and their forced expiratory flow was higher (P = .03). The rest of the lung function was similar in the three groups. Patient 18 presented with altered diffusing capacity, but no emphysema was shown in the resected lobe at CT (Fig 1, A). We did not find any indications of emphysema on inspiratory CT images, such as low-attenuation areas or bullae, nor did we find ground-glass opacities or ill-defined micronodules in our patient population. Bronchial inflammation was evaluated by means of immunohistochemical examination of inflammatory cells, including mast cells (Fig 1, B), monocytes and macrophages, T lymphocytes, polynuclear neutrophils, and eosinophils.


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  		TABLE 1. Participant Characteristics

 


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  		Figure 1. Typical images in a current smoker. A, Expiratory CT scan of the upper lobe delimited according to the contour-tracing algorithm, B, immunohistochemical staining of mast cells in the peripheral bronchi, and C, histologic aspect of type 1 emphysema.

 
As shown in Figure 2, tobacco smoking correlated significantly with the total number of inflammatory cells within the bronchial wall of peripheral airways (n = 22). The subgroup of current smokers demonstrated a significantly higher number of cells per square millimeter than that in the subgroup of ex-smokers (median, 444.3 cells vs 337.6, respectively; P = .03). Figure 3 illustrates the distribution of inflammatory phenotypes in the different layers of small airways (n = 22). Both the proportion and distribution of inflammatory cells were similar in the two subgroups (P > .05). However, the number of immunoglobulin E–positive cells in the whole bronchial wall or within the smooth muscle layer was inversely correlated with the forced expiratory volume in one second (r = -0.51 and P = .02 or r = -0.43 and P = .04, respectively). No other statistically significant correlation was found between inflammatory cell number and in vivo functional parameters. Histologic evaluation findings of microscopic emphysema in the surrounding parenchyma are reported in Table 2. The frequency of alveolar septal dystrophy was higher in the current smoker group (Fig 1, C). No statistically significant correlation was found between these tissue alterations and functional parameters (lung transfer of CO, arterial partial oxygen pressure, and residual volume).



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  		Figure 2. Scatterplot shows correlation between tobacco smoking and inflammatory cellular infiltration in the whole bronchial wall. {circ} = data in ex-smokers, {bullet} = data in current smokers, r = Pearson correlation coefficient.

 


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  		Figure 3a. Cell phenotypes detected by means of monoclonal antibodies directed against specific markers in glycolmethacrylate-embedded specimens. Antibodies are antihuman mast cell tryptase (AA1), antihuman mast cell chymase, antihuman neutrophil elastase (NE), antihuman T lymphocytes (CD3), antihuman monocytes (CD68), and antihuman activated eosinophils (EG2). (a) Box plots represent median with 25% and 75% interquartile ranges; error bars represent 5th and 95th percentiles, and circles represent the minimal and maximal values of cells per square millimeter in the whole tissue section (n = 22). (b) Bar graph shows distribution of each phenotype expressed as a percentage within epithelium (black area), submucosa (white area), and smooth muscle layer (hatched area). The total number of each cell phenotype has been normalized to 100%.

 


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  		Figure 3b. Cell phenotypes detected by means of monoclonal antibodies directed against specific markers in glycolmethacrylate-embedded specimens. Antibodies are antihuman mast cell tryptase (AA1), antihuman mast cell chymase, antihuman neutrophil elastase (NE), antihuman T lymphocytes (CD3), antihuman monocytes (CD68), and antihuman activated eosinophils (EG2). (a) Box plots represent median with 25% and 75% interquartile ranges; error bars represent 5th and 95th percentiles, and circles represent the minimal and maximal values of cells per square millimeter in the whole tissue section (n = 22). (b) Bar graph shows distribution of each phenotype expressed as a percentage within epithelium (black area), submucosa (white area), and smooth muscle layer (hatched area). The total number of each cell phenotype has been normalized to 100%.

 

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  		TABLE 2. Histologic Findings in Ex-smokers and Current Smokers

 
Figure 4 shows typical aspects of inspiratory and expiratory CT images. Measurement of lung attenuation at CT in smokers showed an inspiratory attenuation of -845 HU ± 8.4, an expiratory attenuation of -768 HU ± 13, and a difference of -71 HU ± 7.6 between inspiratory and expiratory attenuation. No significant difference was found between these CT parameters measured in the resected lobe and those in the contralateral lobe (analysis of variance, P > .05). The absolute attenuation difference between inspiration and expiration was significantly higher in control subjects (128 HU ± 11) than in ex-smokers (77 HU ± 10; P = .004) or current smokers (67 HU ± 11; P = .001). No significant difference was found in inspiratory or expiratory attenuation between these groups (data not shown). No statistically significant correlation was found between these CT parameters and in vivo functional parameters.



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  		Figure 4. Typical transverse CT images obtained in, A, B, a current smoker and, C, D, a nonsmoking control subject at end inspiration (A, C) and end expiration (B, D). Note areas of air trapping at expiratory CT that are more visible in the current smoker.

 
Relationship between Lung Attenuation, Tissue Characteristics, and in Vitro Functional Parameters
The number of inflammatory cells within the airway wall and its different layers was plotted against attenuation parameters. Despite the relative weakness of some correlations, the number of cells infiltrating the smooth muscle increased with the decrease in expiratory attenuation (Pearson coefficient, r = -0.46) and the increase in the attenuation difference between inspiratory and expiratory images (r = 0.66). However, no statistically significant correlation was found with the total number of inflammatory cells infiltrating the whole bronchial wall (Table 3).


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  		TABLE 3. Correlation between CT Parameters and Inflammatory Cell Distribution in 22 Patients

 
Among the inflammatory phenotypes present in the smooth muscle layer, the number of both chymase-positive mast cells and neutrophils increased with the increase in attenuation difference between inspiratory and expiratory images (Table 4). Since a significant correlation was found between the independent variables AA1, EG2-positive cells, and neutrophils, multiple regression analysis was performed only with chymase-positive mast cells, neutrophils, T lymphocytes, and macrophages. Stepwise multiple regression analysis indicated that chymase-positive mast cell infiltration of airway smooth muscle was the most important factor in the relationship between inflammation and expiratory attenuation parameters (Table 5). The relationship between chymase-positive mast cells or neutrophils infiltrating the smooth muscle layer and lung attenuation is illustrated in Figure 5.


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  		TABLE 4. Correlation Matrix between Attenuation Parameters and Inflammatory Cell Infiltration in the Smooth Muscle Layer in 22 Patients

 

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  		TABLE 5. Stepwise Multiple Regression Analysis of Attenuation Parameters for Inflammatory Cell Infiltration in the Smooth Muscle Layer

 


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  		Figure 5a. Graph shows results of regression analysis between inflammatory cell infiltration of the smooth muscle layer and (a) expiratory attenuation or (b) the attenuation difference between inspiratory and expiratory images. A log scale was used for the x axes, since inflammatory cell counts have been log transformed for the regression analyses. ß = regression coefficient, solid line = regression line for chymase-positive mast cells, dotted line = regression line for neutrophils.

 


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  		Figure 5b. Graph shows results of regression analysis between inflammatory cell infiltration of the smooth muscle layer and (a) expiratory attenuation or (b) the attenuation difference between inspiratory and expiratory images. A log scale was used for the x axes, since inflammatory cell counts have been log transformed for the regression analyses. ß = regression coefficient, solid line = regression line for chymase-positive mast cells, dotted line = regression line for neutrophils.

 
We compared the different CT parameters in patients with and those without alveolar septal emphysematous dystrophy. No significant difference was found in terms of inspiratory (-852 HU ± 7 vs -836 HU ± 21) and expiratory (-773 HU ± 16 vs -763 HU ± 28) attenuation and the attenuation difference between them (-71 HU ± 13 vs -67 HU ± 9). The presence of respiratory bronchiolitis does not significantly alter the lung attenuation values.

With regard to the in vitro relaxation of isolated airways, the mean plateau of the cumulative concentration-response curve to salbutamol was 947 mg ± 110—that is, 82.7% ± 3.6 of the maximal relaxant response to bamifylline. The geometric mean IC50 was 1.3 x 10-7 (95% CI: 4.5 x 10-8, 3.6 x 10-7). Cellular infiltration of the smooth muscle layer significantly increased with both the increase in sensitivity to salbutamol (IC50, r = 0.83; P = .001) and the decrease in in vitro bronchial reactivity to salbutamol (plateau, r = -0.61; P = .02). However, no statistically significant correlation was found between lung attenuation parameters and in vitro relaxation and between in vivo functional parameters and in vitro relaxation (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
 REFERENCES
 
The results of the present study demonstrate that in patients exposed to cigarette smoking, expiratory lung attenuation correlates negatively with inflammatory cell infiltration in the smooth muscle layer of peripheral airways. Among inflammatory cell phenotypes, chymase-positive mast cells and, to a lesser extent, neutrophils mainly contribute to this relationship.

Decrease in lung attenuation during exhalation appears to correspond to peripheral airway obstruction and has been described in several lung diseases that affect small airways (13,26). Low attenuation areas can be quantified by using a scoring system. However, recent development of thin-section CT techniques allow an objective and observer-independent evaluation of the lung (21,27,28). Parameters that are commonly used are mean attenuation; pixel index, which indicates overall percentage of lung involved; and the lowest 5th or 10th percentile of the frequency distribution. These parameters are related to the physical attenuation of the scanned volume and are dependent on both the level of inflation and the lung tissue in each voxel. Control of the individual’s lung volume is therefore important. In our study, this control was achieved by means of a spirometer connected to the CT scanner that provides spirometrically triggered CT images with high reproducibility (29).

Lung attenuation histograms have recently been used to depict the involvement of peripheral airways in hyperresponsiveness (12) and to evaluate the benefit of corticosteroids on distal airway inflammation (30). In COPD, lung attenuation has mainly been examined to evaluate the extent of emphysema (19,31) but less frequently to analyze peripheral bronchi impairment. Lamers and colleagues (19) reported that lung expiratory attenuation was lower in smokers with chronic bronchitis than in those without bronchitis. In the present work, we have extended this finding by showing that lung expiratory attenuation was significantly lower in current smokers or ex-smokers than in healthy nonsmokers. The present result is in agreement with the findings of Lee et al (17), who demonstrated that the degree of air trapping, measured with a visual scoring system, was higher in smokers than in nonsmoking subjects.

Unlike patients with COPD, most of the participants in our study had lung function within a normal range (Table 1) and did not appear to have emphysema, according to visual inspection of CT images or assessment of diffusing capacity. However, the presence of microscopic emphysema was demonstrated by using histologic evaluation of lung parenchyma in 11 patients. It should be noted that only two patients from the current smokers group showed a significant extension of septal destruction. These emphysematous lesions were not detected at CT, probably because of the low sensitivity ascribed to this imaging technique for diagnosis of mild emphysema. There was no difference in terms of CT lung attenuation parameters between patients with and those without histologic emphysema. It might be expected that a lower lung attenuation is correlated with emphysema, particularly at inspiratory CT. In the present study, the alveolar wall alterations were probably too mild and could not be responsible for an increase in distal airway volume. Thus, in such patients, it is likely that the decrease in lung attenuation parameters measured after exhalation reflect air trapping related to cigarette smoke–induced peripheral airway obstruction that is not detectable with conventional lung function tests. Several patients had respiratory bronchiolitis that probably contributed to the peripheral obstruction but not to a desquamative interstitial pneumonia type of reaction. However, we did not find any significant difference in terms of lung attenuation values between patients with and those without respiratory bronchiolitis, likely as a result of the small size of the groups. In addition, most of the lung specimens with mild emphysema dystrophy also had respiratory bronchiolitis, which, in terms of lung attenuation, may counterbalance emphysema.

The airway inflammatory process associated with cigarette smoking involves both proximal and distal bronchi (32–35). It has been reported that smokers who develop COPD have an increased number of T lymphocytes in peripheral airways, compared with smokers who do not develop the disease (8). Other cell phenotypes have also been implicated in this inflammatory process, including macrophages, neutrophils, eosinophils, and mast cells (9,33–35). In the present work, we found that lung attenuation was negatively correlated with cellular infiltration of the smooth muscle layer of peripheral airways. We also found that, among inflammatory cells, mast cells containing chymase mainly contribute to this relationship. On the one hand, it has been previously shown that mast cells are able to infiltrate airway smooth muscle layer (36) and that the smooth muscle mass is increased in COPD (37). On the other hand, chymase has been implicated in the pathophysiology of COPD through its effect on the regulation of vasoactive neuropeptides (38) and submucosal gland secretion (39). Unlike tryptase (25), however, the actual implication of chymase in airway obstruction has not been clearly established.

Lung attenuation was not correlated with any of the parameters derived from the concentration-response curve for ß2 agonist constructed in isolated peripheral airways. That is, the relaxant response of the smooth muscle from the distal airway was similar whatever the severity of air trapping in the group of current smokers or ex-smokers. Whether the in vitro responses in these patients who were undergoing lung resection do not already demonstrate impaired relaxation compared with those in healthy nonsmoking volunteers is unknown, since such in vitro responses were not determined in the latter group. However, a few studies in the literature indicate that the in vitro response to ß2 agonist is not altered, even in patients with marked COPD (40,41). Therefore, the lack of correlation in the present study between air trapping and cumulative concentration-response curve to salbutamol is not unexpected. This result suggests that airway obstruction responsible for air trapping is not associated with some intrinsic abnormality of airway smooth muscle or at least some impairment of airway smooth muscle relaxation. However, we observed a correlation between the cellular infiltration of the smooth muscle layer and the parameters derived from the cumulative concentration-response curve to salbutamol—that is, salbutamol-induced maximal relaxation response decreased with the increase in smooth muscle inflammatory infiltration. This finding may account for the fact that, in vivo, mediator release from inflammatory cells close to airway smooth muscle contributes to maintained contraction of peripheral airways and thus to lobular air trapping (42).

In conclusion, the results of the present study demonstrate that air trapping in current smokers or ex-smokers is correlated with inflammatory infiltration in the smooth muscle layer of peripheral airways.


   APPENDIX
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 APPENDIX
REFERENCES
 
Stepwise multiple regression analysis was performed to further analyze the relative contribution of each inflammatory cell phenotype (log-transformed cell counts as independent variables) to the relationship between inflammation and expiratory lung attenuation (dependent variable). In this analysis—a modification of the selection technique—independent variables remained in the model until the best model was found according to the following criteria: highest r2, lowest square root of the mean square error, and highest F ratio. This selection technique was used because (a) it is possible to increase r2 by adding more independent variables, but the additional independent variables may cause an increase in the mean square error, which is an unfavorable situation; and (b) the higher the F ratio, the lower the probability of the null hypothesis (that all regression coefficients ß are 0). Results were considered significant when P < .05. All statistical analyses were performed with NCSS 2001 software (Kaysville, Utah).


   ACKNOWLEDGMENTS
 
The writers thank the staff of Service de Chirurgie Thoracique for providing the human lung tissue. We also thank Beatrice Martinez, BS, for technical assistance.


   FOOTNOTES
 
Abbreviation: COPD = chronic obstructive pulmonary disease

Author contributions: Guarantors of integrity of entire study, P.B., F.L., J.M.T.d.L.; study concepts and design, P.B., F.L., J.M.T.d.L.; literature research, P.B., F.L., V.P., R.R., J.M.T.d.L.; clinical studies, P.B., F.L., V.P.; experimental studies, P.B., H.B., R.R., M.M.; data acquisition, P.B., F.L., H.B., V.P., R.R., M.M.; data analysis/interpretation, P.B., F.L., C.R., R.M., J.M.T.d.L.; statistical analysis, P.B., C.R.; manuscript preparation, definition of intellectual content, and editing, P.B., F.L., J.M.T.d.L.; manuscript revision/review, P.B., F.L., M.M., R.M., J.M.T.d.L.; manuscript final version approval, all authors.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 APPENDIX
 REFERENCES
 

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