INTRODUCTION

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Keywords: airway distensibility; airway remodeling; asthma

Asthma is accepted as a disease characterized by airway inflammation and bronchial hyperreactivity (BHR) (1). There is also accumulating evidence that airway structural changes, or "remodeling," occurs, probably secondary to the inflammatory process (1), although this is not a universally reproducible finding (2). Airway remodeling has in turn been linked to the development of physiologic dysfunction, providing a possible mechanism for the development of fixed airflow limitation observed in many patients with asthma (3).

The change in anatomic dead space (VD) with lung volume has long been proposed as a novel, noninvasive index of airway distensibility (VD) or stiffness (4). Using this test the airways of subjects with mild asthma have been shown to be stiffer than normal (5). The initial finding has subsequently been followed up in a larger, more extensive study using improved methodology that is more practicable for the rapid assessment of VD (6). This study confirmed that subjects with asthma had stiffer airways and that measurement of VD was not confounded by lung volume history (6). It has been speculated that the finding of decreased airway distensibility in subjects with asthma is a consequence of airway remodeling (5), but there are as yet no published data that relate airway distensibility to remodeling in asthma.

We have carried out a cross-sectional, pathophysiologic correlation study, to investigate the relationship between airway remodeling, as assessed by subepithelial reticular basement membrane (RBM) thickening, VD, and postbronchodilator large and small airway function, as assessed from a flow-volume curve. Our hypothesis was that airway stiffness would relate to airway remodeling and degree of fixed airflow obstruction.

	METHODS

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This study was approved by the Alfred Hospital (Melbourne, Australia) Ethics Committee and all subjects gave written informed consent.

Patient Selection

Thirty-five subjects with mild to moderate yet symptomatic atopic asthma were recruited. Their mean age was 39 yr (range 20 to 70 yr; SD, 13 yr) and all were nonsmokers, with diagnosed asthma for at least 12 mo. Twenty-four were male. At the time of study all were solely using albuterol for as-needed relief of symptoms and no prophylactic "preventer" medication. No subjects had received treatment with regular inhaled corticosteroids (ICs) or inhaled long-acting ₂ agonists for at least 3 mo before the study. Thirty-four subjects had not received oral corticosteroid treatment for at least 12 mo before the study and 1 subject had received a course of oral corticosteroid treatment 3 mo before the study. Twenty-two normal healthy volunteers were also recruited for the same assessments. None of these normal individuals had any history of respiratory illness and all were nonsmokers; 11 were male and 6 were atopic. Their mean age was 38 yr (range, 19-61 yr; SD, 13 yr).

Study Design

After inclusion in the study there was a 2-wk run-in period during which the subjects with asthma were continued on albuterol alone. At the end of this time physiologic assessments, including flow-volume loops, bronchial responsiveness to methacholine, and airway distensibility measurements were performed. These were followed by bronchoscopy with airway biopsies.

Patient Assessments

Skin tests. Atopic status was assessed by skin prick testing to a panel of seven common aeroallergens and was defined by a response of  3 mm to one or more.

Lung function and methacholine challenge. Flow-volume loops, before and after administration of 400 µg of albuterol, and bronchial responsiveness (BHR) to methacholine were recorded within 1 wk of commencing the study, with both assessed after a bronchodilator-free period of at least 8 h. Predicted values of Knudson and coworkers were used (7). On a separate day, PD₂₀ (provocative dose causing a 20% fall in FEV₁) to methacholine was assessed by a previously described dosimeter technique (8).

Anatomic dead space and airway distensibility. Airway distensibility (VD, ml/L) was measured after administration of 300 µg of albuterol, from the relationship between anatomical dead space (VD) and lung volume as previously described (6). In brief, VD was measured at a number of discrete lung volumes during tidal breathing at progressively decreasing lung volumes from total lung capacity (TLC) to near residual volume (RV), using the Fowler method with carbon dioxide as the indicator gas (4, 6). Each subject sat erect and breathed tidally at FRC for 1 min, and then inhaled to TLC and breathed tidally at progressively diminishing lung volumes until RV was approached. End-inspiratory pause was discouraged by having the subjects breathe in time to a metronome at a breathing frequency of 25 breaths/min. This breathing regimen was performed in triplicate to obtain sufficient data for the determination of VD. VD was measured from the expiratory portion of each expired tidal breath. The lung volume corresponding to each measurement of VD was determined by subtracting the total volume exhaled from TLC measured previously in a body plethysmograph. Measurements were standardized for age and sex using TLC, that is, VD/TLC. The slope of the relationship between standardized VD and lung volume was then calculated by least-squares regression analysis to derive VD.

The coefficient of variation of VD measurements made on three separate days in five normal subjects was 9% (6).

Bronchoscopy and Airway Biopsy

Bronchoscopy and airway biopsy were performed by our standard method (9). Six endobronchial biopsies (EBBs) were taken from around the segmental subcarinae of the right lower lobe of each patient, using alligator forceps (FB 15C; Olympus, Norwood, MA). These were placed in ice-cold acetone-containing protease inhibitors (2 mM phenylmethylsulfonyl fluoride with 20 mM iodoacetamide).

Biopsy processing. Endobronchial biopsies were embedded into the hydrophilic polymer glycol methacrylate. Coded blocks were cut on a semithin resin microtome (RM 2310; Leica, Nussloch, Germany), using glass knives. The 2-µm sections were floated on water and adhered to silanized slides.

Biopsy staining. A modified three-layered immunoperoxidase staining method was used as previously described (9) and sections were stained with goat anti-type I (Southern Biotechnology, Birmingham AL). Slides were assigned a number from a random code and read blind to time, treatment, and patient group.

Biopsy quantification. Subepithelial reticular basement membrane (RBM) thickening was assessed with sections immunostained for collagen I (Figure 1). This method was prospectively decided on after a pilot study comparing EBB sections that were stained with collagen I and collagen III, using immunohistochemistry, as well as sections stained cytochemically with hematoxylin and eosin and toluidine blue. This involved a number of experienced histologists, and there was unanimous agreement that collagen I staining gave the best definition. The correct orientation and adequacy of all specimens were ensured by initial survey sections, which were stained with hematoxylin and eosin. A single experienced observer (M.P.) made measurements of RBM from the base of the bronchial epithelium to its outer limit. The measurement therefore included the "true" basement membrane as well as "reticular components." An image analyzer (Image-Pro Plus, version 4.0; Media Cybernetics, Des Moines, IA) was used to score all of the RBMs in a whole, well-preserved section, by drawing two lines that demarcated the RBMs. The method, which was calibrated against a standard graticule for each scoring session, avoided any potential subjectivity because it quantitated all the available RBM. In all cases a total length of > 2 mm was quantified, giving a sample that substantially exceeds a recommended total of 1 mm for such estimations.

View larger version (139K): [in this window] [in a new window]   Figure 1.   An illustrative photomicrograph (Image Pro Plus video capture) showing (1) subepithelial reticular basement membrane (RBM) thickening as denoted by collagen subtype I staining of an endobronchial biopsy section and (2) diffuse staining in the lamina propria.

Statistical Analyses

Analyses were performed according to the distribution of the data with or without log transformation. PD₂₀ data are presented as geometric means and 95% confidence intervals (CIs). Spirometry and VD measurements are presented as least-square means with SEM and a generalized linear model used for analysis. Univariate correlations were made by using the Pearson correlation.

	RESULTS

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Physiology

The subjects with asthma had mild to moderate degrees of airflow limitation and all had measurable BHR. Mean (± SEM) baseline percent predicted FEV₁ was 91 ± 3%; and baseline percent predicted FEF_25-75% was 56 ± 5%. The geometric mean (range) of PD₂₀ was 25 (1 to 1,578) µg. There was a clinically significant response to bronchodilator (percent predicted FEV₁ postbronchodilator, 103 ± 2%, FEF_25-75% postbronchodilator, 74 ± 6%).

Subepithelial Reticular Basement Membrane Thickening

As expected, RBM thickness was variable in the biopsies assessed. Nevertheless, there was a significant overall increase in RBM thickness in subjects with asthma compared with the normal subjects: 9.1 ± 2.2 versus 7.7 ± 1.2 µm (p < 0.01).

Airway Distensibility

VD was lower in the asthmatic group at baseline compared with the normal group (19.8 ± 1.1 versus 24.1 ± 1.5 ml/L; p = 0.03).

Correlation of VD with FEV₁, Flow Rates, and Reticular Basement Membrane Thickening

There were significant correlations between both the percent predicted FEV₁ and FEF_25-75% and VD postalbuterol (Figures 2 and 3); percent predicted FEV₁ postalbuterol versus VD gave r = 0.59, p < 0.001. Percent predicted FEF_25-75% postalbuterol versus VD gave r = 0.60, p < 0.001. 

View larger version (11K): [in this window] [in a new window]   Figure 2.   Significant correlation between percent predicted FEV1 and VD, after albuterol administration.

View larger version (10K): [in this window] [in a new window]   Figure 3.   Significant correlation between percent predicted FEF25-75% and VD, after albuterol administration.

There was a significant negative correlation between RBM thickening and VD (Figure 4; r = 0.37, p < 0.05).

View larger version (10K): [in this window] [in a new window]   Figure 4.   Significant negative correlation between RBM thickening and VD.

	DISCUSSION

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We have shown that decreased airway distensibility (VD) is related to both pathologic evidence of airway remodeling and postbronchodilator large and small airway function in asthma. This is the first study that we are aware of that has shown such relationships between airway remodeling, airflow limitation, and airway distensibility in human asthma.

It is increasingly accepted that airway remodeling is secondary to inflammation and may be an important mechanism that leads to fixed airflow obstruction in asthma, characterized by a failure to attain predicted lung function levels after bronchodilator therapy (1, 10). As in the present work, two smaller previous independent studies, one from our laboratory, which represent the only other published human data, also found decreased VD in asthma, and it was hypothesized that this might reflect the effects of airway remodeling (5, 6). Neither study included pathologic assessments of airway remodeling and in the absence of such data, other explanations for the findings of decreased VD in asthma, not directly related to remodeling, were also considered. Thus, the potential for inhomogeneous closure of peripheral airways and asynchronous emptying leading to systematic differences in VD in subjects with asthma was considered, although given the dose of ₂ agonist used before the assessment of VD, and the similarity of VD at 50% of TLC, this was thought unlikely (5, 6). More recently a possible role for lung volume history affecting assessments of VD was excluded, in a thorough methodologic evaluation of the rapid VD technique used in the present study (6).

Our finding that decreased VD related to RBM thickening represents an addition to the remodeling literature and the two existing studies evaluating VD measurements in asthma. The present study indicates that physiologic measurements, which do not require airway biopsy, may indeed provide valuable information about airway remodeling. The potential importance of this is underlined by data indicating that airway remodeling may occur early in the pathogenesis of asthma (11), with one report also indicating an intermediate degree of remodeling associated with chronic cough relative to full "classic" asthma and normal measurements (12). The current uncertainty about the response of airway remodeling to treatment, together with the need for more data regarding airway remodeling generally, has identified the requirement for evaluating such potential noninvasive approaches to studying remodeling (13). Our finding, that VD related to predicted values for large and small airway function postbronchodilator, is particularly strong evidence that measurements of VD might provide useful information relevant to the study of airway wall structural change throughout the bronchial tree. The standard approach to remodeling studies, involving invasive, resource-intensive airway biopsy, is practically limited to the larger conducting airways. Transbronchial biopsies have been used in asthma studies but this approach is even more invasive, and frequently fails to sample peripheral airways (14). The potential for VD measurements to give information relevant to both large and small airways might be especially useful in future remodeling studies (13).

The potential mechanism by which epithelial basement membrane thickening contributes to any functionally important changes has been brought into question, however (2, 15), with the potential that RBM does not relate to other "remodeling" changes, which may be functionally more important and may simply be an unrelated epiphenomenon. It is reasonable to consider this, and to recognize that correlation does not imply causation. It is therefore of considerable interest that in a study by James and coworkers, basement membrane thickness was shown to be statistically related to several other parameters of remodeling (16). This study was in the context of whole resected lung specimens, with which this group has provided important pathophysiologic asthma data in previous papers (16). RBM thickening has the considerable advantage of being an index that can be quantified on airway biopsy, and can be monitored in longitudinal studies.

The two previous reports on VD in asthma also made attempts to detect potential relationships with airflow limitation through correlation analyses. The first of these studies (n = 10 subjects with asthma) was unable to show a relationship between VD and airflow limitation. The second, using the same method for VD assessment as the present study, (n = 16 subjects with asthma), found a relationship between percent predicted FEF_25-75% and VD (r = 0.55), similar to that found in our independent study (r = 0.60, n = 35), but with no relationship to FEV₁. We feel that a potential explanation for detection of comprehensive relationships between predicted large and small airway function and VD in the present study was the larger sample size used (n = 35). We also studied a population of subjects with asthma who were prospectively selected as not undergoing treatment with corticosteroids. This was a deliberate strategy because it remains uncertain what the effect of corticosteroids are on airway remodeling, with contradictory findings in the literature (13, 19). There are no published data available on the relationship between VD and corticosteroid therapy and a longitudinal study of VD with other physiologic measurements and airway remodeling before and after inhaled corticosteroid therapy would represent a logical extension to the present work.

In summary, we have confirmed that airway distensibility in subjects with asthma is lower than normal. We have extended our experience with a rapid and simple technique for the assessment of VD (6), and shown that VD relates to airway remodeling. The ease of the measurement of VD makes it amenable to use in the more extensive studies regarding airway remodeling that have been called for (13, 25, 26), including the potential for epidemiologic work. Our present data would indicate that it would be worthwhile to undertake further evaluation of this measure of airway distensibility, and in particular its relationship with changes in airway remodeling in interventional longitudinal studies.