INTRODUCTION

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Nonspecific bronchial hyperresponsiveness (BHR) is a feature of a variety of inflammatory diseases of the lungs and can therefore be associated with airways mucosal inflammation (1). Longitudinal population studies have shown a faster decline of pulmonary function in subjects with BHR, compared with subjects without BHR, independent of symptom status, smoking, atopy, prechallenge FEV₁, age, gender, and height (2). Recent studies have also shown that BHR occurs in subjects without any respiratory symptoms, so-called asymptomatic BHR (2). Interestingly, some studies found that female gender was associated with significantly higher rates of asymptomatic BHR in healthy nonsmokers (3, 4) as well as in smokers with mild chronic obstructive pulmonary disease (COPD) (5). There is increasing evidence that inflammatory changes in bronchioles are an important factor in BHR (6, 7). However, conventional tests of lung function, such as spirometry or body plethysmography, suffer from a lack of sensitivity in detecting such early peripheral lung impairment. Recent studies suggested that physiological tests using monodisperse test aerosols may help to identify early stages of lung disease (8). For example, Kim and coworkers (10, 11) have shown that total lung deposition of 1-µm-diameter particles is increased in asymptomatic smokers or mild bronchitic patients with normal pulmonary function. Similarly, Brand and coworkers (9) demonstrated that the aerosol dispersion test has a higher sensitivity and specificity than conventional lung function tests for the detection of early lung impairment caused by cigarette smoking.

Given the increasing evidence that an inflammatory process in the airway wall is a major component of the underlying pathophysiologic mechanisms of BHR, we investigated the extent to which the presence of asymptomatic BHR alters particle deposition and aerosol bolus dispersion as markers of early lung impairment.

	METHODS

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Aerosol Bolus Deposition and Dispersion

The aerosol bolus method applied in this study was recently reviewed in detail (12). In brief, to perform measurements of aerosol bolus dispersion (D) and deposition (DE) (12), a small volume (bolus) of an aerosol with monodisperse particles is introduced into a single breath of particle-free air, and is inhaled into a preset volumetric lung depth (V_p) and then exhaled. The subjects inhaled from functional residual capacity (FRC) an air volume (V_a) with a constant flow rate. V_a was chosen for each subject to ensure that the end-inspiratory lung volume of all subjects FRC + V_a, was equal to the same fraction F of total lung capacity (70% TLC).

(1)

A narrow bolus of aerosol was introduced at a preselected point in this V_a. The volumetric penetration (V_p) of a bolus, the controlled experimental parameter, was defined as the volume of air inhaled from the volume of maximal particle concentration (bolus mode) to the end-inspiratory lung volume of 70% TLC; thus, V_p is that volume which followed the bolus mode into the lungs. For each subject, boluses were introduced to V_p of 800 cm³ (13). During respiration the particles are subjected to deposition as well as convective gas mixing.

DE is defined as the fraction of inhaled particles that deposits in the lungs during the breathing cycle. DE is given by

(2)

where C is the particle number concentration measured at the mouth as a function of the respired volume, V_i is the inhaled air volume, and V_e is the exhaled air volume.

Aerosol bolus dispersion is defined as the increase of volumetric bolus width during the respiratory cycle which is a marker of convective gas transport. Aerosol bolus dispersion is quantified by:

(3)

where H_50,i is the volumetric half-width of the inhaled and H_50,e that of the exhaled bolus. Half-width is defined as the air volume in which the particle number concentration exceeds half the maximal concentration (13). In this study boluses were inhaled into a volumetric lung depth of 800 cm³.

Instrumental Setup and Inhalation Protocol

The measurements are performed with the Respiratory Aerosol Probe (Pari, Starnberg, Germany) (13). This computer-controlled device combines laser aerosol photometry with pneumotachography in order to measure the number concentration of respired monodisperse particles as a function of the respired air volume. Aerosol supply is provided by a system of pneumatical valves, which allows the inhalation of either particle-free air or aerosol. The required air flow level is controlled by a visual flow signal.

The breathing maneuver for studying particle deposition and aerosol bolus dispersion starts from FRC at which the subjects inhale particle-free air at a flow rate of 250 cm³ s¹ until the lung volume reaches 70% TLC. During inspiration an aerosol bolus with 25 cm³ half-width is inhaled into a V_p of 800 cm³. The subjects then immediately exhale at a flow rate of 250 cm³ s¹ until the aerosol bolus is recovered from the lungs or residual volume (RV) is reached.

Particle Production and Classification

Monodisperse di-2-ethylhexylsebacate (DEHS) droplets suspended in nitrogen are produced by heterogeneous nucleation of DEHS vapor on NaCl nuclei in a MAGE aerosol generator (Lavoro e ambiente, Bologna, Italy). The aerosol is then diluted with particle-free air to obtain a particle number concentration of about 2 · 10⁴ cm³ . The size of the particles is determined from the terminal settling velocity of the particles evaluated within a convection-free sedimentation channel. The mean settling velocity (mean ± SD) of the particles was 26.5 ± 1.8 µm s¹, corresponding to particles of 0.91 ± 0.03 µm.

Pulmonary Function Testing

Body plethysmography and spirometry. Body plethysmography and spirometry were performed using a Jäger-Masterlab (Erich Jäger GmbH, Würzburg, Germany). The following parameters were measured: TLC, vital capacity (VC), intrathoracic gas volume (Vtg), RV, airway resistance (Raw), peak expiratory flow (PEF), forced expiratory volume in one second (FEV₁), and maximal expiratory flows at 25, 50, and 75% vital capacity (MEF₂₅, MEF₅₀, MEF₇₅). Relative values of conventional lung function parameters were calculated by normalization to the reference values proposed by the European Community for Steel and Coal (15). The dead space volume (VD) of the lungs was determined from single-breath washout tracings of argon by the conventional Fowler method.

Methacholine challenge. On a separate day all subjects performed a methacholine challenge testing using a modified dosimeter method that met the guidelines for standardization of bronchial challenges of the European Respiratory Society (16). Aerosols of methacholine were generated by a jet nebulizer (IS-2; Pari; output 83 mg/min; mass median aerodynamic aerosol diameter 3.0 µm; geometric standard deviation 1.8). Prior to the methacholine inhalation the subjects performed at least three satisfactory and two reproducible FEV₁ maneuvers. The largest FEV₁ value from an acceptable maneuver was used as the baseline FEV₁. The subjects then inhaled an aerosol bolus of 0.9% saline and repeated the FEV₁ maneuver 1 min later. Finally the subjects inhaled increasing doses of methacholine up to a cumulative dose of 1.7 mg with repeated measurements of FEV₁ 1 min after each methacholine inhalation. Doses were given at 5-min intervals. Given the nebulizer output of 83 mg/min the cumulative doses of methacholine could be calculated in milligrams for each protocol stage: 0, 0.003, 0.009, 0.028, 0.08, 0.25, 0.76, 1.2, and 1.7 mg. BHR was diagnosed when FEV₁ had fallen by 20% or more at a cumulative provoking dose of methacholine less than 1.7 mg on two occasions 4 wk apart. The results were expressed in cumulative milligrams of methacholine required to cause a 20% fall in FEV₁ (PD₂₀).

Subjects

Thirty-two women with asymptomatic BHR and 60 women without BHR participated in this study. Table 1 (nonsmokers) and Table 2 (smokers) summarize the anthropometric and lung function data of the study population. There were no significant differences in age, height, and lifetime cigarette consumption between subjects with and without BHR. Subjects with BHR showed mild to moderate BHR with a PD₂₀ of 637 ± 425 µg for nonsmokers and 807 ± 451 µg for smokers (p = 0.15). Subjects with BHR did not differ significantly from the control group as to conventional lung function parameters and VD determined from single-breath wash-in tracings of argon. However, the MEF₂₅ showed a trend for decreased values in nonsmokers with BHR compared with nonsmokers without BHR (64 ± 16% of predicted versus 78 ± 24% of predicted; p = 0.03).

Anamnestic data were collected using a questionnaire based on American Thoracic Society recommendations (17). All subjects were interviewed by a pulmonary specialist. In the group of nonsmokers with BHR, two subjects had a history of seasonal allergic rhinitis without pulmonary symptoms, three subjects reported a penicillin allergy, and one subject reported a contact eczema. In the group of smokers with BHR, three subjects had a history of allergic rhinitis without pulmonary symptoms and three subjects reported a contact eczema. Subjects with allergic rhinitis were tested outside of the relevant pollen season, i.e., during the winter months. The smoking habits of the subjects were quantified using the cumulative cigarette consumption expressed as pack-years. Potential participants were excluded if they had a former pulmonary disease or symptoms, a respiratory tract infection 12 wk prior to the study, or were pregnant. All participants were asked to refrain from using caffeine-containing beverages and smokers were asked to refrain from smoking on the day of lung function and provocation testing. Informed written consent was obtained from each eligible subject. The protocol was approved by the ethics committee of the Medical School of the Ludwig-Maximilians-University (Munich, Germany).

Data Analysis

All statistical calculations were performed using the SAS software package (18). The significance of differences between group averages was tested using the t test for independent samples (SAS procedure TTEST). Correlation analysis was performed using the Pearson product-moment correlations (SAS procedure CORR). The requested level of significance was 0.01.

	RESULTS

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Table 3 summarizes particle deposition and aerosol bolus dispersion data of women with and without BHR. Nonsmokers with BHR as well as smokers either with or without BHR showed significantly increased DE values compared with the control group of nonsmokers without BHR (Figure 1). Smokers with BHR showed the highest values of DE (49.2 ± 5.4%). In subjects with BHR there was also a trend for higher D values (nonsmokers: 572 ± 122 cm³; smokers: 587 ± 85 cm³) compared with the control group (542 ± 88 cm³) (p = 0.2). Table 4 summarizes the results of the correlation analysis between DE, anthropometric data, and conventional lung function parameters of the whole study group (n = 93). There was a weak but significant correlation of DE with Raw (r = 0.28; p = 0.008). PD₂₀ did not correlate with DE (r = 0.24; not significant [NS]).

View larger version (20K): [in this window] [in a new window]   Figure 1.   Deposition of particles inhaled into a volumetric lung depth of 800 cm3 in nonsmokers and smokers with and without asymptomatic nonspecific airway BHR. Dotted line is the mean.

	DISCUSSION

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Asymptomatic women with nonspecific BHR, nonsmokers as well as moderate smokers, showed a significantly increased loss of fine aerosol particles in the lung compared with the control group. The striking feature of the results is that nonsmokers with BHR exhibited elevated deposition values comparable to those found in moderate smokers (mean 13.8 pack-years) without BHR. Moderate smokers (mean 12.3 pack-years) with BHR showed the highest aerosol particle deposition being 29% higher than that observed in the control group. From all conventional lung function parameters under consideration subjects with BHR showed a trend for lower MEF₂₅ values and a trend for a higher aerosol bolus dispersion as an early marker for ventilation inhomogeneities. Both findings are indicative of small airway obstruction (13, 19). Given the preferential peripheral lung deposition of fine particles and the well-known pathologic findings in peripheral airways of asymptomatic smokers (20), our data provide support for the hypothesis that asymptomatic nonspecific BHR is also associated with inflammatory airway remodeling.

There is increasing evidence that in subclinical stages or symptom-free periods of asthma and chronic cigarette exposure inflammatory airway changes have already taken place (20). Inflammatory processes in peripheral airways have been shown in asymptomatic asthmatic subjects (23). In particular, one study in asymptomatic hyperresponsive males suggests a relationship between BHR and inflammatory changes in the airways (24). Thus, it is likely that inflammatory changes in the peripheral airways are the underlying mechanisms for BHR in our asymptomatic study population. Inflammatory remodeling processes result in focal hypersecretions, local edema, epithelial damage, and changes in lung elasticity (7, 25, 26). Because deposition of 0.9-µm particles is considered to be located predominately in small conducting airways and acini (27), it appears reasonable to attribute the observed increase in aerosol deposition to subclinical inflammatory changes of peripheral airways. The mechanisms by which inflammatory processes may contribute to increased particle loss are (1) reduction of airway caliber, (2) local flow turbulences, and (3) ventilation inhomogeneities.

Airway Caliber

Airway inflammation may reduce the caliber of small airways because of narrowing of bronchial lumina with mucus, edema, and/or hypertrophy of bronchial smooth muscle (6, 26). Small airways obstruction could be demonstrated in asymptomatic asthmatics who had normal large airways function as judged by spirometry or measurements of airways resistance by using a test of maximal expiratory flow while breathing air and a helium-oxygen mixture (31). Because the deposition of 0.9-µm particles is mainly related to gravitational sedimentation, the deposition efficiency due to sedimentation is dependent on airspace caliber. Thus, a reduction in small airway size is a reasonable explanation for the observed increase in aerosol deposition. In subjects with asymptomatic BHR the demonstrated trend for smaller MEF₂₅ values and higher D values also supports this hypothesis.

Air Flow

Turbulent air flows in central or peripheral airways are dynamic processes which have to be considered as possible factors for the observed enhanced particle loss. In central airways, local obstructions caused by mucus plugs, edema, or epithelial damage may result in turbulent air flow due to increased local air flow velocities. Consequently, local deposition even of particles as small as 1 µm is supposed to be enhanced (10, 11, 32, 33). However, central airway obstruction should be easily detectable by parameters such as Raw and FEV₁. Because these parameters showed no significant differences between subjects with and without BHR, turbulences in central airways may not be important for increased DE in our study groups.

Kim and coworkers (34) postulated that turbulences resulting from subtle focal constrictions in small airways, which can hardly be detected by conventional lung function tests, may lead to increased particle deposition. These investigators measured increased aerosol deposition due to impaction in tubes with focal constriction even at Reynolds (Re) numbers as low as 100. However, in this study measurements were performed with 3-µm particles and it is questionable whether the 0.9-µm particles applied in the present study, which have a 10 times smaller deposition probability due to impaction, may be efficiently deposited in turbulent air flows at low Re numbers. Further, a modeling study by Wiggs and coworkers (35) reported in contracted small airways (10th-15th airway generation) Re numbers < 130 at a flow rate of 1.25 L s¹. Given the flow rate of 250 cm³ s¹ applied in our study the corresponding Re numbers are 5 times lower (Re number < 30). Thus, turbulences in the small airways can be ruled out as a relevant factor of increased particle deposition.

Local Ventilation Inhomogeneities

Inflammatory airway remodeling will rarely be homogeneous and is likely to produce local ventilation inhomogeneities in the lung periphery. According to Otis and coworkers (36) the rate of emptying of a lung region can be characterized by its time constant, that is, the product of its resistance and its compliance. Because local resistance of a lung compartment is supposed to be changed by inflammation, the presence of BHR may cause subtle local ventilation inhomogeneities in the small airways (37). Affected lung regions may exhibit prolonged local time constants and should empty later during expiration. This mechanism in turn increases gravitational particle loss owing to a longer residence time of particles in the affected regions. This hypothesis is supported by Trajan and coworkers (19), who studied quantitatively the relationship between regional ventilation and regional radioaerosol deposition of particles with a mass median aerodynamic diameter of 1.2 µm (g = 1.8). Both in humans and in dogs, an increase in regional time constants, as inferred from a decrease in regional ventilation, was associated with an increase in peripheral aerosol deposition when normalized for ventilation. These investigators concluded that the increased residence time of particles is responsible for the enhanced deposition in regions that received lesser ventilation. Further, increased residence time of particles may also be caused by focal inflammatory airway narrowing (i.e., partial plugging with secretions, focal edema). These obstacles to aerosol particle transport may trigger circulating flows in the longitudinal direction which can act as "dead zones" in which an aerosol is temporarily trapped, thereby enhancing aerosol particle deposition and dispersion (40). A recently published study by Robertson and coworkers (41) observed that the addition of a 1-s inspiratory hold to the ventilatory pattern would cause a 20% increase of aerosol particle loss in the 1.0 µm size range. In addition to our deposition data, the observed trend for increased D values also supports this concept as a marker for ventilation inhomogeneities in subjects with BHR and in smokers (9).

At present there is still uncertainty about the relative importance of these mechanisms with respect to aerosol particle deposition because of the lack of histologic data of subjects with asymptomatic BHR. Finally, it is also not clear whether increased particle deposition is a risk factor because of individual airway geometry that precedes and predisposes to the development of BHR or is instead a manifestation of subclinical airway inflammation caused by exposure to risk factors.

In conclusion, this investigation has shown that particle deposition from inspired aerosol boluses is significantly increased in women with asymptomatic BHR but with normal pulmonary function. Because airways hyperresponsiveness is related to airway inflammation, increased aerosol bolus deposition may be an early marker of subclinical peripheral inflammatory airway remodeling. However, in asymptomatic subjects with BHR histologic studies are needed to provide a greater knowledge about the morphologic basis of the observed altered aerosol particle behavior.