INTRODUCTION

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Our knowledge of airways responsiveness comes largely from spirometric and plethysmographic measurements made during bronchoprovocation. However, the smallest peripheral airways normally contribute little to these measurements due to their low resistance. Woolcock and coworkers referred to these peripheral airways as the "silent zone" of the lung because airflow obstruction within them causes little change in conventional tests of pulmonary function (1). More recently, Mitzner demonstrated that the channels for collateral ventilation, generally regarded as ancillary structures assuming importance only under pathologic conditions, are respiratory bronchioles and alveolar ducts, the smallest airways (2). Hilpert first described a technique to measure the resistance of gas flow through collateral channels (3). With this technique, pressure at the tip of a double-lumen catheter wedged in a subsegment of the lung can be measured while gas flows through the second lumen of the catheter. The flow of gas is unidirectional through the catheter and small airways, exiting through an adjoining bronchus. The major site of resistance through this system is normally in airways less than 0.5 mm diameter (2). The technique has been modified and applied to the study of the lung periphery of man (4), horse (5), sheep (6), dog (7), cat (8), and rabbit (9). The few studies performed with this technique in human subjects have demonstrated that the smallest peripheral airways offer little resistance to air flow in healthy volunteers (10). However, asthmatic subjects demonstrate increased resistance (10) and subjects with emphysema show decreased resistance (4) relative to normal controls. Whether the smooth muscle of these peripheral airways responds to pharmacologic agonists known to constrict large airways, is largely unstudied in human subjects. Two recent studies suggest that these airways constrict after exposure to dry air (11) and bradykinin (12) in asthmatic subjects but not in normal volunteers. The purpose of this study was to determine the peripheral lung responsiveness to histamine, a pharmacologic agonist traditionally used for bronchoprovocation studies and thought to constrict airway smooth muscle by direct mechanisms in the peripheral lung (13). Whether the contractile response differs between asthmatic subjects and normal subjects was also determined.

	METHODS

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Subjects

Eight normal and 11 asymptomatic asthmatic subjects were recruited for this study. Subjects were classified by clinical history and airways responsiveness, with asthmatics demonstrating reactivity to methacholine at a concentration of < 5 mg/ml. Asthmatic subjects were atopic with positive skin test reactivity to one or more common allergens. Subjects refrained from using inhaled -agonists 8 h prior to testing and were taking no other medications. Normal subjects were asymptomatic, skin test negative, and did not respond to the highest concentration of methacholine tested (25 mg/ml). All subjects were free of upper respiratory infections for at least 6 wk prior to testing. None of the subjects had a history of cigarette smoking. This study was approved by the Johns Hopkins Institutional Review Board and informed consent was obtained from each subject.

Pulmonary Function

Measurements of spirometric parameters were made for whole lung methacholine challenge, histamine challenge, and before bronchoscopy. Spirograms were performed in triplicate (Stead-Wells Spirometer; Warren E. Collins, Braintree, MA) and FEV₁ and FVC were measured.

Bronchoprovocation

Histamine and methacholine challenges were performed on separate study days using standard techniques (14). Methacholine hydrochloride or histamine diphosphate was nebulized for 0.6 s by triggering a breath activated dosimeter. After first inhaling a phosphate-buffered saline solution, all subjects received 5 breaths of increasing concentrations of methacholine (0.08, 0.15, 0.31, 0.625, 1.25, 2.5, 5.0, 10.0, and 25.0 mg/ml) or histamine (0.03, 0.06, 0.13, 0.25, 0.5, 1.0, 2.5, 5, 10 mg/ ml). Spirometry was performed during the 5-min intervals between inhalations. The challenge was terminated when a 20% or greater decrease in FEV₁ occurred. Dose-response curves for both agonists were constructed and the cumulative dose in breath units (1 inhalation of 1 mg/ml of agonist) required to cause a 20% decrease in FEV₁ was determined (PD₂₀).

Bronchoscopy

Bronchoscopy was performed on a separate study day and spirograms were obtained prior to the procedure to ensure normal pulmonary function. Subjects were pretreated with atropine (0.6 mg intravenously) and aerosolized lidocaine (5 ml of 4%). An esophageal balloon was inserted through one naris to measure transpulmonary pressure and ensure that all peripheral airway pressure measurements made during bronchoscopy were performed at the same lung volume (FRC). Subjects were sedated with fentanyl (0.1 mg intravenously). With subjects in a supine position, a fiberoptic bronchoscope (Olympus BF10; Olympus Corp., Lake Success, NY) with an outer diameter of 6.0 mm was advanced into the lung as lidocaine (2%, 6-12 ml) was instilled directly into the airway to provide local anesthesia. The bronchoscope channel was cleared free of mucus and airway secretions with a sheathed cytology brush and wedged in the anterior segment of the right upper lobe. A double-lumen catheter (5 FR; Baxter Healthcare Corp., Santa Ana, CA) was inserted through the working channel of the bronchoscope. The catheter was passed through a cap which fit snugly on the port of the working channel to prevent backflow of gas. Air (5% CO₂ in air) flowed through a calibrated flow controller (Sierra, Carmel Valley, CA) and through one lumen of the catheter. Pressure at the tip of the bronchoscope was measured through the other catheter lumen using a Validyne differential transducer (P300D; Validyne Engineering, North-ridge, CA). Histamine aerosol was generated by a DeVilbiss ultrasonic nebulizer (#646; Somerset, PA). Gas flow through a second channel of the flow controller passed through the nebulizer. When challenging the peripheral airways subtended by the bronchoscope, the double-lumen catheter was removed and nebulized histamine passed directly through the working channel. The catheter was removed to ensure adequate aerosol deposition. Pharmacologic agents were nebulized for 90 s at a gas flow of 200 ml/min. Prior measurements of aerosol volumes delivered under these conditions indicated an aerosol delivery of 10 µl as assessed by weight gain of a dry desiccant canister. After the aerosol delivery, the double-lumen catheter was reinserted and pressure measurements were made at constant flow.

To determine the histamine responsiveness of the small airways subtended by the bronchoscope, the following protocol was used. After 4-5 normal tidal breaths, the subject was asked to breath-hold at FRC. Pressure was allowed to reach a stable plateau value after which the subject resumed normal tidal breathing. Duplicate pressure measurements were obtained for each experimental condition, separated by at least 1 min. Pressure was measured under baseline conditions and after aerosol challenge with the saline vehicle, histamine (10, 50, 100 mg/ml) and isoproterenol (2 mg/ml; Winthrop, New York, NY). The next higher concentration of histamine was not given if the plateau pressure achieved during breath-holding was > 30 cm H₂O. Isoproterenol challenge was performed to determine whether histamine-induced constriction could be reversed with this -agonist. Flow was maintained constant at 200 ml/min for all measurements as well as for aerosol delivery. However, because of high baseline pressures, flow was reduced to 100 ml/min in two asthmatic subjects for pressure measurements only.

Data Analysis

Peripheral airways responsiveness is presented as the peripheral airways resistance (Rp) determined by the pressure at the tip of the bronchoscope/gas flow (4). Group data are presented as the mean ± SE. The concentration of histamine that caused a 100% increase in peripheral airways response (PC₁₀₀) was calculated from the slope of the relationship between histamine concentration and the ratio of pressure achieved at each concentration/the pressure after saline challenge (P/Ps). The ratios for each histamine concentration were regressed by least squares analysis through the origin (in this case 1). Doubling this ratio is equivalent to a 100% increase in the response and the inverse slope of the relationship is equal to the PC₁₀₀.

The distribution of the data after a logarithmic transformation was not significantly different from the normal distribution. Thus, parametric statistics were applied on the logarithmic transform of all the data. A comparison of normal and asthmatic subjects' pulmonary function and responsiveness was performed using student's t test for unpaired data. The concentration of histamine that caused a significant increase from the saline control Rp was determined by ANOVA and Duncan's multiple range test. Stepwise multiple regression analysis was used to determine correlations between reactivity and lung function. A p value < 0.05 was accepted as significant.

	RESULTS

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Subject characteristics of the eight normal and 11 asymptomatic asthmatic volunteers are presented in Table 1. Baseline pulmonary function for all subjects was within the normal range. On average, however, the asthmatic subjects demonstrated decreased function compared with normal controls, as evidenced by the significant reduction in FEV₁/FVC (%) and the increase in baseline Rp. Whole lung responsiveness to methacholine and histamine were not demonstrable at the maximum cumulative doses studied for histamine (97 cumulative breath units) and methacholine (225 cumulative breath units) in normal volunteers. However, asthmatics demonstrated marked responsiveness to these agents within a narrow dose range (Figure 1). The individual histamine concentration-response relationships of small airways of normal and asthmatic subjects are presented in Figures 2a and 2b, respectively. Each line connects the absolute Rp values for one subject. Average values of Rp at baseline, after the saline vehicle challenge, the three histamine concentrations, and isoproterenol are presented in Table 2. Baseline Rp of asthmatics (0.041 ± 0.015 cm H₂O/ ml/min; mean ± SE) was significantly greater than normal subjects (0.011 ± 0.003 cm H₂O/ml/min; p = 0.019). On average, normal subjects demonstrated a significant increase in Rp from the saline vehicle control level at a histamine concentration of 50 mg/ml (p < 0.01). However, a clear difference in responsiveness can be seen between the two subject groups. On average, asthmatic subjects demonstrated a significant increase in Rp from saline control at the lowest delivered histamine concentration (10 mg/ml; p < 0.01). A comparison of baseline Rp with that achieved with 50 mg/ml histamine is presented in Figure 3, which demonstrates the difference in responsiveness. At a concentration of 50 mg/ml of histamine, absolute Rp was 0.022 ± 0.003 cm H₂O/ml/min for normal subjects and 0.134 ± 0.018 cm H₂O/ml/min for asthmatics (p < 0.01).

View larger version (10K): [in this window] [in a new window]   Figure 1.   Comparison of whole lung methacholine (PD20) and histamine reactivity (PD20) and peripheral airways histamine responsiveness (PC100) in normal (solid circles) and asthmatic subjects (open circles). PD20 is presented as the cumulative breath units that caused a 20% decrease in FEV1. A breath unit is equal to 1 inhalation of a 1 mg/ml concentration of agonist. PC100 is the histamine concentration that caused a 100% increase in the ratio of peripheral airways pressure/pressure after saline challenge. A significant difference in responsiveness between normals and asthmatics is apparent for whole lung methacholine and histamine challenge (p < 0.001) and peripheral airways responsiveness (p = 0.0114). Points in brackets above the maximum dose represent subjects who failed to respond to the maximum concentration of challenge agonist with a 20% decrease in FEV1.

View larger version (13K): [in this window] [in a new window]   View larger version (18K): [in this window] [in a new window]   Figure 2.   (A) Concentration-response relationship for the peripheral airways of eight normal subjects. Each line connects the peripheral airway resistance (Rp) values for one subject. (B) Concentration-response relationship for the peripheral airways of 11 asthmatic subjects. Each line connects the peripheral airway resistance (Rp) values for one subject.

View larger version (24K): [in this window] [in a new window]   Figure 3.   Comparison of histamine responsiveness (50 mg/ml) to baseline peripheral airway resistance (Rp) in normal (solid circles) and asthmatic (open circles) subjects (#maximum concentration delivered in this subject was 10 mg/ml).

Applying the conventional method for describing the sensitivity of airways to an agonist, the PC₁₀₀ was determined. A graphical presentation of the method by which the PC₁₀₀ was calculated is shown in Figure 4. The ratio of the pressure achieved after each histamine dose divided by the pressure achieved after the vehicle control (P/Ps) is plotted as a function of histamine concentration. Since a doubling of this ratio is equivalent to a 100% increase in the response, the concentration required to achieve this increase is the PC₁₀₀. The inverse slope of this linearized relationship is equal to the PC₁₀₀. The average PC₁₀₀ for the normal subjects was 32.4 ± 5.4 mg/ml and for the asthmatic subjects it was 17.1 ± 5.3 mg/ml (p = 0.0114). Using stepwise multiple regression analysis, asthmatic status (partial r = 0.77; p = 0.0002) and baseline Rp (partial r = 0.70; p = 0.001) were shown to be significant determinants of the log PC₁₀₀. However, the association of Rp and PC₁₀₀ was in the positive direction suggesting that an increased baseline resistance contributed to an increased PC₁₀₀. The range of peripheral airways responsiveness defined by the calculation of PC₁₀₀ was compared with whole lung methacholine and histamine challenge (Figure 1). In the asthmatic subjects, log PC₁₀₀ was significantly correlated with the histamine log PD₂₀ (r = 0.847; p < 0.05). The relationship between histamine log PD₂₀ and log PC₁₀₀ (n = 9) is shown in Figure 5. However, neither histamine log PD₂₀, log PC₁₀₀, nor baseline Rp was significantly correlated with methacholine log PD₂₀. Asthmatic status was associated significantly with low FEV₁/FVC, high baseline Rp, and low PC₁₀₀ (p < 0.05).

View larger version (19K): [in this window] [in a new window]   Figure 4.   Relationship between histamine concentration and the ratio of pressure achieved at each concentration divided by the pressure after the saline vehicle control (P/Ps), in normal subjects (solid circles) and asthmatic subjects (open circles). Solid lines connect the points associated with the response of each normal subject and the dashed lines are associated with asthmatic subject responses.

View larger version (13K): [in this window] [in a new window]   Figure 5.   Relationship between log PD20 histamine (cumulative breath units) and log PC100 histamine (mg/ml) in nine asthmatic subjects, r = 0.847, p < 0.05.

The ability of aerosolized isoproterenol to reverse the histamine-induced constriction of the peripheral airways in individual normal and asthmatic subjects is presented in Figure 6. On average, the increase in Rp of normal subjects after histamine was reversed with the administration of isoproterenol (Table 2). Rp after isoproterenol did not differ from the saline control value. However, within the asthmatic subjects, the delivered dose of isoproterenol was not adequate to completely reverse the constriction. Rp remained significantly elevated over the saline control level (p < 0.01).

View larger version (23K): [in this window] [in a new window]   Figure 6.   Effects of isoproterenol on peripheral airway resistance (Rp) after maximal histamine-induced constriction. Solid circles = normal subjects; open circles = asthmatic subjects.

	DISCUSSION

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Our knowledge of airways reactivity is derived predominantly from tests of whole lung responsiveness to pharmacologic agonists. However, the responsiveness of the smallest airways, potentially most important in the pathogenesis of airways diseases, is minimally represented in these measurements and remains largely unstudied. The results of the present study demonstrate that when measured directly, the smallest airways of normal and asthmatic subjects constrict when challenged locally with histamine. Yet, as expected, only the asthmatic subjects demonstrated whole lung responsiveness to histamine and methacholine. The small airways of normal subjects were surprisingly responsive, demonstrating on average, an approximate 200% increase in resistance when challenged with the highest histamine concentration studied. However, when focusing on the absolute changes in Rp and the magnitude of those changes presented in Figure 3, it is clear that the small airways of the asthmatic subjects studied were much more sensitive to histamine. On average, asthmatic subjects demonstrated a significant increase in Rp after challenge with the lowest histamine concentration studied. Because of high pressures (> 30 cm H₂O), several asthmatic subjects were not given the highest histamine concentration. A 320% increase in Rp over the saline control level was observed in those asthmatic subjects that received the highest histamine concentration (100 mg/ml). Furthermore, when the provocative concentration that caused a 100% increase in peripheral airways response (PC₁₀₀) was calculated, the difference between normal and asthmatic subjects was highly statistically significant. Thus, based both on the extent of constriction after challenge with a particular concentration of histamine as well as the provocative concentration required to cause a 100% increase in resistance, the small airways of the asthmatics were more responsive than those of normal subjects.

Our study of peripheral airways required a method for quantification of peripheral airways responsiveness compar-able to the PD₂₀ used to assess whole lung responsiveness. We elected to use the slope of the histamine dose-response relationship to determine the PC₁₀₀, since this calculation took into account the entire range of the acquired dose-response data. Our reported values for PC₁₀₀ correlated significantly with those obtained by linear interpolation (r = 0.897).

It is interesting to note that baseline Rp was a significant determinant of, and positively correlated with PC₁₀₀. This result likely was due to the three asthmatic subjects that had the highest baseline Rp yet did not readily constrict to histamine. Whether the peripheral airways of these subjects were already maximally constricted or whether the histamine delivery to smooth muscle was limited due to increased constriction or edema, is not known. However, the overall greater PC₁₀₀ of the normal subjects relative to the asthmatic subjects likely is not due to the increased baseline Rp of the asthmatic subjects. These results are in accord with Twentyman and colleagues, who demonstrated that increased baseline airway narrowing does not predict enhanced reactivity (15). These investigators showed that when baseline airway caliber was decreased by methacholine challenge, subsequent contractile responses to histamine were not different from provocation from baseline. Perhaps structural changes within the small airways are induced by inflammatory processes and lead to airway luminal narrowing. However, agonist access and/or smooth muscle contractility may be limited by wall thickening and fibrosis.

We have previously demonstrated a significant correlation between baseline peripheral airways conductance (1/Rp) and whole lung methacholine responsiveness (log PD₂₀) (10). That baseline resistance of these small airways is predictive of whole lung responsiveness to exogenously administered stimuli has been supported by other recent reports. Kaminsky and colleagues demonstrated a significant correlation between Rp and airways responsiveness to exercise (11). In normal subjects, Weinmann and coworkers demonstrated a significant association between Rp and airways responsiveness to O₃ (16). However, in the present study group of asthmatics, baseline Rp did not correlate with whole lung methacholine or histamine reactivity. Subjects with greater extremes of disease severity and/ or reactivity may be required to further confirm the association between baseline Rp and whole lung reactivity.

Histamine has been shown to act directly on airway smooth muscle of small airways in normal subjects (13). This finding was confirmed in animal studies in which vagotomy had no effect on the histamine-induced increase in collateral resistance (17). Histamine H-1 receptors on airway smooth muscle mediate bronchoconstriction and there appears to be no difference in the distribution of these receptors in normal and asthmatic subjects (18). In addition, histamine is known to cause protein and fluid extravasation form the airway vasculature (19). However, it appears unlikely that the enhanced responsiveness observed in the peripheral airways of asthmatics could be related to airway edema based on recent work by Berman and colleagues (12). Using the same methodology as in the present study, bradykinin was shown to cause a dose-dependent increase in Rp in asthmatic subjects but not in normal controls. However, the amount of protein extravasation, assumed to be a marker of airway edema, was the same in the two study groups. Thus it seems unlikely that fluid transudation alone could account for the enhanced histamine responsiveness observed in asthmatics.

We can only speculate as to why isoproterenol reversed completely the constriction induced in the normal subjects but not in the asthmatic subjects. The same single dose of isoproterenol was used for all subjects studied and proved to be inadequate to reverse the high levels of constriction obtained in the asthmatic subjects. Decreased drug delivery and/or access, decreased -receptor number and/or binding affinity, or altered intrinsic properties of smooth muscle relaxation could all have played a role in this observation.

In summary, we have demonstrated that the resistance of the smallest peripheral airways challenged locally with histamine, and measured directly, increases substantially in both normal and asthmatic volunteers. However, the peripheral airways of the asymptomatic asthmatics studied were more sensitive to histamine challenge than those of normal controls.