INTRODUCTION

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IT HAS BEEN RECENTLY SUGGESTED that asthma and rhinitis represent two different phenotypes of the same disease (9). In support of this hypothesis are the observations that experimental exposure to allergen causes bronchial narrowing (2, 6) and inflammation (17) in subjects suffering from rhinitis alone, i.e., without asthma. An important difference between asthma and rhinitis alone is the much greater dose of allergen necessary to cause significant bronchoconstriction in the latter (2), which may explain why some allergic subjects develop asthma symptoms on natural exposure to allergen, whereas others do not. The reasons for a greater bronchial responsiveness to allergen in asthma than in rhinitis are, however, still largely unexplained.

In subjects with a similar type and degree of systemic allergic sensitization, differences in bronchial responsiveness to allergen may be related to differences in bronchial inflammation and remodeling or airway mechanics. It has been previously shown that the bronchi of rhinitic subjects are susceptible to develop an inflammatory response to allergen that is indistinguishable from that of asthmatic bronchi (17). However, only bronchoalveolar lavage (BAL) was used, which precluded any evaluation of bronchial wall inflammation or remodeling. Furthermore, possible differences of airway mechanics in response to allergen between asthmatic and rhinitic subjects were not investigated.

In the present study, we used both BAL and bronchial biopsy (BB) to evaluate bronchial inflammation and remodeling in subjects with mild asthma (with or without rhinitis) and subjects with rhinitis alone. Possible differences in airway mechanics between the two groups were investigated by comparing 1) the bronchial responsiveness to allergen and to methacholine (MCh), and 2) the effects of deep inhalation (DI) on airway caliber (15) during allergen and MCh challenges.

	METHODS

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Subjects. The study was conducted on 17 male, nonsmoking subjects, who were sensitized to house dust mite (HDM), as documented by a third-to-fourth radioallergosorbent test class (Pharmacia, Uppsala, Sweden) and a positive skin prick test. The latter was defined by a wheal response to HDM extract (Lofarma, Milan, Italy) equal to or larger than that caused by 10 mg/ml histamine. Eight subjects (age 25 ± 3 yr) had a history of rhinitis and never experienced symptoms that may be related to asthma or airway hyperresponsiveness, such as wheeze, waking up at night with shortness of breath or chest tightness, or cough or sputum on exposure to house dust (4). Nine subjects (age 24 ± 3 yr) had bronchial asthma, according to the definition of the American Thoracic Society (1). At the time of the study, all subjects were in a stable clinical condition, with a 1-s forced expiratory volume (FEV₁) >70% of predicted (16), and none of them was taking inhaled or oral steroids, cromolyn, antihistamine, or regular bronchodilators. Asthmatic subjects were using short-acting ₂-agonists on demand, which were avoided for 12 h before studies. The study was approved by the institutional ethics committee, and subjects gave written, informed consent.

Protocol. All subjects first underwent a MCh inhalation challenge followed, 24 h later, by BAL and BB. After 1 wk at least, they underwent a HDM inhalation challenge followed, 24 h later, by BAL and BB. Six asthmatic and six rhinitic subjects attended the laboratory on two further occasions to have MCh and HDM challenges repeated to determine the effects of DI.

Lung function measurements. A Vmax 6200 system (SensorMedics, Yorba Linda, CA) was used for baseline pulmonary function tests and MCh challenge. Portable spirometers (Micro Medical, Rochester, Kent, UK) were used to monitor lung function during the allergen challenge, with the same subject using the same instrument at the hospital and at home. On each occasion, the highest of three FEV₁ measurements within 100 ml was retained. The effects of DI during allergen and MCh challenges were determined by comparing expiratory flows at 40% of control forced vital capacity (FVC) during forced expiratory maneuvers started from full inflation (maximal flow) and from ~60% of FVC (partial flow) using dedicated software (Vmax 6200, SensorMedics). Three acceptable sets of partial and maximal flow-volume curves were obtained at control, and one at each step of the challenges. Each set consisted of a forceful expiration from ~60% of FVC to residual volume, followed by a fast inhalation to total lung capacity and, without breath hold, a forceful expiration to residual volume. The bronchodilator effect of DI was inferred from the slope (MP slope) and the intercept (MP intercept) of the regression line of maximal (M) vs. partial (P) flows measured at all steps of the challenges (15). In this analysis, a slope <1 indicates that, for any given decrease of partial flow, the maximal flow decreases less, thus suggesting a bronchodilator effect of DI. The coefficient of variation (CV) of MP slope and MP intercept measurements was determined in a separate group of stable asthmatic subjects to be 14 and 20%, respectively (unpublished data).

MCh challenge. Solutions of MCh were prepared by adding distilled water to dry powder MCh chloride (Laboratorio Farmaceutico Lofarma). Aerosols were delivered by an SM-1 Rosenthal breath-activated dosimeter (SensorMedics) driven by compressed air (30 psi) with 1-s actuations. The aerosol output at the mouth was 10 µl per actuation. Aerosols were inhaled during quiet tidal breathing. After 20 inhalations of saline as a control, the subjects inhaled double-increasing doses of MCh from 0.02 mg until FEV₁, measured 1 min after each dose, decreased below 80% of control value or the maximum dose of 1.2 mg was achieved. The double increments of dose were obtained by using two MCh concentrations (1 and 10 mg/ml) with appropriate numbers of breaths. A 3-min interval was allowed between dose increments. The noncumulative dose causing a 20% reduction of FEV₁ from control (PD₂₀) was calculated by interpolation between two adjacent points of the log dose-response curve. In subjects who did not reach a 20% reduction of FEV₁, the last MCh dose was retained as PD₂₀ for statistical analysis. PD₂₀ values <1.2 mg were considered positive for airway hyperresponsiveness.

HDM challenge. A powder-allergen method was used (13). The HDM extract consisted of a Dermatophagoides pteronyssinus and farinae dry mix (50:50) predosed in arbitrary units (AU) by radioallergosorbent test inhibition technique with an internal (Laboratorio Farmaceutico Lofarma) reference extract to which a value of 1,000 AU had been assigned. One arbitrary unit corresponded to 7.5 IU of the World Health Organization International Union of Immunological Societies Dermatophagoides pteronyssinus International Standard (National Institute for Biological Standards and Control code 82/518). The mean particle size of the allergen powder determined by laser particle sizer (Analysette 22, Fritsch, Idar, Oberstain, Germany), with ethanol as dispersing fluid, was 2.5 µm, with 99.9% of the particles < 18.2 µm, 70% < 3.9 µm, 50% < 2.5 µm, 30% < 1.5 µm, and 10% < 0.7 µm. Micronized allergen in the form of inert lactose powder was contained in rigid gelatin capsules (40 ± 2 mg of powder each). The following doses were made available by the manufacturing firm: 5, 10, 25, 50, 100, 200, and 400 AU. The powder was administered through a turboinhaler device (Schiapparelli-Searle, Torino, Italy) with a series of slow inspiratory capacity maneuvers repeated until the capsule was empty. A control measurement of FEV₁ was obtained 15 min after inhalation of lactose. The challenge was then started from the lowest dose of allergen (5 AU) and stopped when the FEV₁, measured 15 min after the dose, was decreased below 80% of control value. If such a decrease of FEV₁ did not occur with the dose of 790 AU, an additional 400 AU were administered, and the challenge stopped at the cumulative dose of 1,190 AU. In rhinitic subjects, the first three doses (5, 10, and 25 AU) were inhaled consecutively as a unique dose of 40 AU. Cumulative PD₂₀ was calculated by interpolation of the dose-response curve. In subjects who did not reach a 20% reduction of FEV₁, the last allergen dose was retained as PD₂₀ for statistical analysis. FEV₁ measurements were then taken hourly for 24 h, except when the subject was asleep. A decrease of FEV₁ 20% of control value recorded at any time within 24 h after allergen inhalation was considered as evidence of a positive response.

Bronchoscopic procedure. After premedication with atropine (0.5 mg im) and prometazine hydrochloride (50 mg im), lidocaine (2% solution) and adrenaline (0.1:1,000 solution) were instilled into the nostrils. A fiber-optic bronchoscope (Olympus BF, type 1T20) was then passed through the nose or the mouth without endotracheal tube and, after local anesthesia of pharynx and airways, wedged into a subsegmental bronchus of the right middle lobe. Sterile saline (5 aliquots of 20 ml each) was instilled. The BAL fluid was collected by applying a negative pressure (50-120 mmHg) at the proximal port of the bronchoscope. Immediately after BAL, four biopsy specimens were taken from the right upper lobe distal bifurcation.

BAL analysis. After filtration through two layers of sterile gauze, the fluid was centrifuged at 500 rpm for 5 min. The cell pellet was washed once and resuspended in Hanks' balanced salt solution, without Ca²⁺ and Mg²⁺, at a concentration of 10⁶ cells/ml. A small sample of the cell suspension was centrifuged (Cytospin, Shandon Southern Instruments, Sewickley, PA) at 500 rpm for 5 min, spinning ~100,000 cells onto a glass slide. Cells were air dried and stained (Diff-Quik; Merz & Dade, Dudingen, Switzerland) for a differential cell count of 300 cells per slide, by light microscopy. Epithelial cells were not included in the differential count, and their absolute values are reported separately. Cell-free supernatants were assayed for eosinophil cationic protein (ECP) by fluoroimmunoassay (Pharmacia UniCAP System Fluoroenzymeimmunoassay, Pharmacia Diagnostic) and for tumor necrosis factor- (TNF-) content by enzyme immunoassay (Cytelisa, Cytimmune Science, College Park, MD) following the manufacturer's instruction. The sensitivity of ECP and TNF- assay is 2 ng/ml and 4.8 pg/ml, respectively. The albumin concentration in the supernatant was determined by nephelometry. Total and specific IgE to dermatophagoides were measured by reverse enzyme allergosorbent test (Laboratorio Farmaceutico, Lofarma), which is based on the capture of both total and specific IgE by a specific antihuman IgE antibody and the subsequent reaction with streptoavidin-peroxidase and chromogenic substrate (8). The optical density was read on a human IgE reference curve titrated referring to the Second World Health Organization IgE standard 75/502. For both total and specific IgE, the lower and upper detection limits were 0.2 and 100 U/ml, respectively.

BB analysis. Biopsy specimens were fixed in 10% formaldehyde at 4°C for 4 h, embedded in paraffin, cut at 5 µm with a rotative microtome, and stained with hematoxylin and eosin and toluidine blue. Eight too small and incorrectly oriented biopsies were discarded, but for each subject at least one biopsy was available for analysis. Microscopic examination was performed by one independent observer, who was unaware of the aim of the study. For cell counts (eosinophils and mast cells), bronchial mucosa was examined to a depth of 100 µm from the basement membrane over adjacent non-overlapping high-power fields (at ×500 magnification with the aid of an eyepiece graticule) until all of the available area was covered. For each quantification, three sections were examined, and cells were expressed as number per square millimeter of submucosa. Mast cells were also examined at ×1,000 magnification to detect ongoing degranulation (granules surrounding the cell or crossing the cell membrane). The intraobserver mean CV for three repeated measurements was 9% for eosinophils and 8% for mast cells. The thickness of reticular basement membrane (RBM) was measured on hematoxylin- and eosin-stained preparations by light microscope image analysis (Q 500, Leica, Cambridge, UK) at ×400 magnification from the base of bronchial epithelium to the outer limit of the reticular lamina, at regular intervals (20 µm), until 40 readings were obtained. The intraobserver mean CV for three replicate measurements was 3%. The percentage of RBM covered by epithelium was also calculated.

Statistical analysis. Unpaired t-test was used to compare anthropometric and lung function data between groups. Fisher's exact test was used to compare categorical variables. Two-factor, repeated-measure ANOVA with least significant difference post hoc test was used to compare MP slopes and MP intercepts between and within groups. Mann-Whitney U-test was used for comparisons of BAL and BB data between groups before and after allergen challenge. Wilcoxon matched-rank test was used to compare data before and after allergen challenge. P < 0.05 was considered statistically significant. Data are presented as means ± SD or medians with interquartile ranges.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

BAL data. See Table 1. Neither at baseline nor after HDM challenge were the differences in inflammatory cell counts and epithelial cells statistically significant between asthma and rhinitis (P > 0.2, for all comparisons). Total cell number and percentages of eosinophils were increased after HDM challenge significantly in rhinitis and nonsignificantly in asthma, whereas the percentage of macrophages decreased in both groups. The ECP and TNF- contents of supernatant were not significantly different at baseline (P > 0.2), and ECP increased similarly in the two groups after HDM challenge. Total and specific IgE, detectable in five rhinitic and five asthmatic subjects, did not show significant differences between groups (P > 0.3). Also the albumin content of BAL supernatant was not significantly different between groups (P > 0.5).

BB data. See Table 2. There were no significant differences in inflammatory cell numbers between rhinitis and asthma at baseline (P > 0.5 for all comparisons), but the number of eosinophils increased significantly in rhinitis, and the percentage of degranulating mast cells increased in asthma after HDM. Neither RBM thickness nor the percentage of RBM covered by intact epithelium were significantly different between groups (P > 0.7 for both comparisons), although the latter tended (P = 0.06) to be less in asthma after HDM.

Lung function data. See Table 3. Both FEV₁ and its ratio to FVC (FEV₁/FVC) were less in asthma than in rhinitis, suggesting mild airway obstruction at baseline in the former. Individual baseline FEV₁ values on the allergen challenge days were always within ±8% of those on the MCh challenge days, without significant differences between or within groups. Both allergen PD₂₀ and MCh PD₂₀ were significantly less in asthma than in rhinitis. The response to MCh was positive in all asthmatic subjects but in only two rhinitic subjects; this difference was statistically significant (P < 0.01). The response to HDM was positive in all asthmatic subjects and in five out of eight rhinitic subjects, a difference that was not statistically significant (P > 0.08). Of the two rhinitic subjects with positive response to MCh, one (PD₂₀ = 1,200 µg) did respond to HDM, whereas the other (PD₂₀ = 487 µg) did not. The late-phase bronchial response to HDM tended to be more severe in asthma than in rhinitis (maximum late FEV₁ fall: 32 ± 16 vs. 18 ± 11%, P = 0.05), although five asthmatic subjects were given bronchodilator treatment to relieve symptoms.

View larger version (9K): [in this window] [in a new window]   Fig. 1.   Mean regression lines of maximal vs. partial flows during challenges, calculated by solving the linear model y = a + bx, where a is mean intercept and b is mean slope from Table 3. The extreme x values for each line are the mean values of partial flow at control and at challenge end point. Solid lines indicate methacholine challenge; dashed lines indicate house dust mite challenge. A, asthmatic subjects; R, rhinitic subjects. See Table 3 for statistical differences. Note the higher maximal flows for the same partial flows in R than in A during MCh challenge, suggesting a greater bronchodilator effect of deep inhalation. Note also the increase in slope in R during allergen challenge, indicating a progressive reduction of the bronchodilator effect of deep inhalation with increasing allergen dose.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The new findings of this study are that 1) the response to MCh better separated asthmatic from rhinitic subjects than did the response to HDM, 2) the bronchodilator effect of DI was greater in rhinitis than in asthma during MCh challenge, but it progressively decreased in rhinitis during HDM challenge, and 3) bronchial responsiveness to HDM was greater in asthma than in rhinitis, even if baseline airway inflammation and remodeling were similar. In addition, we confirm and extend previous studies (17), showing that the bronchi of rhinitic subjects developed an inflammatory response to inhaled allergen that was similar to that of asthmatic subjects.

Comments on methodology. This study has some limitations. First, inflammatory cell data were obtained only 24 h after the HDM inhalation. Therefore, differences in the timing of inflammatory cell influx into the airways cannot be evaluated. However, enumeration of blood eosinophils in seven subjects (4 with asthma and 3 with rhinitis alone) showed a nonsignificantly different decrease 3 h after HDM inhalation (57 ± 9% in asthmatic subjects vs. 70 ± 30% in rhinitic subjects), suggesting a similar early recruitment of eosinophils from blood in both groups. Second, inflammatory cells lying in the bronchial mucosa deeper than 100 µm below the basement membrane were not evaluated, and differences cannot be excluded. Third, no relationship between inflammatory response and HDM dose could be determined, as it would have been ethically unacceptable to have repeated HDM challenges and bronchoscopies. Fourth, in this study, biopsy specimens were taken at a single site and assumed to be representative of airway remodeling throughout the lung. This assumption seems to be justified by the findings of Bradley et al. (3) and Jeffery et al. (11). Finally, only subjects with mild asthma were studied, and the findings cannot be extrapolated to more severe disease.

Comments on results. In asthma, the degree of allergic sensitization and the degree of bronchial responsiveness are independent determinants of both early and late bronchoconstrictor responses to allergen (5). In the present study, neither blood nor BAL IgE levels were significantly different between asthmatic and rhinitic subjects. For the same degree of systemic allergic sensitization, as revealed by serum-specific IgE level and/or skin test response, the bronchoconstrictor response to allergen may be expected to be determined by local factors, such as baseline bronchial inflammation, allergen-induced bronchial inflammation, and mechanical response.