© 2003 American Thoracic Society
Bronchial Challenge Tests in Patients with Asthma Sensitized to CatsThe Importance of Large Particles in the Immediate ResponseService de Pneumologie, Hôpital Civil and Service de Biostatistiques et Informatique Médicale, Faculté de Médecine, Strasbourg; Institut National de Recherche et de Sécurité, Vandoeuvre; Centre Hospitalier Universitaire Cochin, AP-HP-Université, Paris V, France Correspondence and requests for reprints should be addressed to Frédéric de Blay, M.D., Service de Pneumologie, Hôpital Civil, BP 426, 67091 Strasbourg Cedex, France. E-mail: frederic.deblay{at}chru-strasbourg.fr
Our aim was to compare bronchial responses to major cat allergen (Fel d 1) in individuals with intermittent asthma sensitized to cats (19 subjects) according to the droplet particle size. We used three nebulizers, which delivered particles with mass median aerodynamic diameters of 1.4, 4.8, and 10.3 µm. A dosimeter nebulizer was used. The cat allergen was diluted to obtain the same amount of Fel d 1 per puff with each nebulizer. Each patient underwent three methacholine bronchial challenge tests (BCT), each followed 24 hours later by a cat allergen BCT, each performed with a different nebulizer (randomly selected each time, with patient and tester always blinded). Subjects did not differ for methacholine responsiveness, FEV1, mean forced expiratory flow during the middle half of the FVC (FEF25–75), PEF, or dyspnea (Borg scale) before any of the three cat BCTs. Cat allergen PD20 was 271 ng of Fel d 1 with the 1.4 µm nebulizer, 46 ng with the 4.8 µm nebulizer, and 13.5 ng with the 10.3 µm nebulizer (p = 0.00001). Inhalation of small particles (1.4 µm) resulted in significantly lower FEF25–75 24 hours after provocation than large particles did. In conclusion, immediate bronchial response appears to be localized in large airways, and the use of large particles is more appropriate for cat allergen BCTs.
Key Words: bronchial challenge test • particle size • cat allergen • asthma The particle size of droplets used during bronchial challenge tests (BCT) with allergen has not been substantiated since Tiffeneau (1), and current guidelines on BCT with allergen offer no recommendations concerning particle size (2, 3). Patients appear to recognize the relation between exposure and symptoms more readily for cat than for mite allergens. It has been suggested that the rapidity of bronchial symptoms to cat allergen may be due to its high proportion of small particles (40% <5 µm), which can enter deeply into the lung (4). Moreover, recent studies show that bronchial inflammation is more severe in small than in large airways (5). On the other hand, the dose of cat allergen required to induce symptoms during a standard BCT (with a nebulizer releasing particles ranging from 3 to 5 µm) has been reported to be 5 to 300 times higher than the doses needed to induce symptoms at home (6–8). One possible explanation among several hypotheses (e.g., during BCT, aqueous concentrated allergen extract is delivered, inhalation is only oral, and cofactors are absent) is the absence of large particles (10 µm or larger) during a standard BCT. In contrast, during domestic exposure, cat allergen is inhaled on a wide range of particle sizes—39% on particles measuring 6 to 20 µm (9, 10). To assess the bronchial effect of cat allergen according to the size of the particles nebulized, we used three different aerosols with mass median aerodynamic diameters of 1.4, 4.8, and 10.3 µm to compare bronchial response in individuals with intermittent asthma sensitized to cats.
Measurement of Particle Size Laser particle size analysis (MasterSizer X; Malvern Instruments Ltd, Worcestershire, UK) (11) was used to help select three air-driven nebulizers of different sizes. Cat extract (Stallergenes Laboratories, Antony, France) was nebulized, and particle size distribution was measured with a cascade impactor (Andersen 2000 Inc., Schaefer, Germany) (12). The cat major allergen Fel d 1 was measured with a two-site monoclonal antibody ELISA (Indoor Biotechnology, Charlottesville, USA) (10).
Patients
The Study Design
Methacholine BCTs They were performed with a nebulizer (Mediprom FDC 88, Paris, France) that administered doubling doses of a 1% methacholine solution, from 25 to 3,200 µg (cumulative), as described previously (13).
Cat BCTs
Statistical Analysis
Measurement of Particle Size The particle actually delivered by each of the three air-driven nebulizers chosen (Microcirrus and Cirrus; Intersurgical Laboratories, Fontenay-sous-bois, France and Pari Tia; Pari Aerosol Research Institute, Munich, Germany) was measured repeatedly by laser particle size analysis and with the cascade impactor (Table 1) (18). There was almost no overlap between the three size distributions. The mass median aerodynamic diameters obtained with the cascade impactor were comparable with those measured with the laser particle size analysis.
Patients Nineteen patients (11 women and 8 men, mean age 27 years) participated in the study, after giving written informed consent (Table 2) . No detectable levels of airborne Fel d 1 were found in the homes of 15 patients, and the levels were close to the detection limit in the other 4. The median time from exposure to onset of cat-related symptoms at home was 30 minutes. Those sensitized to tree and grass pollen were studied out of pollen season. Smokers and nonsmokers did not differ significantly for any of the parameters measured (methacholine responsiveness, PEF, FEV1, FEF25–75, or dyspnea Borg scale); we note that none of the smokers smoked more than 5 cigarettes per day.
Dyspnea (Borg Scale), Pulmonary Functions, and Methacholine Challenge Test before the Cat BCTs Table 2 reports preinclusion FEV1 and FEF25–75. The patients did not differ significantly in their dyspnea (Borg scale), FEV1, FEF25–75, or PEF before the three cat BCTs (p = 0.21, p = 0.55, p = 0.19, and p = 0.78, respectively). The methacholine PD20 did not differ significantly before any of the three cat BCTs (p = 0.127) (Table 3) .
Effect of Particle Size on Fel d 1 PD20 PD20 for cat allergen decreased from a geometric mean value of 271 ng of Fel d 1 with the 1.4 µm nebulizer to 46.0 ng with the 4.8 µm nebulizer and 13.5 ng with the 10.3 µm nebulizer (p = 0.00001; after adjustment for prechallenge FEV1) (Table 3). This result was obtained for all patients, except Patient 5, whose PD20 for Fel d 1 was the same with the 4.8 and 10.3 µm nebulizers but dramatically different with the smallest droplets (1.4 µm). All patients had a positive bronchial response to the 4.8 and 10.3 µm particles but six had a negative cat BCT with the 1.4 µm aerosol.
Effect of Particle Size on Estimated Deposition of Fel d 1 after Cat BCT
Effect of Particle Size on Immediate Bronchial Response to Cat Allergen Because 6 of the 19 patients had no immediate bronchial response to the 1.4 µm particles, the FEV1 and FEF25–75 were closer to their predicted value at the end of the cat BCT for the 1.4 µm particles than for the other particle sizes (p = 0.019 for FEV1 and p = 0.0009 for FEF25–75) (Figures 2A and 2B) ; this was also the case for dyspnea (p = 0.013) (Figure 2C).
Effect of Particle Size on Late Bronchial Response to Cat Allergen At 6 hours, the frequency of a late 15% drop in FEV1 did not differ significantly for any of the three particle sizes (i.e., 2 after 1.4 µm, 4 after 4.8 µm, and 1 after 10.3 µm aerosol). FEV1 values did not differ significantly by particle size at 3, 6, or 24 hours after the end of the cat BCT (Figure 2A). FEF25–75 did not differ according to particle size at 6 hours (p = 0.919); after 24 hours, however, it was significantly lower with the 1.4 µm particles (p = 0.006) (Figure 2B). In addition, its difference between before and 24 hours after the cat BCT was significantly greater with the 1.4 µm aerosol than with the others (means of 8.6% [95% confidence interval: 3.7; 13.7]; -1.2% [-5.4; 3] and -3.4% [-7.9; 1.1] with 1.4, 4.8, and 10.3 µm particles, respectively; p = 0.003)(Figure 2B). Six hours after the end of the BCT, Borg scale values for dyspnea were significantly higher with the 1.4 µm aerosol than with the other two (p = 0.02) (Figure 2C). Moreover, the difference between these Borg values before and 6 hours after cat BCT also differed according to particle size (means of 0.3 cm [95% confidence interval: 0; 0.6]; 0.15 cm [-0.22; 0.52] and -0.6 cm [-1.06; -0.14] with 1.4, 4.8, and 10.3 µm particles, respectively; p = 0.007). The 6 patients with negative cat BCTs (with the 1.4 µm aerosol) underwent methacholine challenge tests the next day: methacholine responsiveness did not differ from that measured the day before the cat BCT (p = 0.25; Student's t test). The mean PEF values measured during the week after the cat BCT with all three nebulizers did not differ significantly from those recorded the week before. Moreover, the aerosol particle size had no effect on the PEF response during the week after the BCT (p = 0.46).
Our study has demonstrated that individuals with intermittent asthma sensitized to cats developed an immediate bronchial response with dramatically smaller amounts of major cat allergen (Fel d 1) when it was inhaled on large (10.3 µm mass median aerodynamic diameter) compared with smaller (1.4 and 4.8 µm mass median aerodynamic diameter) particles. To ensure that particle size was the only difference between the three BCTs, we used two different methods described in the European Norm project for nebulizing systems (19)—cascade impaction and laser diffraction—to check the particle size of the nebulizers. The mass median aerodynamic diameters were comparable with both methods, and there was limited overlap between the three particle size distributions. Accordingly, we obtained three different patterns of deposition in the respiratory tract. We also verified that the inhalation method was reproducible. We performed BCT with an inhalation dosimeter, using a quantitative method, so that we could measure the PD (3). Because the breathing method is also known to be important for bronchial particle deposition (20), we trained our patients to standardize their breathing method to avoid any such bias: they inhaled cat allergen from RV to total lung capacity, with a 2-second breath hold after each inhaled puff. The inhalation dosimeter had a control system for inspiration flow (set at 0.5 l/second) and for puff duration (0.8 seconds). Before the cat BCTs, all the patients were comparable for methacholine PD20, dyspnea (Borg scale), FEV1, FEF25–75, and PEF. We showed that the dose of Fel d 1 provoking immediate bronchial symptoms was 20 times smaller when the allergen was carried on 10.3 µm particles than on 1.4 µm particles. With the DEVORE software, we estimated that 12.4% of the 10.3 µm aerosol Fel d 1 mass was deposited in the distal airways. The small airways do not, however, seem to be the major site for immediate bronchial response to cat allergen: 30 times more Fel d 1 was deposited there with the 1.4 µm aerosol than with the 10.3 µm aerosol at the end of cat BCT, and six patients had no significant immediate bronchial response to the 1.4 µm aerosol. In contrast, all patients responded immediately to the 4.8 and 10.3 µm doses. Accordingly, we considered that the dose that induced an immediate bronchial response in our patients appears to be the dose deposited in the proximal airways. According to DEVORE software, the dose of Fel d 1 deposited in proximal airways necessary to induce an immediate bronchial reaction varied from 1.5 to 4.4 ng according to particle size. The role of central airways in the reversal of bronchoconstriction was also found by Usmani and colleagues (21): inhalation of salbutamol, the reference treatment for reversing immediate bronchial response to specific and nonspecific agents, was more effective with the larger particles (3 µm), producing greater clinical response and improvement in FEV1 than did the small particles (1.5 µm). Moreover, the Fel d 1 PD20 of the cat BCT with the large particles seemed closest to the levels of airborne Fel d 1 that induce symptoms at home. Mean airborne exposure to cat allergen in 50 homes with a cat in Strasbourg has been measured at 14 ng Fel d 1/m3 (22), and among our 19 subjects, the median time to onset of developing bronchial symptoms was 30 minutes in the presence of a cat: the PD inducing asthma in real life was 4.2 ng of Fel d 1. This differed dramatically from previous results in which the dose of cat allergen inducing symptoms during BCT with 5 µm particles was 5 to 300 times higher (6, 7). It thus appears to us that large particles are very important in the occurrence of immediate bronchial response. BCT with a 10.4 µm aerosol carrying cat allergen corresponds better to exposure in daily life than BCT with smaller particles. Late asthmatic responses assessed with FEV1 were not more frequent with any particle size and no isolated late asthmatic response was observed. The results of the Borg scale for dyspnea at 6 hours and of FEF25–75 at 24 hours indicate, however, that small particles did induce late symptoms and decreased peripheral airway caliber. The six patients with a negative BCT with the 1.4 µm aerosol did not have any change in FEV1 at 6 and 24 hours, and all had decreased FEF25–75 at 24 hours (compared with baseline). Although the increase in the Borg scale for dyspnea at 6 hours was relatively slight (7%) with small particles, it may be clinically relevant: the coefficient of variation of the visual analog scale measurement of dyspnea has been reported to be 6% (23, 24), close to our coefficient of variation on dyspnea scale. Because late asthmatic response is known to be mediated by eosinophils, our results are consistent with those of Hamid and coworkers, who reported more activated eosinophils in the small than in the large airways (5); these eosinophils may induce a late inflammatory process in small airways. Moreover, inhalation challenge of guinea pigs (25) with a small aerosol (1.6 µm) of diphenyl-methane-4,4'-diisocyanate provoked greater recruitment of eosinophilic granulocytes in bronchial tissue taken 24 hours after the challenge than did a similar challenge with large-sized (5.1 µm) diphenyl-methane-4,4'-diisocyanate aerosol. In conclusion, our results demonstrated that, although inhalation of aqueous allergen extract for a short period of time during BCT is artificial compared with natural exposure, particle size seems to be important: the Fel d 1 PD20 obtained in BCT with a 10.3 µm aerosol carrying cat allergens approaches most closely the PD inducing asthma in real life. This suggests that immediate bronchial response to cat allergen is localized in the large airways. Consequently, immediate bronchial reaction in patients allergic to cats may be studied most usefully with BCT with large particles.
The authors thank F. Frey, M. J. Hoffner, J. Linder, L. Mahr, B. Sbinne, A. Vérot, and R. Wrobel for their excellent technical assistance and Dr. M. Ciobanu, C. Favre, and C. Mbazoa for their contribution. They thank Pr. P. Diot for fruitful discussions and Stallergènes Laboratories for providing the reagents.
Supported by 3M Santé Laboratories, Cergy Pontoise, France. Dr. Lieutier-Colas was supported by a grant from the Conseil Régional Alsace. Received in original form April 25, 2002; accepted in final form January 22, 2003
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TOP
ABSTRACT
METHODS
Twenty-four hours later, FEF25–75 decreased significantly more with small particles (means of 71.9% with 1.4 µm, 76.3% with 4.8 µm, and 81.5% with 10.3 µm aerosol; p = 0.006). Mean within-subject CV for FEF25–75 was 6% (confidence interval 4.52–6.68, range 2.7–12%). (C) Dyspnea (Borg scale) results before each cat BCT, at the end of the test, at 6, and 24 hours. At the end of the test, dyspnea was less important with the 1.4 µm aerosol (2.9 cm with 1.4 µm, 4.5 cm with 4.8 µm, and 4.4 cm with 10.3 µm aerosol; p= 0.013). 
