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American Journal of Respiratory and Critical Care Medicine Vol 166. pp. 294-300, (2002)
© 2002 American Thoracic Society

Original Article

Asymptomatic Worsening of Airway Inflammation during Low-Dose Allergen Exposure in Asthma

Protection by Inhaled Steroids

Josephine de Kluijver, Christine E. Evertse, Jasmijn A. Schrumpf, Hilly van der Veen, Aeilko H. Zwinderman, Pieter S. Hiemstra, Klaus F. Rabe and Peter J. Sterk

Departments of Pulmonology and Medical Statistics, Leiden University Medical Center, Leiden, The Netherlands

Correspondence and requests for reprints should be addressed to Josephine de Kluijver, Lung Function Laboratory, C2-P-62 Leiden University Medical Center, P.O. Box 9600, NL-2300 RC, Leiden, The Netherlands. E-mail: j.de_kluijver{at}lumc.nl


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Asthma is a chronic inflammatory disease that persists even during adequate therapy and asymptomatic episodes. We questioned whether "silent" chronic allergen exposure can induce and maintain airway inflammation and whether this still occurs during regular treatment with inhaled steroids. Twenty-six patients with house dust mite allergy and mild asthma (dual responders) participated in a parallel, double-blind study. All patients inhaled a low-dose of allergen on 10 subsequent working days (Days 1–5, 8–12). They were treated with 400 µg budesonide once daily (n = 13) or placebo (n = 13) from Days -3 to 19. At baseline (Day -6) and on Days 5, 12, and 19 we measured the provocative concentration of methacholine causing a 20% fall in FEV1 (PC20), and percent eosinophils, interleukin (IL)-5/interferon-{gamma} messenger RNA ratio (in sputum cells by real-time reverse transcription-polymerase chain reaction [RT-PCR]), and eosinophilic cationic protein (ECP) in induced sputum. Symptoms, peak expiratory flow (PEF), FEV1, and exhaled nitric oxide (NO) were recorded repeatedly during the study. In the placebo group, repeated low-dose allergen exposure resulted in a significant increase in sputum eosinophils (p = 0.043), ECP (p = 0.011), IL-5/IFN-{gamma} messenger RNA ratio (p = 0.04), and in exhaled NO (p = 0.001), without worsening of symptoms, PEF, or baseline FEV1 (p > 0.07). In the budesonide group, the changes in PC20, sputum ECP, and exhaled NO were significantly different as compared with the placebo group (p < 0.03). We conclude that repeated low-dose allergen exposure in asthma can lead to airway inflammation without worsening of symptoms, which can be prevented by inhaled steroid treatment. This suggests that antiinflammatory therapy is beneficial during allergen exposure, even during asymptomatic episodes.

Key Words: asthma • airway hyperresponsiveness • induced sputum • eosinophils • inhaled steroids


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Asthma is a chronic disease, characterized by episodes of chest symptoms and airways obstruction (1). Despite variability between patients in the onset and clinical course of asthma, the disease persists in virtually all patients (2). Such persistence can also be demonstrated for the accompanying hyperresponsiveness to inhaled irritants (3) as well as for eosinophilic airway inflammation (4). Remarkably, these abnormalities seem to be maintained even during long-term asymptomatic episodes of clinical remission (5, 6).

The mechanisms that are responsible for the persistence of asthma are largely unknown. Either the inflammatory process becomes self-sustained after an inducing event or ongoing environmental exposures are chronically driving the cellular pathways involved. It has been postulated that repeated, relatively low-dose allergen exposure is playing a role in the development of atopic asthma (7), and it is not unlikely that such chronic exposure can also be at least one of the determinants of the maintenance of the disease (8).

Indeed, repeated low-dose allergen exposure has been shown to worsen most of the disease features in patients with preexisting atopic asthma. For instance, this has been demonstrated with respect to increased airway hyperresponsiveness (9–15) as well as for an increase in the percentage of eosinophils, levels of the T helper (Th) 2-cytokine interleukin (IL)-5, and eosinophilic cationic protein (ECP) in induced sputum (13). The patients in most of these studies also became clinically symptomatic, as appeared from an increase in chest symptoms (11, 13), increased ß2-agonist usage (13, 14), and airways obstruction as measured by peak flow rates or FEV1 (10, 11, 14). Hence, the low doses of allergen were well perceived by the patients and led to a flare-up of their disease. We questioned whether the persistence of clinically stable asthma can be driven by similar repeated exposures. In that case, even unperceived "silent" allergen exposure would suffice to induce airway inflammation during asymptomatic episodes.

At present, inhaled steroids are the most effective therapy to control the clinical symptoms of asthma (1). Inhaled steroids also protect against the worsening of airway hyperresponsiveness and airway inflammation following single, high-dose allergen challenge (16), or after 5 days of symptomatic, low-dose allergen exposure (17). Nevertheless, it appears that airway inflammation is often not effectively suppressed during regular therapy in asthma, even when clinical symptoms are adequately controlled (18, 19). Therefore, we aimed to address whether unperceived allergen exposure can still drive airway inflammation when patients are treated with inhaled steroids.

In the present study, we first postulated that even in the absence of symptomatic worsening, repeated low-dose allergen exposure increases airway inflammation in asthma, as measured by, e.g., exhaled nitric oxide (NO), and numbers of eosinophils and Th2/Th1 cytokine ratio (IL-5/interferon-{gamma} [IFN-{gamma}] messenger RNA [mRNA] ratio) in induced sputum. Second, we investigated whether this still occurs in the presence of maintenance therapy with inhaled steroids. To that end, atopic asthmatic subjects were exposed to 10 unperceived doses of allergen during a 2-week period, while being treated with inhaled budesonide or placebo in a parallel, randomized controlled study. The outcome parameters included: chest symptoms, ß2-usage, peak flow rates, spirometry, methacholine responsiveness, exhaled NO, and inflammatory indices (eosinophils, neutrophils, ECP, neutrophil elastase [NE], and IL-5/IFN-{gamma} mRNA ratio using real-time reverse transcription-polymerase chain reaction [RT-PCR]) in induced sputum, which were recorded before, during, and after the low-dose allergen exposure.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
The extended version of the methods is available in the online data supplement. Twenty-six atopic patients with mild asthma participated in this study (Table 1) . The Medical Ethics Committee of the Leiden University Medical Center approved the study, and the subjects gave written informed consent before entering. The study had a placebo-controlled, double-blind, parallel, two-armed design. The 26 patients were randomly assigned to either low-dose allergen exposure with inhaled steroid treatment (n = 13; 400 µg inhaled budesonide once daily, which was given from Day -3 until Day 19), or low-dose allergen with placebo (n = 13).


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  		TABLE 1. Subject's characteristics

 
Screening high-dose allergen challenges were performed according to a standardized protocol (20–22), with purified aqueous allergen extract of Dermatophagoides pteronyssinus (SQ 503; Vivodiagnost, ALK, The Netherlands). Subjects had a documented early and late response, which was defined as a fall in FEV1 of more than 15% between 3 and 7 hours following allergen. The noncumulative dose of allergen causing a fall in FEV1 of 5% from postdiluent baseline during this allergen challenge was selected as the low dose to be used for repeated allergen exposure. The patients inhaled this as a single dose for two consecutive weeks (Table 2) . Spirometry was done before and 10, 20, and 30 minutes after inhalation on each occasion.


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  		TABLE 2. Study schedule

 
FEV1, PC20 to methacholine (20), exhaled NO measurements (23), and sputum inductions were performed according to the study schedule represented in Table 2. Sputum inductions were performed 1 hour after the methacholine challenges (24). Asthma symptom scores, short-acting ß2-agonist usage, and peak expiratory flow (PEF) measurements were recorded three times daily in diary cards during the 4 weeks of the study (25, 26).

Sputum was induced according a protocol that has been validated in our laboratory (27). After administration of salbutamol, the patients inhaled hypertonic saline aerosols (NaCl 4.5% wt/vol) during three to four 5-minute intervals. Whole sputum samples were processed. Differential cell counts of eosinophils and neutrophils were performed on May–Grünwald–Giemsa-stained cytospins. Cell counts are expressed as a percentage of cells excluding squamous cells. If more than 80% of the cells consist of squamous cells, the quality of the sample for cellular analysis was judged dissatisfactory and was excluded from analysis. In the sputum supernatant, concentrations of ECP and levels of neutrophil elastase were determined by fluoroimmunoassay (27) and sandwich enzyme-linked immunosorbent assay (28), respectively. Specific IgE to D. pteronyssinus in peripheral blood was measured on Days -4 and 15, by fluoroimmunoassay.

Cells obtained from processed sputum were lysed in Solution D (29) and stored at -80°C. Total cellular RNA of the sputum cells was isolated using TRIzol (Life Technologies, Breda, The Netherlands), according to manufacturer's instructions, and converted to cDNA using M-MLV reverse transcriptase (Life Technologies). The ratio of IL-5 and IFN-{gamma} cDNA present in the samples was determined with the real-time PCR method, using the ABI Prism 7,700 Sequence Detector (Perkin Elmer Applied Biosystems, Foster City, CA). Oligonucleotide primer pairs and fluorogenic probe for IL-5 were designed on the basis of known sequences of human IL-5 cDNA (30) with the use of Primer Express software (Perkin Elmer). The IFN-{gamma} primer and probe sequences were used as previously described by Härtel and coworkers (31). Real-time PCR was performed using a PCR reaction kit (qPCR Core Kit; Eurogentec, Seraing, Belgium) according to the manufacturer's protocol. Standard curves were generated using plasmids (pGEM T-easy; Promega, Madison, WI), containing either IL-5 or IFN-{gamma} inserts. The comparative CT method was used to determine relative quantities of IL-5 and IFN-{gamma} cDNA in cell samples, and by interpolation from the standard curves, IL-5/IFN-{gamma} mRNA ratios were obtained.

Mixed-model analysis of variance (ANOVA) and Student's t tests were used on asthma symptoms, ß2-agonist usage, baseline FEV1, and exhaled NO measurements. PEF- and log-transformed PC20 values were analyzed using Student's paired and unpaired t tests, and sputum data were analyzed using Wilcoxon signed ranks and Mann–Whitney U tests to test within- and between-group differences, respectively.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Acute Response to Low-Dose of Allergen Exposure
All patients completed the study and the procedures were well tolerated. There were no differences in baseline values of any of the measurements between the groups (p > 0.1). The mean maximal fall in FEV1 (± SEM) within 30 minutes after low-dose allergen exposure was 4.81% (± 0.82) and 3.88% (± 0.70) in the placebo and budesonide groups, respectively. There was no significant difference in fall in FEV1 between the two groups, except for Day 11 (mean% ± SEM, placebo group: 6.50 ± 0.80, budesonide group: 3.38 ± 1.20, p = 0.04) (Figure 1) .



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  		Figure 1. Acute response (0–30 minutes) to low dose of allergen exposure (mean fall in FEV1 from preallergen baseline ± SEM). The open and closed bars represent the placebo and budesonide groups, respectively. There was no significant difference in fall in FEV1 between the two groups, except for Day 11 (p = 0.04).

 
Asthma Symptoms, ß2-Agonists, and PEF
There were no significant differences in total daily symptom scores within and between the two groups during the whole study period using mixed-model ANOVA (p = 0.46) (Figure 2A) . However, there was a difference in ß2-agonists usage between both groups during the study (p = 0.027), which was mainly based on elevated ß2-agonists usage in the placebo group on Day 2 (Figure 2B). PEF measurements did not change significantly in either group during the study, nor were there significant differences between the groups (p > 0.07).



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  		Figure 2. (A) Total daily symptom score (mean score ± SEM). The open and closed symbols represent the placebo and budesonide groups, respectively. The maximal score was 30, which is depicted on the y-axis. Mixed-model ANOVA showed no significant differences between the groups during the study. (B) Total daily usage of ß2-agonist puffs (100 µg). The open and closed symbols represent the placebo and budesonide groups, respectively.

 
Baseline FEV1 and Airway Hyperresponsiveness
There were no significant differences in FEV1 within or between both groups during the two allergen weeks (p = 0.38) (Figure 3A) . In the placebo group, PC20 decreased 1.8-fold after 2 weeks of allergen exposure, which did not reach significance (geometric mean ± geometric SEM, baseline: 0.43 ± 1.28 mg/ml, Day 12: 0.24 ± 1.28 mg/ml, p = 0.1) (Figure 3B). However, there was a significant increase in PC20 in the budesonide group on Day 5 (geometric mean ± geometric SEM: 0.96 ± 1.39 mg/ml) compared with baseline (0.57 ± 1.27 mg/ml; p = 0.034). Moreover, significant differences were found in the levels as well as in the changes in PC20 from baseline between the groups (Figure 3B).



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  		Figure 3. (A) Baseline FEV1 (mean FEV1 in liters ± SEM). The open and closed symbols represent the placebo and budesonide groups, respectively. Mixed-model ANOVA showed no significant differences between the groups during the study. (B) PC20Methacholine (geometric mean in mg/ml ± geometric SEM). The open and closed symbols represent the placebo and budesonide groups, respectively. Significant differences in PC20 level between the groups on Days 5, 12, and 19 are depicted by asterisks, and differences in changes in PC20 from baseline on the same days are depicted by open triangles.

 
Sputum Eosinophils, Neutrophils, ECP, Neutrophil Elastase, and IL-5/IFN-{gamma} mRNA Ratio
There was a significant increase in sputum eosinophils on Day 5 as compared with baseline in the placebo group (p = 0.043), with a subsequent decrease on Day 19 compared with Day 12 (p = 0.017) (Figure 4A) . The differences between the two groups did not reach significance (p > 0.09). Sputum ECP increased significantly in the placebo group on Day 12 (p = 0.011) as compared with baseline, which persisted until Day 19 (p = 0.028). The changes in ECP levels were significantly different between the groups on Days 12 (p = 0.007) and 19 (p = 0.026) (Figure 4C). We did not observe significant differences or changes in the percentage of sputum neutrophils (p > 0.1) and in the sputum levels of neutrophil elastase (p > 0.3) (Figures 4B and 4D). In the placebo group, a significant increase in IL-5/IFN-{gamma} ratio was detected on Day 19 (median ratio [range]: 0.14 [0–254.26]) compared with Day 12 (0.06 [0–0.44], p = 0.04) (Figure 5) . There were no significant increases in specific IgE to D. pteronyssinus in peripheral blood, either in the placebo-treated group (mean IU/ml ± SEM, Day -4: 79.3 ± 10.2, Day 15: 77.6 ± 10.0) or in the budesonide-treated group (mean IU/ml ± SEM, Day -4: 122.8 ± 34.3, Day 15: 124.9 ± 35.4).



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  		Figure 4. (A) Sputum eosinophils (mean% eosinophils ± SEM). The open and closed symbols represent the placebo and budesonide groups, respectively. In the placebo group, there was a significant increase in sputum eosinophils on Day 5 compared with baseline (p = 0.043), with a subsequent decrease on Day 19 compared with Day 12 (p = 0.017). (B) Sputum neutrophils (mean% neutrophils ± SEM). The open and closed symbols represent the placebo and budesonide groups, respectively. (C) Sputum ECP (mean concentration of ECP in ng/ml ± SEM). The open and closed symbols represent the placebo and budesonide groups, respectively. Compared with baseline, sputum ECP levels increased significantly in the placebo group on both Days 12 (p = 0.011) and 19 (p = 0.028). Significant differences between the changes from baseline between both groups are depicted by open triangles. (D) Sputum neutrophil elastase (mean concentration of NE in ng/ml ± SEM). The open and closed symbols represent the placebo and budesonide groups, respectively.

 


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  		Figure 5. Sputum IL-5/IFN-{gamma} mRNA ratio (individual data with the median depicted by a horizontal line). The open and closed symbols represent the placebo and budesonide groups, respectively. In the placebo group, a significant increase in IL-5/IFN-{gamma} ratio was detected on Day 19 compared with Day 12 (p = 0.04).

 
Exhaled NO
Significant differences were detected between both groups with respect to the changes in exhaled NO during the study period (p < 0.001) (Figure 6) . During the first week, exhaled NO increased in both groups, whereas the further increase during the second week in the placebo group did not occur in the budesonide group. The changes between the two groups were significantly different between the first and the second week (p = 0.003).



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  		Figure 6. Exhaled NO (mean exhaled NO in ppb ± SEM). The open and closed symbols represent the placebo and budesonide groups, respectively. Mixed-model ANOVA showed significant differences between the groups during the study (p < 0.001). Significant differences in NO levels within the placebo group (compared with baseline) are depicted by thetas, between the groups by asterisks, and between the changes from baseline between both groups by open triangles.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of the present study demonstrate that 2 weeks of low-dose allergen exposure can lead to an increase in airway inflammation as represented by an increase in sputum eosinophils, ECP, IL-5/IFN-{gamma} mRNA ratio, and levels of exhaled NO in patients with asthma. Interestingly, this even occurs in the absence of worsening of chest symptoms. The unperceived proinflammatory effects of repeated allergen exposure can largely be prevented by inhaled steroid therapy. Our findings suggest that allergen-induced deterioration of airway inflammation can be silent, which may have implications for the monitoring and management of asthma.

This study shows that repeated allergen exposure can occur asymptomatically, yet promote features of airway inflammation as measured in induced sputum in patients with asthma. Our study confirms that repeated low-dose allergen exposure has proinflammatory effects (10, 11, 13, 15, 17, 32–34), represented not only by an almost 10-fold increase in sputum eosinophils and a fivefold increase in sputum ECP, but also by an increase in Th2/Th1 cytokine ratio and a rise in exhaled NO. Even at 24 hours after exposure to low doses of allergen, we found an increase in sputum eosinophils and ECP. We purposely chose to perform these tests 1 day after the preceding low-dose allergen inhalation, to avoid any potential interference with an ongoing late asthmatic response (13). This timing may partly explain the nonsignificant decrease in PC20 in our study, even though the magnitude of change in the PC20 at 24 hours following low-dose allergen in the placebo group (almost twofold decrease) was of the same order of magnitude as observed by others at 7 hours (13). In contrast to what was reported in earlier studies (13, 14), in the present study it appears that the increase in eosinophilic inflammation was neither associated with an increase in symptoms nor with changes in PEF measurements or spirometry. These data extend those from a previous study, showing that asymptomatic repeated exposure to cat allergen was able to induce increases of ECP in serum and bronchoalveolar lavage fluid in asthma (15). Hence, it appears that symptoms do not necessarily accompany chronic allergen exposure inducing an increase in eosinophilic airway inflammation in asthma.

The optimal low dose of allergen appeared to be the noncumulative dose causing a 5% fall in FEV1 during a screening high-dose allergen challenge. In a pilot study, this dose did not lead to worsening of symptoms during the 2-week exposure period. In view of the perennial exposure to indoor allergens, it was not feasible to challenge patients outside allergen seasons (9, 10, 14). The low-dose allergen exposure was considered to be an additional inducer of inflammation, thereby accepting that it was not feasible to fully exclude concurrent influences by other (outdoor and indoor) exposures. Finally, the present dose of inhaled budesonide was based on the recommendations for mild persistent asthma (1). This relatively low dose of steroids (400 µg once daily) was considered to be appropriate when examining its potential protection against allergen exposure during asymptomatic episodes.

How can the present results be explained? In vivo and in vitro studies, both in animals and in humans, have shown that repeated allergen exposure can stimulate multiple immunologic and inflammatory cascades. Our observations regarding sputum eosinophils, ECP, and IL-5/IFN-{gamma} mRNA ratio are in line with previous studies showing that repeated exposure to low doses of allergen causes eosinophilic airway inflammation (10, 11, 13, 15, 17, 32) and a shift in the balance between mucosal Th2 and Th1 cells (13, 32, 33, 35). However, the increase in mRNA ratio reached significance only after the allergen exposure already was stopped, suggesting that this increase could not be the cause of the increased number of eosinophils during exposure. Other factors, such as epithelial-derived chemokines, might also be involved in such recruitment (36). Recently, Palmans and coworkers (37) demonstrated, in rats, that the increase in eosinophilic infiltrate, as induced by repeated allergen exposure, is sustained over a longer period of up to 12 weeks. Together with the present findings, this suggests that repeated low-dose allergen exposure may indeed contribute to the maintenance of airway inflammation in stable asthma (18, 19, 38), although the development of long-term immunologic tolerance cannot be excluded by the present study's duration (39, 40).

We did not observe a significant worsening in airway hyperresponsiveness after low-dose allergen exposure. Even though hyperresponsiveness might be associated with airway eosinophilia (18), it may also be associated with airway structural changes. The latter have indeed been observed after repeated allergen exposure in experimental animals (37, 41, 42). It cannot be excluded that the limited effects of repeated allergen exposure on airway hyperresponsiveness in the current study are due to the compensatory mechanisms against structural changes, which appear to be operative during repeated allergen exposure in animal models (37). The presently observed increase in exhaled NO is in keeping with observations after acute allergen exposure in asthma and compatible with increased activity of inducible NO synthase (43, 44). However, during prolonged allergen exposure in experimental animals such increased NO production seems to wear off (44). We did not observe a drop in exhaled NO at later time points.

Inhaled steroids provided adequate protection against most of the proinflammatory effects of repeated allergen exposure in our patients. This was most noticeable during the second week of exposure, when the patients had been treated for more than 1 week. These findings extend those by Gauvreau and coworkers (17), who observed protection by 400 µg budesonide daily intake against the rise in PC20 and in sputum eosinophils after low-dose allergen challenges on four consecutive days. Notably, asymptomatic and more prolonged activation of airway inflammation can also be adequately suppressed by inhaled steroids. However, it is unknown yet whether steroids prevent the induction of airway structural changes by repeated allergen exposure, as suggested by animal models (45, 46). Therefore, the protective effects of inhaled steroids against ongoing "silent" allergen exposure still needs to be examined on the long-term clinical and histologic outcomes.

In conclusion, it appears that chronic allergen exposure can occur "silently" in patients with mild persistent asthma. Unnoticed by the patient, such exposure can intensify airway inflammation. Relatively low doses of inhaled steroid treatment can prevent the proinflammatory effects by ongoing allergen exposure, which suggests that prophylactic treatment could be of value in patients during asymptomatic episodes. It still needs to be evaluated whether such treatment strategy leads to a better long-term outcome of the disease.


   Acknowledgments
 
The authors would like to thank A. C. van der Linden and P. van Noort of the Department of Pulmonology, J. J. Baelde of the Department of Pathology, and R. A. van Soest of the Department of Haematology, Leiden University Medical Center, for their expertise and assistance. They also thank all the volunteers for their cooperation.

Supported by The Netherlands Asthma Foundation and by AstraZeneca, The Netherlands.


   FOOTNOTES
 
This article has an online data supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Received in original form December 31, 2002; accepted in final form May 1, 2002


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 DISCUSSION
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