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British Journal of Clinical Pharmacology logoLink to British Journal of Clinical Pharmacology
. 2015 Feb 20;79(3):523–532. doi: 10.1111/bcp.12508

High- and low-dose allergen challenges in asthmatic patients using inhaled corticosteroids

Wha-Yong Lee 1,2,3,✉, Thomas Southworth 1,2,3, Steven Booth 1,2,3, Dave Singh 1,2,3
PMCID: PMC4345962  PMID: 25214200

Abstract

Aims

The inhaled allergen challenge model has been used previously to investigate the effects of novel anti-inflammatory drugs in inhaled corticosteroid (ICS)-naïve asthmatics. The aim of this study was to characterize high- and low-dose allergen challenges in asthmatic patients using ICS.

Methods

Twenty-eight asthmatic patients taking ICS (beclomethasone equivalent <1000 μg day−1) were recruited for high-dose allergen challenge, of whom 10 subsequently also had a repeat low-dose challenge comprising seven allergen challenges. Induced sputum was collected for measurements of cell counts and supernatant biomarkers.

Results

The high-dose allergen challenge caused an early and late asthmatic response in 19 of 28 patients; the mean maximal fall in the forced expiratory volume in 1 s (FEV1) was 29.1% (SD 6.2%) and 25.1% (SD 9.6%), respectively. There was also an increase in sputum eosinophils of 6.2% (P = 0.0004), as well as supernatant eosinophil cationic protein levels. The low-dose allergen challenge caused an acute fall in FEV1, but had no effect on FEV1 at 24 h after challenge or sputum measurements.

Conclusions

The high-dose allergen challenge in asthmatics using ICS induces a late asthmatic response associated with an increase in eosinophilic airway inflammation. This may be a suitable model for studying the effects of novel anti-inflammatory drugs added to maintenance ICS treatment.

Keywords: allergen challenge, asthma, inhaled corticosteroids, induced sputum

What is Already Known about this Subject

  • Eosinophilic airway inflammation is a feature of allergic asthma.

  • Inhaled allergen challenge studies in inhaled corticosteroid-naïve asthmatics have been used to assess the effects of novel anti-inflammatory drugs on eosinophilic airway inflammation.

What this Study Adds

  • High-dose allergen challenge causes eosinophilic airway inflammation in patients taking inhaled corticosteroids, and may be a suitable model for investigating novel anti-inflammatory drugs in this population.

Introduction

The characteristic features of asthma are variable airflow obstruction, airway inflammation and bronchial hyper-reactivity. Inhaled corticosteroids (ICS) are the most commonly used anti-inflammatory therapy for patients with asthma 1. However, many patients remain symptomatic despite ICS therapy 2. Novel anti-inflammatory therapies are being developed as add-on treatments for patients who have poor asthma control despite ICS treatment.

Allergic asthma is associated with increased numbers of eosinophils and mast cells in the airways, and an increase in the levels of T-helper 2 (Th2) cytokines such as interleukin (IL)-4, IL-5 and IL-13 in the airways 3,4. The inhaled allergen challenge model causes immediate bronchoconstriction, known as the early asthmatic response (EAR), followed by a late asthmatic response (LAR) characterized by an increase in eosinophilic airway inflammation 5–10. The allergen challenge model has been used to assess the efficacy of novel anti-inflammatory drugs 10–12. These trials have enrolled patients with ICS-naïve asthma or have withdrawn ICS for at least 4 weeks prior to the study, because it is possible that the anti-inflammatory effect of maintenance ICS treatment would diminish the magnitude of the LAR 13,14; this would result in a smaller window of lung function change for the evaluation of the effectiveness of novel anti-inflammatory drugs on the LAR. On the contrary, one could argue that it is highly relevant to study the effect of novel anti-inflammatory drugs when administered to patients taking ICS, because this may be the intended use of the drug in clinical practice.

Inhaled allergen challenges are most commonly performed by the administration of a single high dose 5–14. These challenges use concentrations of allergen that are not commonly encountered in the environment in real life; it is more common for patients to be exposed to lower allergen concentrations for consecutive days. The low-dose allergen challenge model has been developed in order to administer a more physiologically relevant concentration of allergen repetitively over a period of days. Low-dose allergen challenge increases airway inflammation and bronchial hyper-reactivity 15–17 and is inhibited by ICS and antileukotriene drugs in ICS-naïve asthma patients 15,18. The low-dose allergen challenge model has not been evaluated in patients taking ICS.

The aim of this study was to characterize the response to inhaled allergen in patients taking ICS as maintenance treatment. We have used both the high- and the low-dose challenge models in patients taking ICS regularly. We were interested to understand the changes in lung function and sputum inflammatory biomarkers after allergen challenge during treatment with ICS.

Methods

Subjects

Twenty-eight atopic asthmatic patients taking ICS only at a dose of <1000 μg day−1 beclomethasone dipropionate equivalent, with a positive skin test to house dust mite, grass pollen or cat allergen, were recruited for high-dose allergen challenge. Patients were all Global Initiative for Asthma (GINA) Step 2 and Step 3 according to the GINA treatment classification guidelines 1 and were not using long-acting β-agonists, leukotriene receptor antagonists, theophylline or nasal corticosteroids. Patients were lifelong nonsmokers with a forced expiratory volume in 1 s (FEV1) ≥70% predicted. Ten patients who demonstrated both an EAR and LAR to the high-dose allergen challenge participated in a low-dose allergen challenge. All patients provided written informed consent. The study was approved by the local research ethics committee (Liverpool Central 11/NW/0219).

Study design

Subjects attended for a screening visit, at which spirometry, asthma controlled questionnaire (ACQ), fractional exhaled nitric oxide (FeNO), skin prick tests and sputum induction were performed, followed by a high-dose allergen challenge. Induced sputum was collected at 24 h after the allergen challenge. The EAR was defined as a fall in FEV1 of ≥20% from the postdiluent value, on at least one occasion, between 5 and 30 min after the final concentration of allergen. The LAR was defined as a fall in FEV1 of ≥10% from the postdiluent value, on at least three occasions, between 4 and 10 h after the final concentration of allergen. Previous studies in ICS-naïve subjects have used the same LAR criteria, except that the fall in FEV1 was defined as ≥15% from the postdiluent values 17,19; we reduced this to 10% to account for the protective effect of ICS, which make it less likely that large reductions in FEV1 will occur during the LAR.

Ten subjects who demonstrated an EAR and LAR after the high-dose challenge then proceeded to the low-dose allergen challenge protocol 2 weeks later. Seven low-dose allergen challenges were inhaled in the morning on five consecutive days from Monday to Friday, then again on Monday and Tuesday the following week (challenge 1–7; Table S1). The FEV1 was measured before and at 10, 20 and 30 min after allergen inhalation on each of these days. The FeNO was measured pre-allergen inhalation on every low-dose challenge day. Induced sputum was collected 1 h after challenges 3, 5 and 7. Inhaled corticosteroids were continued on the day of the high-dose challenge and during the entire period of the low-dose challenge.

High-dose allergen challenge

The high-dose allergen challenge was performed as we have previously described 12 using a Mefar Dosimeter (Marcos-Mefar, Bovezzo, Italy). Allergen for skin prick tests (Soluprick SQ; ALK Abelló, Horsholm, Denmark) was stored at 4°C; each subject was assessed for sensitivity to house dust mite, cat and grass pollen, alongside positive and negative controls. The allergen for inhalation was selected according to the largest skin test wheal (positive >3 mm) and clinical history. Fresh solutions of inhaled allergen (Aquagen; ALK Abelló) were made up in diluent in doubling concentrations from 250 to 32 000 SQU ml−1. Incremental doses of allergen were inhaled until an EAR was observed. Measurements of FEV1 were made at 5, 10, 20, 30, 45 and 60 min and, thereafter, at 30 min intervals up to 10 h.

Low-dose allergen challenge

The low-dose allergen challenges were also performed using the Mefar dosimeter (Marcos-Mefar). The allergen dose was calculated from the high-dose allergen challenge using linear extrapolation (GraphPad software, San Diego, CA, USA); the cumulative dose causing a fall in FEV1 of 5% (PD5) from postdiluent in the high-dose allergen challenge was used.

Fractional exhaled nitric oxide

The FeNO was measured using the Niox MINO (Aerocrine, Solna, Sweden). Three acceptable readings were recorded from each subject, and the mean was used for analysis.

Induced sputum

Sputum was induced and processed initially with phosphate-buffered saline to obtain supernatants, followed by dithiothreitol to obtain cells as previously described 20. Supernatants were stored at −80°C for later analysis. Cytospin preparations were made (Cytospin 4; Shandon, Runcorn, UK) and stained with Rapi-diff (Triangle, Skelmersdale, UK). Four hundred nonsquamous cells were counted and differential cell counts obtained as a percentage of the total nonsquamous cells. Cell viability was analysed by trypan blue exclusion.

Phosphate-buffered saline-processed sputum supernatants were analysed for eosinophil cationic protein (ECP) using MESACUP ECP kit (MBL international, Aichi, Japan; lower limit of detection 0.125 ng ml−1) and for neutrophil elastase (NE) by Luminex assay (Merck-Millipore, Watford, UK; lower limit of detection 20 pg ml−1) on a Magpix analyser (Luminex, Austin, Texas, USA). Sputum supernatants at baseline and after the high-dose challenge and low-dose challenges 5 and 7 were analysed.

For sputum supernatants with measurements less than the lower limit of quantification, half the lower limit was assigned 21.

Statistical analysis

Normality was assessed using the Kolmogorov–Smirnov test. The FEV1, age, ICS dose, FeNO, ACQ and sputum eosinophil, neutrophil and macrophage percentage cell counts were parametrically distributed. All other sputum cell measurements were nonparametrically distributed. Sputum supernatant ECP data were parametric, while sputum supernatant NE data were parametric after logarithmic transformation.

The EAR is expressed as the area under the curve (AUC) of the percentage change in FEV1 during first 2 h (AUC0–2h) and maximal fall in FEV1 during this time. The LAR is expressed as the AUC of the percentage change in FEV1 during first 4–10 h (AUC4–10h) and maximal fall in FEV1 during this time. The mean EAR AUC0–2h, LAR AUC4–10h and maximal fall in FEV1 during the EAR and LAR were parametrically distributed.

Student's paired t tests and Wilcoxon's tests were used for parametric and nonparametric data, respectively, to compare baseline and post high-dose allergen challenge end-points. Comparisons of changes in measurements during the low-dose allergen challenge were made using ANOVA for parametric data and the Kruskal–Wallis test for nonparametric data. A post hoc analysis comparing the high-dose allergen challenge EAR and LAR from this study conducted in patients using ICS with our previous published data from ICS-naïve asthmatics 19 was performed using Student's unpaired t test. Analyses were conducted using GraphPad software (San Diego, CA, USA). Differences were considered to be statistically significant when P < 0.05.

Results

High-dose allergen challenge

Patient recruitment for the high- and low-dose allergen challenges and the number of induced sputum samples analysed are summarized in Figure 1. Twenty-eight patients underwent the high-dose allergen challenge, of whom 19 patients demonstrated both an EAR and LAR. Induced sputum samples were obtained at both baseline and following the challenge from 13 of the 19 patients who demonstrated both EAR and LAR. The demographics of the 19 patients demonstrating EAR and LAR, along with the nine patients who did not, are shown in Table 1. There were no significant differences in the age, FEV1 predicted, ICS dose and FeNO between all three cohorts. However, the patients without both EAR and LAR showed evidence of better asthma control, with a lower ACQ score in comparison to subjects with both EAR and LAR (P = 0.014).

Figure 1.

Figure 1

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Flow chart showing recruitment of patients. Figure shows patient recruitment for the high- and the low-dose allergen challenges, and the numbers of adequately induced sputum and sputum supernatants analysed. Abbreviations are as follows: EAR, early asthmatic response; ECP, eosinophil cationic protein; LAR, late asthmatic response; NE, neutrophil elastase

Table 1.

Subject demographics

Parameter High-dose allergen challenge cohort (n = 19) Low-dose allergen challenge cohort (n = 10) No EAR and LAR cohort (n = 9)
Age (years) 38 (11) 44 (8) 43 (11)
Gender (male/female) 12/7 7/3 8/1
FEV1 (% predicted) 89.0 (14.0) 90.6 (16.9) 94.2 (9.9)
ICS dose (μg BDP equivalent) 397 (235) 455 (259) 317 (218)
FeNO (ppb) 57 (37) 55 (28) 49 (42)
ACQ Score 1.4 (0.7) 1.6 (0.5) 0.8 (0.4)
Allergen used for bronchial challenge 13 house dust mite, 3 grass mix, 3 cat 6 house dust mite, 2 grass mix, 2 cat 7 house dust mite, 1 grass mix, 1 cat
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Mean (SD) values are shown for age, FEV1, ICS dose, FeNO and ACQ.

ACQ, asthma control questionnaire; BDP, beclomethasone dipropionate; EAR, early asthmatic response; FeNO, fractional exhaled nitric oxide; FEV1, forced expiratory volume in 1 second; ICS, inhaled corticosteroid; LAR, late asthmatic response.

Lung function

The effect of the high-dose allergen challenge on FEV1 in those patients showing both EAR and LAR (n = 19) is shown in Figure 2. The mean (SD) maximal fall in FEV1 was 29.1% (6.2%) and the mean (SD) AUC0–2h was 23.9 (8.7) ΔFEV1% h–1 for the EAR. The mean (SD) maximal fall in FEV1 was 25.1% (9.6%) and the mean (SD) AUC4–10h was 89.1 (46.9) ΔFEV1% h–1 for the LAR. Thirteen of the 19 patients demonstrated an LAR defined as ≥15% from the postdiluent values.

Figure 2.

Figure 2

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The effect of the high-dose allergen challenge on forced expiratory volume in 1 s (FEV1). Lung function was measured up to 10 h after the allergen challenge. Results are expressed as percentage change in FEV1 from the postdiluent value over time, with data points showing the mean ± SEM for n = 19 patients

Sputum measurements

There was a significant increase in sputum eosinophil counts at 24 h after allergen challenge (Table 2); the mean percentage of eosinophils and mean absolute eosinophil count increased by 6.2% and 8-fold, respectively (P = 0.0004 and P = 0.0002, respectively). There were no significant changes in any other sputum cellular measurements.

Table 2.

Sputum cell counts at baseline and after high-dose allergen challenge (n = 13)

Baseline After high-doseallergen challenge P Value
Sputum total cell count (×106 g−1)† 1.32 1.04 P = 0.41
(0.55–1.73) (0.49–3.76)
Neutrophil (%)* 40.92 51.48 P = 0.18
(24.88) (21.86)
Eosinophil (%)* 0.85 7.04 P = 0.0004
(0.92) (4.94)
Macrophage (%)* 47.79 38.08 P = 0.18
(23.23) (22.09)
Lymphocyte (%)* 0.04 0.02 P = 0.77
(0–0.25) (0–0.00)
Absolute neutrophil count (×106 g–1 sputum)† 0.33 0.62 P = 0.19
(0.15–1.31) (0.19–2.01)
Absolute eosinophil count (×106 g–1 sputum)† 0.01 0.08 P = 0.0002
(0–0.02) (0.02–0.19)
Absolute macrophage count (×106 g–1 sputum)† 0.68 0.35 P = 0.95
(0.22–0.88) (0.22–0.60)
Absolute lymphocyte count (×106 g–1 sputum)† 0.00 0.00 P = 0.50
(0–0.00) (0–0.00)
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*

Parametric data are presented as the mean (SD) and analysed by Student's paired t test. †Nonparametric data are presented as the median (interquartile range) and analysed by the Wilcoxon rank signed test.

Baseline and post high-dose allergen-induced sputum NE and ECP levels were analysed in all the 13 patients. Figure 3 shows that there was a significant increase in ECP levels after high-dose allergen challenge (P = 0.006). There was no significant changes in NE levels (P = 0.15).

Figure 3.

Figure 3

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Effect of high-dose allergen challenge on sputum eosinophil cationic protein (ECP) and neutrophil elastase (NE) levels. The expression of ECP (in nanograms per millilitre) and NE (in picograms per millilitre) in sputum following the high-dose allergen challenge is shown in A and B, respectively. Individual data and the mean (ECP) or geometric mean value (NE) are shown. Comparisons of baseline and postchallenge levels were by Student's paired t test. *P < 0.05; NS, not significant

Low-dose allergen challenge

Lung function

The absolute and percentage reduction in FEV1 immediately after low-dose allergen challenge and effect on the morning baseline (pre-allergen challenge) FEV1 measurements are shown in Figure 4A–C. The greatest absolute and percentage fall in FEV1 (SD) were observed after the second challenge; they were 0.3 (0.1) l and 8.5 (2.2)%, respectively. The mean (SD) of the maximal percentage fall in FEV1 for the low-dose allergen challenges was 6.8 (1.0)%. The repeated low-dose allergen challenge had no statistically significant effect on the morning baseline FEV1 measurements (ANOVA, P = 0.99).

Figure 4.

Figure 4

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Effect of the low-dose allergen challenge on FEV1. (A) The maximal absolute fall in FEV1 from baseline (in litres) after the low-dose allergen challenge. (B) The maximal percentage fall in FEV1 from baseline within 30 min after the low-dose allergen challenge. (C) Morning baseline FEV1 (in litres) prior to the low-dose challenges. (D) The mean FEV1 at baseline (pre-allergen challenge; in litres), 30 min and 10 h after HDC; and the mean FEV1 at baseline (pre-allergen challenge and after LDC on each challenge day. Data points are mean ± SD values for n = 10 patients. *Significant difference (P < 0.05) in changes in FEV1 from baseline and post-HDC and post-LDC. Abbreviations are as follows: B, baseline pre-allergen challenge; FEV1, forced expiratory volume in 1 s; HDC, high-dose challenge; LDC, low-dose challenge

The effect of the repeated low-dose challenge and the high-dose challenge on FEV1 in the 10 patients who completed the low-dose challenge protocol is shown in Figure 4D. The high-dose challenge caused a significant reduction in the mean FEV1 from baseline (pre-allergen challenge), with changes at 30 min and 10 h postchallenge shown. The low-dose challenge did not cause any change in the FEV1 values in the morning prior to the next challenge, although FEV1 was reduced immediately after the low-dose challenge.

Fractional exhaled nitric oxide

The FeNO measured daily prior to the low-dose challenges showed no change in FeNO during the low-dose allergen challenge (ANOVA, P = 0.99; Figure S1).

Sputum measurements

The sputum cell counts from the low-dose allergen challenge are shown in Table 3. There were no changes in sputum percentage or absolute cell counts at any of the sampling time points during the low-dose challenge period. Sputum NE and ECP measurements did not change following low-dose challenge (Table S2).

Table 3.

Sputum cell counts after low-dose allergen challenge (n = 10)

Baseline Challenge 3 Challenge 5 Challenge 7 ANOVA
Sputum total cell count (×106 g−1)† 1.31 1.31 0.82 1.12 P = 0.65
(0.58–1.46) (0.72–3.16) (0.55–1.56) (0.77–3.41)
Neutrophil (%)* 39.70 (24.62) 61.83 (23.59) 46.40 (20.57) 47.90 (23.73) P = 0.34
Eosinophil (%)* 1.45 (1.28) 1.75 (2.00) 2.53 (2.07) 2.23 (1.77) P = 0.56
Macrophage (%)* 51.91 (25.48) 33.23 (21.57) 44.08 (20.43) 37.03 (23.48) P = 0.43
Lymphocyte (%)* 0.00 0.00 0.25 0.13 P = 0.31
(0.00–0.15) (0.00–0.56) (0.00–0.38) (0.00–0.50)
Absolute neutrophil count (×106 g–1 sputum)† 0.30 0.77 0.38 0.62 P = 0.34
(0.16–0.65) (0.32–2.40) (0.23–0.70) (0.20–1.51)
Absolute eosinophil count (×106 g–1 sputum)† 0.01 0.01 0.02 0.02 P = 0.57
(0.00–0.02) (0.00–0.09) (0.00–0.05) (0.00–0.07)
Absolute macrophage count (×106 g–1 sputum)† 0.54 0.32 0.42 0.32 P = 0.92
(0.21–0.99) (0.26–0.54) (0.18–0.83) (0.20–1.64)
Absolute lymphocyte count (×106 g–1 sputum)† 0.00 0.00 0.00 0.00 P = 0.60
(0.00–0.00) (0.00–0.01) (0.00–0.00) (0.00–0.00)
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*

Parametric data are presented as the mean (SD) and analysed by Student's paired t test. †Nonparametric data are presented as the median (interquartile range) and analysed by the Wilcoxon rank signed test.

Post hoc comparison with high-dose allergen challenge in ICS-naïve asthmatics

We have previously reported the effects of high-dose allergen challenge using the same protocol in 14 ICS-naïve asthmatics 19. The EAR and LAR values from this previous study were compared with the results reported here in order to assess any possible effect of ICS use; see Table 4. The EAR responses were similar in the two populations. In contrast, the LAR was significantly reduced in asthmatic patients taking ICS; the mean maximal fall in FEV1 and mean AUC4–10h were lower in patients taking ICS (P = 0.001 and P = 0.002, respectively).

Table 4.

Post hoc analysis of high-dose allergen challenge in ICS-naïve asthmatics (n = 14) and asthmatics on ICS (n = 19)

Parameter ICS-naïve asthmatics Asthmatics using ICS P Value
Age (years)* 38.0 (10.8) 38.4 (11.4) 0.93
Baseline FEV1 (% predicted)* 89.5 (10.2) 90.0 (14.0) 0.91
ACQ Score* 1.4 (0.9) 1.4 (0.7) 0.91
Total allergen dose (SQU ml−1)† 10514 (4770–23176) 6259 (3049–12848) 0.32
Allergen used 9 house dust mite, 4 grass mix, 1 cat 13 house dust mite, 3 grass, 3 cat
Maximal reduction in FEV1 (% change from baseline)
EAR* 29.7 (7.9) 29.1 (6.2) 0.80
LAR* 38.4 (11.3) 25.1 (9.6) 0.001
AUC [% change in FEV1 (h)−1]
AUC0–2h* 26.3 (9.1) 23.9 (8.7) 0.46
AUC4–10h* 147.5 (52.7) 89.1 (46.9) 0.002
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*

Parametric data are presented as the mean (SD) and analysed by Student's unpaired t test. †Nonparametric data are presented as the geometric mean (95% confidence interval) and analysed by the Mann–Whitney U test.

ACQ, asthma control questionnaire; AUC, area under the curve; EAR, early asthmatic response; FEV1, forced expiratory volume in 1 second; ICS, inhaled corticosteroid; LAR, late asthmatic response.

Discussion

A high-dose allergen challenge in patients with asthma taking ICS caused an LAR, associated with increased eosinophilic airway inflammation. The inhalation of a single high dose of allergen is therefore sufficient to cause eosinophilic airway inflammation, despite the use of ICS. In contrast, the low-dose allergen challenge did not cause eosinophilic inflammation; this suggests a protective effect of ICS against repetitive low-dose allergen exposure. It appears that the protective effect of maintenance ICS treatment in human allergen challenge models is critically dependent on the dose of allergen used. Furthermore, the high-dose allergen challenge model may have utility in testing the effects of novel anti-inflammatory drugs in patients already taking ICS.

High-dose allergen challenge in ICS-naïve asthmatics increases the sputum eosinophil percentage, ranging from ∼4- to 11-fold change from baseline 22–24. We observed a similar increase in sputum eosinophil percentage; an ∼8-fold increase. The reduction in FEV1 during the LAR, coupled with the increase in sputum eosinophils, indicates that maintenance ICS usage does not prevent allergic inflammation after high-dose allergen challenge in these patients.

We performed a post hoc analysis, comparing the data in this study performed on asthmatic patients taking ICS with previous data in asthmatic patients not taking ICS 19. These studies were performed using the same high-dose challenge protocol. Inhaled corticosteroids are known not to inhibit the EAR 14, and our analysis showed no difference in the EAR between populations. The LAR was reduced in patients taking ICS, compatible with clinical trials showing that ICS intervention reduces the LAR 13,14. However, the magnitude of the LAR observed in asthmatic patients taking ICS was still relatively large, because the mean maximal fall in FEV1 was 25.1%. This effect size would allow the actions of novel drugs to be studied in this model.

In previous repeated low-dose allergen challenge studies, ICS-naïve asthmatics demonstrated small reductions in FEV1 immediately after allergen exposure (ranging from 6.50 to 9.2%) without any significant effect on FEV1 measured the next morning 16–18,25–29; this is similar to our findings. However, these previous studies also demonstrated increased airway inflammation; FeNO, sputum measurements of eosinophils and sputum Th2 mediators increased after low-dose challenge 18,27,28. We did not observe any upregulation of airway inflammation. This contrasts with the high-dose challenge results and suggests that the ICS maintenance treatment controlled inflammation after repetitive low-dose allergen challenge but not after single high-dose allergen challenge.

Previous low-dose challenge studies in patients not taking ICS have measured FEV1 and FeNO in the morning prior to the repeated low-dose challenge as a measure of inflammation caused by the LAR 18,27,28, with no significant effect on morning baseline FEV1 and a progressive increase in FeNO levels observed 27,28. We took the same approach, but there was no change in FeNO levels. Likewise, an increase in sputum eosinophils has been observed after repeated low-dose challenge 22, but again we could not reproduce this finding, suggesting an inhibition of airway inflammation caused by low-dose challenges due to ICS.

Clinical trials in ICS-naïve asthmatics have shown that ICS inhibit the increase in sputum eosinophils in both the low- and the high-dose allergen challenge models 13–15. It is clear that ICS block allergic inflammation, and our results strongly suggest that the inhibitory effect of ICS on allergen-induced eosinophilic airway inflammation can be overcome by an increase in the inhaled allergen load.

There is no standardized procedure for the low-dose allergen challenge model. We used PD5 as the selected allergen dose for 7 days based on previous publications where this dose increased airway hyper-responsiveness and sputum eosinophils in ICS-naïve subjects 15,28,29. However, this allergen dose was insufficient to induce eosinophilic airway inflammation in asthmatics taking ICS; probably, a higher dose of allergen is required to achieve this. The high-dose allergen challenge was designed to cause a 20% decrease in FEV1; this also increased sputum eosinophils in this study. Perhaps an allergen dose causing PD10 or PD15 would have been more successful in the low-dose model.

In ICS-naïve asthmatics, an increase in the Th2-mediated biomarkers, IL-5, eotaxin-3 and ECP, has been demonstrated in sputum supernatant following the high-dose allergen challenge 7,30–33. We also observed a Th2-mediated response following the high-dose allergen challenge; there was an increase in eosinophils and ECP.

What could be the potential use of the high-dose allergen challenge model in asthmatics taking ICS? We have shown clear reductions in lung function and increases in sputum eosinophils and ECP after high-dose allergen challenge in these patients. Phase 2 studies of the effects of novel anti-inflammatory drugs could be performed in this population, because the high-dose allergen challenge model could provide very important information on the potential effectiveness of these drugs on allergic inflammation when administered in addition to ICS. There are a number of novel therapies in development that target Th2 inflammation, such as anticytokine monoclonal antibodies and small molecule inhibitors, such as chemoattractant receptor expressed on T-helper type 2 (CRTh2) antagonists 34–37. The potential therapeutic effectiveness of these therapies can be tested at an early stage of clinical development in a small number of subjects using the model and end-points described here.

In summary, we evaluated both the high-dose and the low-dose allergen challenge models in asthmatics on ICS. The high-dose allergen challenge induced an LAR associated with an increase in eosinophilic airway inflammation, but this inflammatory response was not observed in the PD5 low-dose allergen model. The high-dose allergen challenge model may be useful for investigating the effects of novel anti-inflammatory drugs when used in addition to ICS.

Competing Interests

All authors have completed the Unified Competing Interest form at http://www.icmje.org/coi_disclosure.pdf (available on request from the corresponding author). DS has received sponsorship to attend international meetings, honoraria for lecturing or attending advisory boards and research grants from various pharmaceutical companies including Almirall, AstraZeneca, Boehringer Ingelheim, Chiesi, CIPLA, Forest, Genetech, GlaxoSmithKline, Merck, Novartis, Pfizer and Takeda. WYL, TS and SB have no financial relationships with any organizations that might have an interest in the submitted work in the previous 3 years; no other relationships or activities that could appear to have influenced the submitted work.

The authors gratefully acknowledge support from the Medicines Evaluation Unit for the excellent patient recruitment and technical support.

Supporting Information

Figure S1 The effect of the low-dose allergen challenge on fractional exhaled nitric oxide (FeNO). The figure shows the FeNO (in parts per billion) measured prior to the low-dose allergen challenges. Data points are the mean ± SD values for the patients (n = 10)

bcp0079-0523-sd1.gif (7.5KB, gif)

Table S1 Low-dose allergen challenge study design

Table S2 Sputum eosinophil cationic protein (ECP) and neutrophil elastase (NE) levels during low-dose allergen challenge

bcp0079-0523-sd2.docx (18.3KB, docx)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1 The effect of the low-dose allergen challenge on fractional exhaled nitric oxide (FeNO). The figure shows the FeNO (in parts per billion) measured prior to the low-dose allergen challenges. Data points are the mean ± SD values for the patients (n = 10)

bcp0079-0523-sd1.gif (7.5KB, gif)

Table S1 Low-dose allergen challenge study design

Table S2 Sputum eosinophil cationic protein (ECP) and neutrophil elastase (NE) levels during low-dose allergen challenge

bcp0079-0523-sd2.docx (18.3KB, docx)

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