Allergy
ORIGINAL ARTICLE
AIRWAY DISEASES
Airway responsiveness to mannitol 24 h after allergen challenge in atopic asthmatics B. E. Davis1,2, D. O. Amakye2 & D. W. Cockcroft1,2 1
Division of Respirology, Critical Care and Sleep Medicine, Department of Medicine, University of Saskatchewan; 2Department of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, SK, Canada
To cite this article: Davis BE, Amakye DO, Cockcroft DW. Airway responsiveness to mannitol 24 h after allergen challenge in atopic asthmatics. Allergy 2015; 70: 682–688.
Keywords allergen; asthma; mannitol; refractoriness. Correspondence Dr. Beth E. Davis, Division of Respirology, Critical Care and Sleep Medicine, Royal University Hospital, 103 Hospital Drive, Ellis Hall, 5th Floor, Saskatoon, SK S7N 0W8, Canada. Tel.: 306 844 1444 Fax: 306 844 1532 E-mail: [email protected] Accepted for publication 18 February 2015 DOI:10.1111/all.12601 Edited by: De Yun Wang
Abstract Background: Airway responsiveness to indirect stimuli correlates positively with airway inflammation. In atopic asthmatics, allergen inhalation is associated with an influx of inflammatory cells and increased responsiveness to the direct-acting stimuli methacholine at 3 and 24 h after exposure. We have shown mannitol responsiveness decreases 3 h after allergen inhalation. The current investigation assessed mannitol responsiveness 24 h after allergen challenge. Methods: Eleven mild atopic asthmatics completed allergen challenges on two separate occasions. In random order, methacholine or mannitol challenges were performed 24 h pre- and post–allergen challenge. Levels of fractional exhaled nitric oxide were also measured. Results: Allergen challenge increased airway responsiveness to methacholine 24 h postchallenge; the geometric mean (95% CI) methacholine PC20 decreased from 5.9 mg/ml (1.8–19.4) to 2.2 mg/ml (0.81–5.89); P = 0.01. This coincided with a significant increase (P = 0.02) in FeNO levels. Conversely, allergen challenge decreased airway responsiveness to mannitol; geometric mean (95% CI) dose– response ratio was significantly higher after allergen exposure (57 mg/% FEV1 fall [27–121] to 147 mg/% FEV1 fall [57–379]; P = 0.03), and FeNO levels were not significantly increased (P = 0.054). Conclusion: Allergen-induced changes in airway responsiveness to direct and indirect stimuli are markedly different. The loss in responsiveness to mannitol is likely not explainable by a refractory state. The effect(s) of allergen exposure on airway responsiveness to indirect-acting stimuli require further investigation.
A well-studied phenomenon in individuals with atopic asthma is the increase in airway responsiveness to directacting stimuli following allergen exposure. The increased sensitivity has been shown as early as 3 h (1) post–allergen challenge and can last for at least 7 days (2). We also know that inflammatory cells are recruited to the airway following allergen challenge (3, 4) and that eosinophilic airway inflammation correlates better with airway hyperresponsiveness (AHR) to indirect stimuli vs AHR to direct stimuli (5). The relationship between airway responsiveness to indirect stimuli and airway inflammation is also supported by decreases in responsiveness to AMP (6) and mannitol (7) following administration of inhaled glucocorticosteroid. We recently reported a significant decrease in mannitol responsiveness at 3 h post–allergen challenge in a group of
682
mild allergic asthmatics (8) and questioned whether the 3 h time point was too early to observe an increase, possibly due to a cross-refractory mechanism. Additionally, although there is evidence to support an increase in the number of activated eosinophils at 3 h post–allergen challenge (9), we did not assess allergen-induced changes in airway inflammation in the earlier study. Given the relationship between airway responsiveness to indirect stimuli and airway inflammation, a lack of recruitment of inflammatory cells at 3 h post–allergen challenge may have influenced our previous results. Therefore, our current hypothesis is that the allergeninduced change in airway responsiveness to mannitol would increase to a greater extent than that which has been shown for methacholine at 24 h post–allergen exposure.
Allergy 70 (2015) 682–688 © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Davis et al.
Allergen induced changes in AHR
performed. Data were collected from November 2013 to March 2014. Table 2 provides a study schematic.
Materials and methods Study participants Individuals of either gender between the ages of 18 and 65 with a history of atopic asthma and no other significant medical comorbidity were eligible for screening (Table 1). Additional inclusion criteria included baseline FEV1 ≥ 70% predicted, methacholine PC20 ≤ 16 mg/ml, positive skin prick test to at least one inhalable allergen, and no requirement for controller asthma treatment (i.e. salbutamol prn only). Pregnant or lactating females were excluded. The University of Saskatchewan Biomedical Research Ethics Board approved the study, and all participants consented to participation prior to the conduct of any study procedures.
Study design The study was conducted as a randomized crossover trial and included an initial screening visit followed by two testing triads. The triads were comprised of three consecutive days of testing in which Visit 2 was either a methacholine or mannitol challenge, Visit 3 was an allergen inhalation challenge, and Visit 4 matched Visit 2. A 2-week minimum washout between allergen inhalation challenges was required. If methacholine challenges were performed during the first triad, then mannitol challenges were performed during the second triad (Visits 5 and 7) and vice versa. Skin prick testing and fractional exhaled nitric oxide measurements were also
Skin prick testing Skin prick testing was performed to confirm hypersensitivity to one or more allergens and to identify an appropriate allergen for inhalation challenge. Drops of a variety of common allergens (pollens, animal danders, molds, foods) were placed on the volar surface of the forearm and introduced to the body by a tenting of the skin with a lancet or small beveled needle. Responses were observed after 10-15 min. The allergen producing the largest wheal was chosen for inhalation challenge testing unless clinical history (admission of symptom manifestation upon exposure or the type of allergen producing the largest wheal (e.g. food)) suggested otherwise. Standardized stock allergen extracts of cat hair (10 000 AU/ ml), grass pollen (100 000 BAU/ml), and house dust mite (pteronyssinus and farinae: both 30 000 AU/ml) were used for the allergen inhalation challenges (Table 1).
Fractional exhaled nitric oxide Measurements of fractional exhaled nitric oxide were collected to assess changes in airway inflammation and were performed prior to spirometry and bronchoprovocation testing at each visit. A 7-h post–allergen challenge measurement was also made prior to the collection of the 7-h FEV1. Data
Table 1 Participant demographics Subject
Age (years)
Gender
Height (cm)
FEV1 (l)
FEV1 (% predicted)
Allergen inhaled
1 2 3 4 5 6 7 8 9 10 11
28 37 27 29 23 30 22 21 26 19 22
M M F M F F F M F F F
185 178 160 180 168 160 180 183 155 157 168
4.71 3.54 2.74 4.57 3.19 2.24 4.04 4.57 3.01 3.06 3.13
96 82 88 99 93 77 101 93 101 97 89
Cat hair Cat hair HDM-DP Grass pollen HDM-DF Grass pollen HDM-DF Cat hair Cat hair Cat hair Cat hair
M, male; F, female; FEV1, forced expiratory volume in 1 s; HDM-DP, house dust mite dermatophagoides pteronyssinus; HDM-DF, house dust mite dermatophagoides farinae.
Table 2 Study schematic Visit 1
Visit 2
Visit 3
Visit 4
Washout
Visit 5
Visit 6
Visit 7
Consent MCh SPT
FeNO MCh or mannitol challenge
FeNO Allergen challenge FeNO*
FeNO MCh or mannitol challenge (same as Visit 2)
14 days minimum
FeNO MCh or mannitol challenge (opposite of Visit 2)
FeNO Allergen challenge FeNO*
FeNO MCh or mannitol challenge (same as Visit 5)
SPT, skin prick testing; FeNO, fractional exhaled nitric oxide; FeNO*: captured at 7 h post–allergen challenge; MCh, methacholine challenge.
Allergy 70 (2015) 682–688 © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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Allergen induced changes in AHR
Davis et al.
were captured using the NIOX MINO instrument (Aerocrine AB, Solna, Sweden). Participants inhaled fully on the filter mouthpiece and then exhaled at a constant flow rate (50 ml/ s) for 10 s. Nose clips were not used. Two reproducible measurements (10%) were required. Methacholine challenge Methacholine challenges were conducted according to the standardized 2-min tidal breathing methodology (10). In brief, reproducible, full flow-volume spirograms preceded bronchoprovocation challenge to assess baseline lung function and airway stability. An aerosol of normal saline was then inhaled over the course of 2 min of tidal breathing via face mask and with nose clip on. The jet nebulizer was calibrated to deliver aerosol at a rate of 0.13 ml/min. With nose clip in place, spirometric maneuvers were then performed at 30 and 90 s postinhalation to capture FEV1 data only. The lower of these values was retained for calculating the decrease in FEV1 following the inhalation of increasing (doubling) concentrations of methacholine solutions. Methacholine PC20 values (the concentration of methacholine that causes a 20% decrease in FEV1) were calculated by either interpolation (11) or extrapolation (12) from the log concentration vs response curve. Mannitol challenge Mannitol challenges were performed per standardized methodology (13). Single-use challenge kits (Pharmaxis Ltd, Frenchs Forest, NSW, Australia) containing the delivery device and all capsules required to deliver a 635-mg cumulative dose were used for each challenge. The capsule(s) are placed in the device and punctured, and the contents are then inhaled via slow deep inhalation. A 5-s breath hold followed the inhalation. The challenges began with the inhalation of a 0-mg (empty) capsule. After an additional 55 s, two FEV1 measurements were performed. The higher of these two measurements was used to calculate the fall in FEV1 after subsequent inhalations. The test was complete when a 15% fall in FEV1 occurred, or when there was a 10% incremental fall in FEV1 between doses or when the highest dose (635 mg) had been administered. Responses were assessed as the dose– response ratio (DRR). Allergen challenge Allergen challenges were performed as previously described (14). In brief, participants performed routine baseline spirometry to obtain three reproducible baseline full flow-volume measurements. Aerosolized allergen was administered by way of the Wright Nebulizer (Roxon Medi-Tech, Montreal, QC, Canada) fitted with a two-way Hans-Rudolph valve and calibrated to deliver an output of 0.13 ml/min. By way of mouthpiece and with nose clips in place, participants inhaled doubling concentrations of aerosolized allergen during 2 min of tidal breathing. Ten minutes later, two FEV1 measurements were obtained and the highest was used to calculate the
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fall in FEV1 from the highest baseline FEV1. The challenge was stopped when the FEV1 had fallen 20%. Lung function was then monitored (FEV1 only) at various time points up to 7 h. There was a washout period of at least 2 weeks between allergen challenges. The total dose of allergen administered during the second challenge was identical to that administered during the first challenge (i.e. same starting concentration and number of concentrations administered). Analysis Bronchoprovocation data (methacholine PC20 and mannitol DRR), and FeNO data were log-transformed and compared using the paired t-test (STATISTIX 10; Analytical Software, Tallahassee, FL, USA). A P-value < 0.05 was considered significant.
Results Study participants Eleven mild atopic asthmatics completed the study (Table 1). Two participants had methacholine PC20 results >16 mg/ml during screening at Visit 1. These two individuals, however, had documented (within 1 year) methacholine PC20 test results 15% during the 3– 7 h postchallenge) during both allergen challenges. Ten had isolated early responses. Methacholine and mannitol bronchoprovocation Airway responsiveness to methacholine significantly increased 24 h after allergen challenge; the geometric mean methacholine PC20 (95% CI) decreased from 5.9 mg/ml (1.8–19.4) to 2.2 mg/ml (0.81–5.89); P = 0.01. Conversely, airway responsiveness to mannitol significantly decreased 24 h after allergen challenge; the geometric mean DRR (95% CI) was higher after allergen challenge (147 mg/% FEV1 fall [57–379] vs 57 mg/% FEV1 fall [27–121]; P = 0.03) (Fig. 2A,B, respectively). Fractional exhaled nitric oxide Twenty-four-hour post–allergen challenge FeNO levels were significantly increased (P = 0.02) in the methacholine arm (Fig. 3) vs 24-h preallergen challenge levels. FeNO levels in the mannitol arm also increased 24 h after allergen challenge, but the increase was not statistically significant (P = 0.054).
Allergy 70 (2015) 682–688 © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Davis et al.
Allergen induced changes in AHR
Figure 1 Mean data for airway responses to inhaled allergen. Similar responses to allergen challenge were observed for each testing arm. Open squares – methacholine arm. Closed circles – mannitol arm.
A
Figure 2 Individual methacholine PC20 (concentration of methacholine causing a 20% fall in FEV1) (A) and mannitol dose–response
Discussion We have shown a significant increase in airway responsiveness to methacholine 24 h after allergen challenge which coincided with significantly higher levels of FeNO. In contrast, however, airway responsiveness to mannitol significantly decreased 24 h after allergen challenge and this coincided with a nonsignificant increase in FeNO. The decrease in airway responsiveness to mannitol at 24 h post–allergen challenge is likely not explained by a refractory or cross-refractory mechanism as the reported duration of refractory periods ranges from 1 to 2 h for exercise-induced bronchoconstriction (15), 2–8 h for adeno-
B
ratio (B) data pre- and post–allergen challenge. Single data points with error bars represent the geometric means and standard error.
sine-50 -monophosphate (AMP) (16), and up to 2 h for mannitol (17, 18). We are unaware of a refractory state with respect to repeat allergen exposure. We do know that daily low-dose allergen exposure, enough to cause a 5% fall in FEV1, results in increased responsiveness to directacting stimuli (e.g. histamine or methacholine), increased FeNO, and increased sputum eosinophils (19, 20). The recruitment of inflammatory cells following repeated lowdose allergen challenge supports our hypothesis that mannitol responsiveness should increase 24 h after allergen challenge. Interestingly however, the Ihre et al. study (19) also noted airway responsiveness to AMP to be unchanged 48 h after the last low dose of allergen. The lack of
Allergy 70 (2015) 682–688 © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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Allergen induced changes in AHR
Davis et al.
Figure 3 Geometric mean and standard error for FeNO data at time points pre- and post–allergen challenge. The data are grouped
by testing arm. Mannitol data are on the left and methacholine data are on the right.
responsiveness to the indirect stimuli AMP supports our current findings that allergen challenge decreases airway responsiveness to indirect-acting stimuli, but there are methodological differences between the studies, including sample size and the time at which the measurement was made. In the Palmqvist et al. study (20) repeated low-dose allergen challenge diminished the LAR of a high-dose allergen challenge despite an increase in AHR to methacholine and an increase in airway inflammation. In the absence of an effect on the EAR, the inhibition of the LAR could be interpreted as a ‘delayed refractory period’ and this could point to a cross-refractory mechanism between allergen and mannitol, two stimuli that constrict the airway via a similar mechanism (i.e. mast cell mediator release). The loss in airway responsiveness may also be dependent on the order in which the indirect stimuli are administered as the response to allergen has been shown to increase when the airway is in an AMP refractory state (21). It may be, however, that repeated low-dose allergen exposure decreases airway responsiveness to indirect-acting stimuli, including a subsequent allergen challenge, by a mechanism that is not refractory by definition. Our data are most comparable with the Aalbers et al. (22) data that looked at allergen-induced changes in methacholine and AMP at three and 24 h post–allergen challenge. They showed an increase in airway responsiveness to methacholine at both time points but found an increase in AMP responsiveness at 3 h only. In our investigations, not only did we not find an increase in responsiveness to an indirect stimulus at 3 and 24 h postallergen, we found a significant decrease at
both time points. Notably, all participants in the Aalbers study were late responders and this differs from our study population which included mostly (10/11) isolated early responders. However, in a study by Evans et al. (23), allergen challenge failed to increase airway responsiveness to metabisulfite (MBS) at 3 and 24 h postchallenge in a predominantly (14/18) late responder population. The study also showed an apparent trend toward a significant loss in MBS sensitivity at 24 h, and this signal supports our findings despite the difference in the number of late responders studied. Both allergen and mannitol act by causing the release of mast cell mediators. An intuitive explanation for how the response to mannitol is decreased by prior allergen challenge is therefore the depletion of mast cell mediators. Larsson et al. tested this hypothesis during an investigation of mannitol refractoriness. Not only did they find no decrease in the urine content of bronchoconstricting mast cell mediators following repeat mannitol challenge, it was observed that those who were most refractory had significantly higher levels of mast cell mediators (PGF2 and LTE4) after repeat testing (18). A number of additional hypotheses regarding potential mechanisms of the refractory state have been proposed and recently reviewed (24). In the current investigation, we used standardized allergen challenge triad methodology to induce an early asthmatic response, monitor the development of a late asthmatic response, and assess allergen-induced changes in airway inflammation and airway responsiveness to methacholine and mannitol at 24 h post–allergen challenge. Ideally, our study population would have included a higher number of
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Allergy 70 (2015) 682–688 © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Davis et al.
Allergen induced changes in AHR
individuals with dual or late airway responses. This was not a requirement for study entry, however, and only one participant developed an LAR after allergen challenge. This could be viewed as a limitation, but we did observe increases in FeNO at 24 h post–allergen challenge. Therefore, a signal for airway inflammation was apparent at a time when the change in airway responsiveness to direct or indirect stimuli was being made. The observation that FeNO was significantly higher during the methacholine testing arm but not during the mannitol testing arm may also be viewed as a study limitation, but we propose this may be a signal for a mechanistic finding that could explain the observed mannitol nonresponsiveness following allergen challenge. Based on our hypothesis, we also did not incorporate the requirement for a positive mannitol challenge at study entry anticipating that allergen exposure would increase airway responsiveness to mannitol and potentially shift a negative or near-positive response preallergen to a positive response postallergen. As a result of the high number of mannitol nonresponders (eight of 11), we analyzed the data as a ratio of cumulative dose administered to airway response at that dose, the DRR. The decrease in the DRR was statistically significant and was seen in 10 of 11 participants. The outcomes of allergen exposure on airway responsiveness to indirect stimuli are not well described and may in fact
be very different from that which can be hypothesized based on current knowledge. We conclude from our investigation that allergen-induced changes in airway responsiveness to direct and indirect-acting stimuli are markedly different and that the unexpected loss in airway sensitivity to mannitol requires further investigation. Acknowledgments We would like to acknowledge and thank Pharmaxis Ltd., NSW, Australia, for supplying the mannitol test kits. Author contributions Beth E. Davis performed study design, data collection, and data analysis and helped in drafting, reviewing, and approving the manuscript. Daniel O. Amakye performed study design, data collection, and data analysis and helped in reviewing and approving the manuscript. Donald W. Cockcroft performed study design and data analysis and helped in reviewing and approving the manuscript. Conflicts of interest The authors declare that they have no conflicts of interest.
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the PC20 of methacholine. Am J Respir Crit Care Med 2001;164:1127–1132. Brannan JD, Koskela H, Anderson SD, Chan HK. Budesonide reduces sensitivity and reactivity to inhaled mannitol in asthmatic subjects. Respirology 2002;7:37–44. Amakye DO, Davis BE, Martin AL, Peters GE, Cockcroft DW. Refractoriness to inhaled mannitol 3 hours after allergen challenge. Ann Allergy Asthma Immunol 2013;111:182–184. Aalbers R, Kauffman HF, Vrugt B, Koeter GH, de Monchy JG. Allergen-induced recruitment of inflammatory cells in lavage 3 and 24 h after challenge in allergic asthmatic lungs. Chest 1993;103:1178–1184. Cockcroft DW, Killian DN, Mellon JJA, Hargreave FE. Bronchial reactivity to inhaled histamine: a method and clinical survey. Clin Allergy 1977;7:235–243. Cockcroft DW, Murdock KY, Mink JT. Determination of histamine PC20: comparison of linear and logarithmic interpolation. Chest 1983;84:505–506. Jokic R, Davis EE, Cockcroft DW. Extrapolation of methacholine PC20. Chest 1998;103:1796–1797. Anderson SD, Brannan J, Spring J, Spalding N, Rodwell LT, Chan K et al. A new method for bronchial-provocation testing in asthmatic subjects using a dry powder of mannitol. Am J Respir Crit Care Med 1997;156:758–765.
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14. Davis BE, Illamperuma C, Gauvreau GM, Watson RM, O’Byrne PM, Deschesnes F et al. Single-dose desloratadine and montelukast and allergen-induced late airway responses. Eur Respir J 2009;33:1302–1308. 15. Edmunds AT, Tooley M, Godfrey S. The refractory period after exercise-induced asthma: its duration and relation to the severity of exercise. Am Rev Respir Dis 1978;117:247–254. 16. Daxun Z, Rafferty P, Richards R, Summerell S, Holgate ST. Airway refractoriness to adenosine 50 -monophosphate after repeated inhalation. J Allergy Clin Immunol 1989;83:152–158. 17. Suh DI, Lee JK, Kim JT, Koh YY. Airway refractoriness to inhaled mannitol after repeated challenge. Pediatr Pulmonol 2011;46:1007–1014. 18. Larsson J, Perry CP, Anderson SD, Brannan JD, Dahlen SE, Dahlen B. The occurrence of refractoriness and mast cell mediator release following mannitol-induced bronchoconstriction. J Appl Physiol 2011;110:1029– 1035. 19. Ihre E, Gyllfors P, Gustafsson LE, Kumlin M, Dahlen B. Early rise in exhaled nitric oxide and mast cell activation in repeated low-dose allergen challenge. Eur Respir J 2006;27:1152–1159. 20. Palmqvist M, Cui Z-H, Sjostrand A, Linden A, Lotvall J. Reduced late asthmatic
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response by repeated low-dose allergen exposure. Eur Respir J 2001;17:872–880. 21. Phillips GD, Bagga PK, Djukanovic R, Holgate ST. The influence of refractoriness to adenosine 50 -monophosphate on allergen-provoked bronchoconstriction in asthma. Am Rev Respir Dis 1989;140: 321–326.
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22. Aalbers R, Kauffman HG, Koeter GH, Postma DS, De Vries K, de Monchy JGR. Dissimilarity in methacholine and adenosine 50 -monophosphate responsiveness 3 and 24 h after allergen challenge. Am Rev Respir Dis 1991;144:352–357. 23. Evans DJ, Coulby L, O’Connor BJ. Effect of allergen challenge on airway responsive-
ness to histamine and sodium metabisulphite in mild asthma. Thorax 1996;51:1185–1191. 24. Larsson J, Anderson SD, Dahlen SE, Dahlen B. Refractoriness to exercise challenge. A review of the mechanisms old and new. Immunol Allergy Clin North Am 2013;33:329–345.
Allergy 70 (2015) 682–688 © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
ORIGINAL ARTICLE
AIRWAY DISEASES
Airway responsiveness to mannitol 24 h after allergen challenge in atopic asthmatics B. E. Davis1,2, D. O. Amakye2 & D. W. Cockcroft1,2 1
Division of Respirology, Critical Care and Sleep Medicine, Department of Medicine, University of Saskatchewan; 2Department of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, SK, Canada
To cite this article: Davis BE, Amakye DO, Cockcroft DW. Airway responsiveness to mannitol 24 h after allergen challenge in atopic asthmatics. Allergy 2015; 70: 682–688.
Keywords allergen; asthma; mannitol; refractoriness. Correspondence Dr. Beth E. Davis, Division of Respirology, Critical Care and Sleep Medicine, Royal University Hospital, 103 Hospital Drive, Ellis Hall, 5th Floor, Saskatoon, SK S7N 0W8, Canada. Tel.: 306 844 1444 Fax: 306 844 1532 E-mail: [email protected] Accepted for publication 18 February 2015 DOI:10.1111/all.12601 Edited by: De Yun Wang
Abstract Background: Airway responsiveness to indirect stimuli correlates positively with airway inflammation. In atopic asthmatics, allergen inhalation is associated with an influx of inflammatory cells and increased responsiveness to the direct-acting stimuli methacholine at 3 and 24 h after exposure. We have shown mannitol responsiveness decreases 3 h after allergen inhalation. The current investigation assessed mannitol responsiveness 24 h after allergen challenge. Methods: Eleven mild atopic asthmatics completed allergen challenges on two separate occasions. In random order, methacholine or mannitol challenges were performed 24 h pre- and post–allergen challenge. Levels of fractional exhaled nitric oxide were also measured. Results: Allergen challenge increased airway responsiveness to methacholine 24 h postchallenge; the geometric mean (95% CI) methacholine PC20 decreased from 5.9 mg/ml (1.8–19.4) to 2.2 mg/ml (0.81–5.89); P = 0.01. This coincided with a significant increase (P = 0.02) in FeNO levels. Conversely, allergen challenge decreased airway responsiveness to mannitol; geometric mean (95% CI) dose– response ratio was significantly higher after allergen exposure (57 mg/% FEV1 fall [27–121] to 147 mg/% FEV1 fall [57–379]; P = 0.03), and FeNO levels were not significantly increased (P = 0.054). Conclusion: Allergen-induced changes in airway responsiveness to direct and indirect stimuli are markedly different. The loss in responsiveness to mannitol is likely not explainable by a refractory state. The effect(s) of allergen exposure on airway responsiveness to indirect-acting stimuli require further investigation.
A well-studied phenomenon in individuals with atopic asthma is the increase in airway responsiveness to directacting stimuli following allergen exposure. The increased sensitivity has been shown as early as 3 h (1) post–allergen challenge and can last for at least 7 days (2). We also know that inflammatory cells are recruited to the airway following allergen challenge (3, 4) and that eosinophilic airway inflammation correlates better with airway hyperresponsiveness (AHR) to indirect stimuli vs AHR to direct stimuli (5). The relationship between airway responsiveness to indirect stimuli and airway inflammation is also supported by decreases in responsiveness to AMP (6) and mannitol (7) following administration of inhaled glucocorticosteroid. We recently reported a significant decrease in mannitol responsiveness at 3 h post–allergen challenge in a group of
682
mild allergic asthmatics (8) and questioned whether the 3 h time point was too early to observe an increase, possibly due to a cross-refractory mechanism. Additionally, although there is evidence to support an increase in the number of activated eosinophils at 3 h post–allergen challenge (9), we did not assess allergen-induced changes in airway inflammation in the earlier study. Given the relationship between airway responsiveness to indirect stimuli and airway inflammation, a lack of recruitment of inflammatory cells at 3 h post–allergen challenge may have influenced our previous results. Therefore, our current hypothesis is that the allergeninduced change in airway responsiveness to mannitol would increase to a greater extent than that which has been shown for methacholine at 24 h post–allergen exposure.
Allergy 70 (2015) 682–688 © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Davis et al.
Allergen induced changes in AHR
performed. Data were collected from November 2013 to March 2014. Table 2 provides a study schematic.
Materials and methods Study participants Individuals of either gender between the ages of 18 and 65 with a history of atopic asthma and no other significant medical comorbidity were eligible for screening (Table 1). Additional inclusion criteria included baseline FEV1 ≥ 70% predicted, methacholine PC20 ≤ 16 mg/ml, positive skin prick test to at least one inhalable allergen, and no requirement for controller asthma treatment (i.e. salbutamol prn only). Pregnant or lactating females were excluded. The University of Saskatchewan Biomedical Research Ethics Board approved the study, and all participants consented to participation prior to the conduct of any study procedures.
Study design The study was conducted as a randomized crossover trial and included an initial screening visit followed by two testing triads. The triads were comprised of three consecutive days of testing in which Visit 2 was either a methacholine or mannitol challenge, Visit 3 was an allergen inhalation challenge, and Visit 4 matched Visit 2. A 2-week minimum washout between allergen inhalation challenges was required. If methacholine challenges were performed during the first triad, then mannitol challenges were performed during the second triad (Visits 5 and 7) and vice versa. Skin prick testing and fractional exhaled nitric oxide measurements were also
Skin prick testing Skin prick testing was performed to confirm hypersensitivity to one or more allergens and to identify an appropriate allergen for inhalation challenge. Drops of a variety of common allergens (pollens, animal danders, molds, foods) were placed on the volar surface of the forearm and introduced to the body by a tenting of the skin with a lancet or small beveled needle. Responses were observed after 10-15 min. The allergen producing the largest wheal was chosen for inhalation challenge testing unless clinical history (admission of symptom manifestation upon exposure or the type of allergen producing the largest wheal (e.g. food)) suggested otherwise. Standardized stock allergen extracts of cat hair (10 000 AU/ ml), grass pollen (100 000 BAU/ml), and house dust mite (pteronyssinus and farinae: both 30 000 AU/ml) were used for the allergen inhalation challenges (Table 1).
Fractional exhaled nitric oxide Measurements of fractional exhaled nitric oxide were collected to assess changes in airway inflammation and were performed prior to spirometry and bronchoprovocation testing at each visit. A 7-h post–allergen challenge measurement was also made prior to the collection of the 7-h FEV1. Data
Table 1 Participant demographics Subject
Age (years)
Gender
Height (cm)
FEV1 (l)
FEV1 (% predicted)
Allergen inhaled
1 2 3 4 5 6 7 8 9 10 11
28 37 27 29 23 30 22 21 26 19 22
M M F M F F F M F F F
185 178 160 180 168 160 180 183 155 157 168
4.71 3.54 2.74 4.57 3.19 2.24 4.04 4.57 3.01 3.06 3.13
96 82 88 99 93 77 101 93 101 97 89
Cat hair Cat hair HDM-DP Grass pollen HDM-DF Grass pollen HDM-DF Cat hair Cat hair Cat hair Cat hair
M, male; F, female; FEV1, forced expiratory volume in 1 s; HDM-DP, house dust mite dermatophagoides pteronyssinus; HDM-DF, house dust mite dermatophagoides farinae.
Table 2 Study schematic Visit 1
Visit 2
Visit 3
Visit 4
Washout
Visit 5
Visit 6
Visit 7
Consent MCh SPT
FeNO MCh or mannitol challenge
FeNO Allergen challenge FeNO*
FeNO MCh or mannitol challenge (same as Visit 2)
14 days minimum
FeNO MCh or mannitol challenge (opposite of Visit 2)
FeNO Allergen challenge FeNO*
FeNO MCh or mannitol challenge (same as Visit 5)
SPT, skin prick testing; FeNO, fractional exhaled nitric oxide; FeNO*: captured at 7 h post–allergen challenge; MCh, methacholine challenge.
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were captured using the NIOX MINO instrument (Aerocrine AB, Solna, Sweden). Participants inhaled fully on the filter mouthpiece and then exhaled at a constant flow rate (50 ml/ s) for 10 s. Nose clips were not used. Two reproducible measurements (10%) were required. Methacholine challenge Methacholine challenges were conducted according to the standardized 2-min tidal breathing methodology (10). In brief, reproducible, full flow-volume spirograms preceded bronchoprovocation challenge to assess baseline lung function and airway stability. An aerosol of normal saline was then inhaled over the course of 2 min of tidal breathing via face mask and with nose clip on. The jet nebulizer was calibrated to deliver aerosol at a rate of 0.13 ml/min. With nose clip in place, spirometric maneuvers were then performed at 30 and 90 s postinhalation to capture FEV1 data only. The lower of these values was retained for calculating the decrease in FEV1 following the inhalation of increasing (doubling) concentrations of methacholine solutions. Methacholine PC20 values (the concentration of methacholine that causes a 20% decrease in FEV1) were calculated by either interpolation (11) or extrapolation (12) from the log concentration vs response curve. Mannitol challenge Mannitol challenges were performed per standardized methodology (13). Single-use challenge kits (Pharmaxis Ltd, Frenchs Forest, NSW, Australia) containing the delivery device and all capsules required to deliver a 635-mg cumulative dose were used for each challenge. The capsule(s) are placed in the device and punctured, and the contents are then inhaled via slow deep inhalation. A 5-s breath hold followed the inhalation. The challenges began with the inhalation of a 0-mg (empty) capsule. After an additional 55 s, two FEV1 measurements were performed. The higher of these two measurements was used to calculate the fall in FEV1 after subsequent inhalations. The test was complete when a 15% fall in FEV1 occurred, or when there was a 10% incremental fall in FEV1 between doses or when the highest dose (635 mg) had been administered. Responses were assessed as the dose– response ratio (DRR). Allergen challenge Allergen challenges were performed as previously described (14). In brief, participants performed routine baseline spirometry to obtain three reproducible baseline full flow-volume measurements. Aerosolized allergen was administered by way of the Wright Nebulizer (Roxon Medi-Tech, Montreal, QC, Canada) fitted with a two-way Hans-Rudolph valve and calibrated to deliver an output of 0.13 ml/min. By way of mouthpiece and with nose clips in place, participants inhaled doubling concentrations of aerosolized allergen during 2 min of tidal breathing. Ten minutes later, two FEV1 measurements were obtained and the highest was used to calculate the
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fall in FEV1 from the highest baseline FEV1. The challenge was stopped when the FEV1 had fallen 20%. Lung function was then monitored (FEV1 only) at various time points up to 7 h. There was a washout period of at least 2 weeks between allergen challenges. The total dose of allergen administered during the second challenge was identical to that administered during the first challenge (i.e. same starting concentration and number of concentrations administered). Analysis Bronchoprovocation data (methacholine PC20 and mannitol DRR), and FeNO data were log-transformed and compared using the paired t-test (STATISTIX 10; Analytical Software, Tallahassee, FL, USA). A P-value < 0.05 was considered significant.
Results Study participants Eleven mild atopic asthmatics completed the study (Table 1). Two participants had methacholine PC20 results >16 mg/ml during screening at Visit 1. These two individuals, however, had documented (within 1 year) methacholine PC20 test results 15% during the 3– 7 h postchallenge) during both allergen challenges. Ten had isolated early responses. Methacholine and mannitol bronchoprovocation Airway responsiveness to methacholine significantly increased 24 h after allergen challenge; the geometric mean methacholine PC20 (95% CI) decreased from 5.9 mg/ml (1.8–19.4) to 2.2 mg/ml (0.81–5.89); P = 0.01. Conversely, airway responsiveness to mannitol significantly decreased 24 h after allergen challenge; the geometric mean DRR (95% CI) was higher after allergen challenge (147 mg/% FEV1 fall [57–379] vs 57 mg/% FEV1 fall [27–121]; P = 0.03) (Fig. 2A,B, respectively). Fractional exhaled nitric oxide Twenty-four-hour post–allergen challenge FeNO levels were significantly increased (P = 0.02) in the methacholine arm (Fig. 3) vs 24-h preallergen challenge levels. FeNO levels in the mannitol arm also increased 24 h after allergen challenge, but the increase was not statistically significant (P = 0.054).
Allergy 70 (2015) 682–688 © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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Figure 1 Mean data for airway responses to inhaled allergen. Similar responses to allergen challenge were observed for each testing arm. Open squares – methacholine arm. Closed circles – mannitol arm.
A
Figure 2 Individual methacholine PC20 (concentration of methacholine causing a 20% fall in FEV1) (A) and mannitol dose–response
Discussion We have shown a significant increase in airway responsiveness to methacholine 24 h after allergen challenge which coincided with significantly higher levels of FeNO. In contrast, however, airway responsiveness to mannitol significantly decreased 24 h after allergen challenge and this coincided with a nonsignificant increase in FeNO. The decrease in airway responsiveness to mannitol at 24 h post–allergen challenge is likely not explained by a refractory or cross-refractory mechanism as the reported duration of refractory periods ranges from 1 to 2 h for exercise-induced bronchoconstriction (15), 2–8 h for adeno-
B
ratio (B) data pre- and post–allergen challenge. Single data points with error bars represent the geometric means and standard error.
sine-50 -monophosphate (AMP) (16), and up to 2 h for mannitol (17, 18). We are unaware of a refractory state with respect to repeat allergen exposure. We do know that daily low-dose allergen exposure, enough to cause a 5% fall in FEV1, results in increased responsiveness to directacting stimuli (e.g. histamine or methacholine), increased FeNO, and increased sputum eosinophils (19, 20). The recruitment of inflammatory cells following repeated lowdose allergen challenge supports our hypothesis that mannitol responsiveness should increase 24 h after allergen challenge. Interestingly however, the Ihre et al. study (19) also noted airway responsiveness to AMP to be unchanged 48 h after the last low dose of allergen. The lack of
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Figure 3 Geometric mean and standard error for FeNO data at time points pre- and post–allergen challenge. The data are grouped
by testing arm. Mannitol data are on the left and methacholine data are on the right.
responsiveness to the indirect stimuli AMP supports our current findings that allergen challenge decreases airway responsiveness to indirect-acting stimuli, but there are methodological differences between the studies, including sample size and the time at which the measurement was made. In the Palmqvist et al. study (20) repeated low-dose allergen challenge diminished the LAR of a high-dose allergen challenge despite an increase in AHR to methacholine and an increase in airway inflammation. In the absence of an effect on the EAR, the inhibition of the LAR could be interpreted as a ‘delayed refractory period’ and this could point to a cross-refractory mechanism between allergen and mannitol, two stimuli that constrict the airway via a similar mechanism (i.e. mast cell mediator release). The loss in airway responsiveness may also be dependent on the order in which the indirect stimuli are administered as the response to allergen has been shown to increase when the airway is in an AMP refractory state (21). It may be, however, that repeated low-dose allergen exposure decreases airway responsiveness to indirect-acting stimuli, including a subsequent allergen challenge, by a mechanism that is not refractory by definition. Our data are most comparable with the Aalbers et al. (22) data that looked at allergen-induced changes in methacholine and AMP at three and 24 h post–allergen challenge. They showed an increase in airway responsiveness to methacholine at both time points but found an increase in AMP responsiveness at 3 h only. In our investigations, not only did we not find an increase in responsiveness to an indirect stimulus at 3 and 24 h postallergen, we found a significant decrease at
both time points. Notably, all participants in the Aalbers study were late responders and this differs from our study population which included mostly (10/11) isolated early responders. However, in a study by Evans et al. (23), allergen challenge failed to increase airway responsiveness to metabisulfite (MBS) at 3 and 24 h postchallenge in a predominantly (14/18) late responder population. The study also showed an apparent trend toward a significant loss in MBS sensitivity at 24 h, and this signal supports our findings despite the difference in the number of late responders studied. Both allergen and mannitol act by causing the release of mast cell mediators. An intuitive explanation for how the response to mannitol is decreased by prior allergen challenge is therefore the depletion of mast cell mediators. Larsson et al. tested this hypothesis during an investigation of mannitol refractoriness. Not only did they find no decrease in the urine content of bronchoconstricting mast cell mediators following repeat mannitol challenge, it was observed that those who were most refractory had significantly higher levels of mast cell mediators (PGF2 and LTE4) after repeat testing (18). A number of additional hypotheses regarding potential mechanisms of the refractory state have been proposed and recently reviewed (24). In the current investigation, we used standardized allergen challenge triad methodology to induce an early asthmatic response, monitor the development of a late asthmatic response, and assess allergen-induced changes in airway inflammation and airway responsiveness to methacholine and mannitol at 24 h post–allergen challenge. Ideally, our study population would have included a higher number of
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individuals with dual or late airway responses. This was not a requirement for study entry, however, and only one participant developed an LAR after allergen challenge. This could be viewed as a limitation, but we did observe increases in FeNO at 24 h post–allergen challenge. Therefore, a signal for airway inflammation was apparent at a time when the change in airway responsiveness to direct or indirect stimuli was being made. The observation that FeNO was significantly higher during the methacholine testing arm but not during the mannitol testing arm may also be viewed as a study limitation, but we propose this may be a signal for a mechanistic finding that could explain the observed mannitol nonresponsiveness following allergen challenge. Based on our hypothesis, we also did not incorporate the requirement for a positive mannitol challenge at study entry anticipating that allergen exposure would increase airway responsiveness to mannitol and potentially shift a negative or near-positive response preallergen to a positive response postallergen. As a result of the high number of mannitol nonresponders (eight of 11), we analyzed the data as a ratio of cumulative dose administered to airway response at that dose, the DRR. The decrease in the DRR was statistically significant and was seen in 10 of 11 participants. The outcomes of allergen exposure on airway responsiveness to indirect stimuli are not well described and may in fact
be very different from that which can be hypothesized based on current knowledge. We conclude from our investigation that allergen-induced changes in airway responsiveness to direct and indirect-acting stimuli are markedly different and that the unexpected loss in airway sensitivity to mannitol requires further investigation. Acknowledgments We would like to acknowledge and thank Pharmaxis Ltd., NSW, Australia, for supplying the mannitol test kits. Author contributions Beth E. Davis performed study design, data collection, and data analysis and helped in drafting, reviewing, and approving the manuscript. Daniel O. Amakye performed study design, data collection, and data analysis and helped in reviewing and approving the manuscript. Donald W. Cockcroft performed study design and data analysis and helped in reviewing and approving the manuscript. Conflicts of interest The authors declare that they have no conflicts of interest.
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