Detrimental effects of albuterol on airway responsiveness requires airway inflammation and is independent of β-receptor affinity in murine models of asthma

Abstract

Background: Inhaled short acting β2-agonists (SABA), e.g. albuterol, are used for quick reversal of bronchoconstriction in asthmatics. While SABA are not recommended for maintenance therapy, it is not uncommon to find patients who frequently use SABA over a long period of time and there is a suspicion that long term exposure to SABA could be detrimental to lung function. To test this hypothesis we studied the effect of long-term inhaled albuterol stereoisomers on immediate allergic response (IAR) and airway hyperresponsiveness (AHR) in mouse models of asthma.

Methods: Balb/C mice were sensitized and challenged with ovalbumin (OVA) and then we studied the IAR to inhaled allergen and the AHR to inhaled methacholine. The mice were pretreated with nebulizations of either racemic (RS)-albuterol or the single isomers (S)- and (R)-albuterol twice daily over 7 days prior to harvest.

Results: We found that all forms of albuterol produced a significant increase of IAR measured as respiratory elastance. Similarly, we found that AHR was elevated by albuterol. At the same time a mouse strain that is intrinsically hyperresponsive (A/J mouse) was not affected by the albuterol isomers nor was AHR induced by epithelial disruption with Poly-L-lysine affected by albuterol.

Conclusions: We conclude that long term inhalation treatment with either isomer of albuterol is capable of precipitating IAR and AHR in allergically inflamed airways but not in intrinsically hyperresponsive mice or immunologically naïve mice. Because (S)-albuterol, which lacks affinity for the β2-receptor, did not differ from (R)-albuterol, we speculate that isomer-independent properties of the albuterol molecule, other than β2-agonism, are responsible for the effect on AHR.

Figure 1

Timelines of the experiments. A) To measure the immediate allergic airways response (IAR) Balb/C mice were immunized with OVA + Alum i.p. on days 0 and 14. On days 21-26, animals were exposed for 30 minutes to 1% aerosolized OVA; controls received PBS aerosol. Four different groups of mice were treated with nebulized albuterol, (R)- (2.5 mg/ml), (S)- (2.5 mg/ml), (RS)- (5 mg/ml) or control PBS, twice daily for 20 minutes in the morning and in the afternoon on days 21-27. Lung mechanics was measured on day 28 following inhalation of 5% OVA aerosol. B) To measure the effect of albuterol on allergen induced AHR, Balb/C mice were immunized with OVA + Alum i.p. on days 0 and 14. On days 21-23 animals were exposed for 30 minutes to 1% aerosolized OVA; controls received PBS aerosol. The mice were treated with nebulized albuterol, (R)- (2.5 mg/ml), (S)- (2.5 mg/ml), (RS)- (5 mg/ml) or control PBS, twice daily for 20 minutes in the morning and in the afternoon on days 18-24. AHR was assessed by measuring Z_rs at increasing doses of inhaled methacholine (MCh).

Figure 2

Effect of albuterol on respiratory mechanics in mice sensitized and challenged with OVA. Balb/C mice were anesthetized and connected to a flexiVent and then received inhalation challenges of aerosolized OVA. The OVA inhalation was repeated 4 times and the respiratory impedance was measured after each challenge as indicated by arrows and numbers on the X-axis as follows: Following OVA inhalation at indicator 1, 2, 3 and 4 respiratory mechanics was measured every 10 seconds for 1 minute; following OVA inhalation at indicator 5 mechanics was measured once every minute for 20 minutes. Parameters from fitting the constant phase model to input impedance data are shown. R_n is Newtonian resistance of the conducting airways; H is lung elastance and G is tissue resistance. Animals were treated with (RS)-, (R)- or (S)- albuterol (n = 14, 13 and 15) twice daily for seven days with the last administration 18 hours before experiment. PBS (n = 9) was used as vehicle control. Both H and G were significantly elevated by (RS)-, (R)- and (S)- albuterol compared with control PBS over the 30 - 60 minutes interval (*** p < 0.001). Changes in R_n were not statistically significant (p > 0.05).

Figure 3

Cell differentials, cytokine titers, plasma indicators from BALF and histology scores. Following euthanasia, lungs were lavaged with 1 ml of PBS, and cells were counted, and cytospin slides were stained with H&E (n = 5 in each group). No statistically significant differences were found between treatment groups (p > 0.05). Cytokine concentrations were measured from the BALF supernatant using Bio-Plex^®. * p < 0.05, ** p < 0.01, (n = 10 in each group). Results of scoring of PAFS stained histological sections of lungs; no statistical difference was found between groups. Results of scoring of CCSP staining; no statistical difference was found between groups (n = 5 in each group). Total BALF protein was significantly increased in mice treated with (RS)-albuterol compared with (R)- and (S)-albuterol (* p < 0.05) whereas IgG1 in BALF was not affected by either treatment (n = 10 - 18 in each group).

Figure 4

Effect of albuterol on AHR in allergic mice. Respiratory mechanics time course following methacholine challenge. Parameters from fitting the constant phase model to input impedance data; R_n is Newtonian resistance of the conducting airways; H is lung elastance and G is tissue resistance. Left column: Allergic Balb/C mice; AHR measured as dose-response time-course to increasing doses of methacholine inhalation in OVA sensitized Balb/C mice. Animals were treated with (RS)-, (R)- or (S)- albuterol (n = 11, 12 and 10) twice daily for seven days with the last administration 18 hours before experiment. PBS (n = 10) was used as vehicle control. H was significantly elevated over PBS control at the 12.5 and 50 mg/ml doses of methacholine by (RS)-, (R)- and (S)- albuterol. * p < 0.05, ** p < 0.01 and *** p < 0.001. Right column: Naïve Balb/C mice; AHR assessment in naïve Balb/C mice. Animals were treated with (RS)-, (R)-, (S)- albuterol or control PBS, n = 8 per group.

Figure 5

Effect of albuterol on AHR in non-allergic mice. Respiratory mechanics time course following methacholine challenge. Animals were treated with albuterol twice daily for seven days with the last administration 18 hours before experiment. Parameters from fitting the constant phase model to input impedance data; R_n is Newtonian resistance of the conducting airways; H is lung elastance and G is tissue resistance. Left column: AHR assessment in naïve C57Bl/6 mice, treated with (RS)-, (R)-, (S)- albuterol or control PBS (n = 8, 6, 7 and 8). Middle column: AHR assessment in naïve A/J mice. Animals were treated with (RS)-, (R)-, (S)- albuterol or control PBS (n = 8, 5, 7 and 7). Right column: AHR assessment in Balb/C mice pretreated oropharyngeally with Poly-L-lysine (PLL) (50 μg in 50 μl PBS). PLL was administered once daily for 4 consecutive days before the assessment of respiratory mechanics with methacholine. Animals were treated with (RS)-, (R)-, (S)- albuterol or control PBS (n = 6, 6, 7 and 8).