We have performed a detailed assessment of the effects of racemic albuterol as well as its separate isomers on the respiratory phenotype. In particular we focused on the effects of albuterol isomers on allergen and methacholine perturbed respiratory mechanics following an extended period of pretreatment with inhaled albuterol. We were interested to investigate if albuterol might induce effects that would persist beyond termination of administration, therefore the study was designed in such a manner that drugs were delivered twice daily over seven days and then stopped 18 hours before analysis. With this approach, the drug had time to wash out and we were studying only the sequelae of the treatment and not the direct effect of the drug, such as bronchial relaxation. First, we studied whether albuterol affects allergen induced responses in the lung. We found that the IAR in terms of H and G were increased. With this piece of information, we then speculated that AHR might also be affected. Hence, we studied the effect of albuterol on allergen-induced AHR and discovered that AHR in terms of H was elevated by treatment with (RS)-, (S)- and (R)- albuterol. Finally we tested whether the AHR could be due to epithelial disruption or effects on the smooth muscle and found that neither could explain the increase in AHR caused by extended albuterol treatment.
We triggered the IAR by administering nebulized OVA to allergic mice and then immediately started tracking the respiratory mechanics. We expected the OVA to trigger a constriction of airway smooth muscle that would be seen as an increase in R
. The responses in R
elicited by OVA were generally small, but repeatable and seemed to be inhibited by (R)-albuterol, although not to a statistically significant extent (Figure 2). If we compare the amplitude of the responses in R
following an OVA challenge with the response seen in lungs challenged with methacholine in Figures 4 and 5, we conclude that the airway constriction elicited by inhaled allergen is very small and probably does not carry much biological significance in the airways of mice. The increase in H and G following the allergen challenge, on the other hand, were much more pronounced over time in the presence of (RS)-, (S)- or (R)- albuterol. These observations illustrate that mice are capable of generating a smooth muscle response in the conducting airways when exposed to allergen, however, the muscle response was small and the result demonstrate that the conducting airways are probably not the location in which most of the activity of the allergen takes place. Instead, the allergen induced effects in the lung periphery (H and G) were augmented with (RS)-, (S)- or (R)- albuterol likely due to closure of peripheral airways .
Inhalation of allergen is a common trigger of asthma and instigates an immediate release of mediators from mast cells that have the capacity to activate a number of pathways that lead to lung inflammation and AHR . Some of the mast cell mediators, e.g. histamine and serotonin, have the capacity to stimulate smooth muscles to contract, whereas other mediators are involved in the cascade that leads to overt inflammation, including recruitment of leucocytes, plasma leakage and eventually AHR [32, 33]. The immediate response to an allergen challenge is usually manifest as a bronchoconstriction of the conducting airways leading to a reduction of airflow and shortness of breath . Typically, this IAR can be successfully treated with inhaled bronchodilators such as albuterol. The notion that β-agonists can cause a decline in lung function is neither new nor is it limited to observations in animal models. It was noted in a year-long study that asthmatic patients treated as needed with racemic fenoterol resulted in more exacerbations, a significant decline in baseline lung function, and an increase in airway responsiveness to methacholine, but did not alter bronchodilator responsiveness . As indicated by our results, one explanation to the deteriorating lung function in patients could be that the albuterol treatment increased the propensity for airway closure following allergen challenge.
We next addressed the cause of airway closure exacerbated by prolonged albuterol treatment by exploring two alternative hypotheses. The first is that increased mucus production from the epithelial cells is promoted by albuterol treatment. The second is that albuterol treatment increases plasma leakage into the lung. We studied the mucus producing epithelial cells in a semi-quantitative manner and found that the score of PAFS positive cells was not augmented by any treatment. We then focused on quantification of extravasation in the BALF and used IgG1 and total protein in BALF as indicators of plasma extravasation. The increase in total protein in the (RS)-albuterol treated mice was small but significant compared with (R)- and (S)-albuterol treated mice, suggesting that (R)- and (S)-albuterol, which otherwise had no significant effect on plasma extravasation on their own, may have mild detrimental effects on plasma extravasation when administered simultaneously as a racemic mixture. IgG1 extravasation into the lung, on the other hand, was not affected by albuterol. A recent study from our group demonstrated that AHR induced by acute acid aspiration correlates with BALF protein, whereas this correlation was lost over time, possibly due to healing of the acid induced epithelial injury . The techniques we used to study extravasation herein do not directly measure plasma leakage, hence, we are unable to completely rule out the possibility that plasma leakage did occur. Notwithstanding this uncertainty, our data do not support plasma extravasation as a mechanism for why the isomers of albuterol and the racemic mixture produced similar degrees of airway closure.
We performed an extensive analysis of BALF cytokines one hour post allergen challenge. While the concentrations of most cytokines did not change and the titers were generally low, we found that IL-4, IL-5 and IL-13 were significantly increased in mice treated with (RS)-albuterol. These cytokines are conventionally considered as Th2 cytokines and thought to promote the asthma phenotype . Chronic administration of various racemic β2-agonists have been shown to induce increased production of pro-inflammatory IL-13 in Th2 cells from asthmatic patients in vitro, which was suggested to be independent of the isomer of albuterol. In this context, it is interesting to note that in our study the single isomer (R)-albuterol did not significantly induce inflammatory cytokines. However, when (S)-albuterol was present in the form of (RS)-albuterol, the picture changed in the direction of more Th2 cytokines being produced. The significant decreases in IL-12p40 in the BALF from mice receiving (RS)-albuterol may partially explain the observed increases in Th2 cytokines from these same mice, as IL-12p40 acts as a negative regulator of IL-12p70 signaling , which itself functions to promote Th1 responses that antagonize Th2. The increase in Th2 cytokines did not seem to affect respiratory mechanics, as we did not measure any difference between (RS)-albuterol and the pure isomers when the mice were challenged with allergen. Studies in vitro have shown that (S)-albuterol may activate mast cells and enhance release of histamine and IL-4 , which could adversely affect patients.
The total cell number present in lavageable airspaces appeared increased in all treatment groups although not statistically significant (Figure 3) and the cell differentials revealed that the inflammation was dominated by eosinophils.
A significant problem in asthma is the hyperresponsiveness to various inhaled stimuli [40, 41]. Testing patients for hyperresponsiveness helps in setting the diagnosis of asthma. As it has been suggested that extensive β2-agonist treatment might contribute to the development of hyperresponsiveness, we designed experiments to address this issue in vivo in different animal models. We found that pretreatment with either compound had an effect on methacholine induced hyperresponsiveness in allergic mice (Figure 4). This was evidenced by a significant increase in H commensurate with an increase in lung de-recruitment . From these data, we draw the conclusion that β-receptor independent properties of albuterol appear to augment the AHR in allergic mice. We also found that (S)-albuterol did not affect H neither in a strain known to be genetically hyperresponsive (A/J (Figure 5) nor in normal responsive animals (non-allergic Balb/C and C57Bl/6 (Figure 4, 5)). A/J mice exhibit AHR as an increase in R
, which in turn depends on the airway smooth muscle having a higher shortening velocity in the A/J compared to that of most other mouse strains [26, 42]. Since AHR was not affected by albuterol in A/J mice (Figure 5), this suggests that the AHR increase in OVA sensitized mice was probably not due to effects on the airway smooth muscle. Thus, it appears that preexisting lung inflammation is necessary for albuterol to cause further negative effects on the hyperresponsiveness of the respiratory system. Since each of the isomers of albuterol, as well as the racemic mixture, increased AHR, the mechanism must be β-receptor independent.
When comparing the results obtained with IAR and AHR we noticed a qualitative difference in that inhaled OVA (Figure 2) generated an increase in both G and H, whereas inhaled methacholine (Figure 4) produced only an increase in H. We speculate that these differences are explained by the different modes of action of methacholine and OVA. Methacholine stimulates airway smooth muscle directly via muscarinic receptors, accounting for the effect on R
. Methacholine is also a secretagogue with the capacity to trigger epithelial cells to expel mucus  which might account for airway closure and the increase in H. OVA, on the other hand, acts more indirectly via intermediary resident and inflammatory leukocytes (i.e. mast cells)  that conceivably could trigger both mucus secretion and alterations in the visco-elastic properties of the lung, thereby leading to a more complex response including both G and H.
It is, of course, difficult to compare clinical asthma with our mouse model particularly since we used a long-term treatment protocol followed by a wash-out period. While only a few clinical studies with (S)-albuterol have been performed the results have been mixed. Two crossover trials failed to detect any increase in AHR with a single dose of 100 μg (S)-albuterol [44, 45], whereas another study detected an increase in AHR, albeit after a much higher single dose of (S)-albuterol, (5 mg) . Taken together, this might suggest that either high doses or sustained treatment with albuterol is needed to reveal any adverse effects on AHR.
We administered a model cationic protein, PLL, that mimics MBP from eosinophils, which has been shown to induce increased permeabilization of the epithelial lining  with subsequent hyperresponsiveness to inhaled methacholine, which in turn is probably due to increased epithelial permeability primarily affecting the conducting airways [30, 48]. It has also been shown that salmeterol prevents compromise of the airway epithelial barrier when histamine-1 receptor or Protease Activated Receptor-2 were activated in primary airway epithelium . We used PLL expecting that it would reveal effects of the long-term treatment with albuterol isomers on the smooth muscle. The hypothesis was that the smooth muscle would normally be protected by an intact epithelium disguising the effect of methacholine. We found that PLL induced a robust response to methacholine comparable to what has been shown before by our group , however, pretreatment with albuterol did not affect the response in any manner. Since albuterol did not affect AHR in Poly-L-lysine treated mice (Figure 5) nor in non-allergic mice (Figure 4 and 5), we conclude that the AHR in OVA allergic mice was probably not due to changes in epithelial permeability.