help button home button
AJRCCM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Vanacker, N. J.
Right arrow Articles by Kips, J. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Vanacker, N. J.
Right arrow Articles by Kips, J. C.
American Journal of Respiratory and Critical Care Medicine Vol 166. pp. 1128-1134, (2002)
© 2002 American Thoracic Society

Articles

Effect of Combining Salmeterol and Fluticasone on the Progression of Airway Remodeling

Nele J. Vanacker, Els Palmans, Romain A. Pauwels and Johan C. Kips

Department of Respiratory Diseases, Ghent University Hospital, Ghent, Belgium

Correspondence and requests for reprints should be addressed to Nele Vanacker, B.Pharm., Department of Respiratory Diseases, Ghent University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium. E-mail: Nele.Vanacker{at}barclab.com

ABSTRACT

In subjects insufficiently controlled with low to moderate doses of inhaled corticosteroids, adding ß-agonists is clinically more beneficial than increasing the dose of inhaled corticosteroids. In the present study, we investigated the effect of adding salmeterol to fluticasone on allergen-induced airway inflammation and remodeling. Sensitized rats, in which characteristics of remodeling had been induced by ovalbumin exposure every 2 days from Days 14 to 28, were further exposed to ovalbumin or PBS from Days 29 to 42. During the last 2 weeks, before allergen exposure, rats were treated with aerosolized fluticasone propionate (10 mg), salmeterol (1 mg), salmeterol (1 mg) plus fluticasone propionate (10 mg), or placebo. After 4 weeks of ovalbumin exposure, the airways showed inflammatory changes, goblet cell hyperplasia, and enhanced fibronectin and collagen deposition. Salmeterol in monotherapy decreased bronchoalveolar lavage fluid eosinophil number but had no influence on structural changes. Combining salmeterol with fluticasone propionate counteracted goblet cell hyperplasia, but increased the amount of fibronectin and collagen in the airway wall. These effects of salmeterol did not influence airway responsiveness. We conclude that the combination of salmeterol and fluticasone propionate enhances aspects of allergen-induced airway remodeling. This is not accompanied by changes in airway responsiveness.

Key Words: asthma • corticosteroids • long-acting ß-agonists • remodeling • salmeterol

The combination of inhaled corticosteroids (ICS) and long-acting ß-agonists has gained increasing popularity. Several studies indicate that, irrespective of the degree of asthma severity, adding long-acting ß-agonists results in a better improvement in clinical and physiologic indices than increasing the dose of ICS within a two- to fourfold range (1–5). The influence of combination treatment on the underlying airway inflammation and remodeling, however, is incompletely understood. ICS are known to decrease inflammation and to have a more variable, dose-dependent effect on aspects of airway remodeling (6–8). Data, mainly derived from in vitro and in vivo animal studies, suggest that long-acting ß-agonists might also have some anti-inflammatory potential (9–14). To what extent this contributes to their therapeutic activity in humans remains to be fully resolved, as does their effect on airway remodeling. Biopsies of patients treated with salmeterol for 6 weeks showed no changes in sub-basement membrane collagen or tenascin deposition (15). The effect of concomitant treatment with ICS on airway changes again is unclear. Although somewhat disputed (16–18), evidence exists of a favorable bidirectional interaction between ß-agonists and steroids on various pathophysiologic aspects of asthma (19–21). No difference in sputum eosinophil count was observed in patients treated with formoterol combined with budesonide (200 µg) compared with monotherapy with budesonide (800 µg) (3). The benefit of the combination of ICS and ß-agonists on airway remodeling, however, needs to be further investigated.

We have developed an in vivo rat model of airway remodeling (22). In this model, inhaled fluticasone was shown to partly prevent the development and, especially, inhibit the progression of allergen-induced structural airway changes (23, 24). We have now investigated whether the combination of an ICS and a long-acting ß-agonist had additional benefit on the progression of ovalbumin (OA)-induced airway changes, when introduced at a stage when airway remodeling was already present.

METHODS

Animals and Study Design
Specific pathogen-free, male Brown Norway rats (n = 10 per experimental group) (Harlan, Zeist, The Netherlands), were sensitized intraperitoneally on Days 0 and 7 with 1 mg of OA (Grade III; Sigma, Poole, UK) and 200 µg of Al(OH)3 in 0.5 ml of sterile saline. Rats were exposed for 30 minutes to aerosolized 1% (w/v) OA, every 2 days from Day 14 to Day 28 (Table 1) . From Day 29 to Day 42, rats were further exposed every 2 days to OA or to PBS. Thirty minutes before each exposure, animals were treated with aerosolized salmeterol (SLM) (30 ml), fluticasone propionate (FP) (30 ml), SLM + FP, or placebo (Plac) (GlaxoSmithKline, Stockley Park, UK). SLM was given at a concentration of 1 mg/30 ml of PBS, and FP was given at a dose of 10 mg/30 ml of 0.1% ethanol/PBS. In the SLM + FP groups, animals were treated consecutively with 30 ml of SLM and 30 ml of FP. As in previous experiments, adding 0.1% ethanol had no discernible effect; PBS was administered as placebo.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Study design

 
Outcome Measures
On Day 43, 24 hours after the last aerosol exposure, airway responsiveness to carbachol was assessed as described previously (22). The concentration of carbachol causing a 50% increase in lung resistance (PC50RL) was calculated. Subsequently bronchoalveolar lavage (BAL) was performed via the tracheal cannula. The total number of leukocytes in the BAL fluid was determined with a Bürker chamber. A differential cell count was performed on cytocentrifuged preparations (Cytospin 2; Shandon, Runcorn, UK) stained with May–Grünwald–Giemsa, and was based on application of standard morphologic criteria to at least 400 cells (22).

Lung Histology
After lavage, 4% paraformaldehyde-fixed lungs were embedded in paraffin and cut into 2-µm-thick sections. Eosinophil counts and morphometric analyses were performed on Congo red-stained sections. Goblet cells were quantified by periodic acid–Schiff staining. Fibronectin in the airway wall was stained by the streptavidin–biotin–peroxidase method and the amount of collagen was determined with Sirius red (22). Quantitative measurements were performed with a computerized image analysis system (KS400; Zeiss, Oberkochen, Germany). For quantification of fibronectin and collagen in the airway wall, the pixels of each color image were divided into three color components (hue, saturation, and intensity). The threshold for each color component for the brown stain (fibronectin) or the red stain (collagen) was defined and kept constant throughout analysis. The area in the airway wall covered by the stain was determined by the software and its value was calculated.

For each experimental group, 2 or 3 lung sections of each of 6 to 10 animals were analyzed. As a result, a mean of 22 large (length of basement membrane [Pbm] > 2,000 µm; cf. Sapienza and coworkers [25]) airways per group was evaluated. The total bronchial wall area (WAt) was normalized to the squared length of basement membrane (P2bm]). Fibronectin and collagen were quantified in total airway wall (WFt and WCt, respectively) and expressed per micrometer of basement membrane (Pbm).

To avoid observer bias, all microscope slides were coded before analysis by one observer and read in a blinded fashion.

Data Analysis
Reported values are expressed as means ± standard error of the mean (SEM). Cellular composition of BAL fluid, goblet cells in the epithelium, peribronchial eosinophils, and PC50RL for the different experimental groups were compared by Kruskal–Wallis test for multiple comparisons. Pairwise comparisons were made by means of a Mann–Whitney U test with the Bonferroni correction. For analysis of the morphometric measurements and the measurements of fibronectin and collagen deposition, data for the rats in a particular experimental group were pooled. To ensure that a similar range of airway size was being compared, we compared the frequency distribution of Pbm for the different experimental groups, using the Kolmogorov–Smirnov test. WAt was normalized to P2bm and WFt and WCt were normalized to Pbm and were compared between experimental groups by one-way analysis of variance with post hoc (least significant difference and Scheffé) tests. p Values less than 0.05 were considered significant.

RESULTS

Inflammatory Changes
Exposure of sensitized rats to OA for 4 weeks caused a significant increase in the number of infiltrating eosinophils around the airways, compared with rats that were exposed to OA for 2 weeks followed by 2 weeks of PBS exposure (OA-Plac/OA versus OA-Plac/PBS, p < 0.0001) (Table 2) . Treatment with fluticasone during the last 2 weeks of allergen exposure totally inhibited the increase in eosinophil infiltration (OA-FP/OA versus OA-Plac/OA, p < 0.0001), whereas salmeterol did not have any influence. The combination of salmeterol with fluticasone also decreased eosinophil number to baseline levels (OA-SLM+FP/OA versus OA-Plac/OA, p < 0.0001). Salmeterol did not influence eosinophil number in PBS exposed animals.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Peribronchial eosinophil number, total airway wall area, and pc50rl

 
Similarly, in BAL fluid (Table 3) , eosinophil numbers were suppressed to baseline level by combined therapy (OA-SLM+FP/OA versus OA-Plac/OA, p < 0.0001). In addition, fluticasone and salmeterol had an anti-eosinophil effect on their own (OA-FP/OA versus OA-Plac/OA, p < 0.003; OA-SLM/OA versus OA-Plac/OA, p < 0.02).


View this table:
[in this window]
[in a new window]
  		TABLE 3. Total cell count and cell differentiation in bronchoalveolar lavage fluid

 
Lymphocyte numbers in BAL fluid were significantly increased by prolonged OA exposure (OA-Plac/OA versus OA-Plac/PBS, p < 0.0001). Monotherapy with salmeterol or fluticasone had no effect whereas the combination significantly reduced lymphocyte count (OA-SLM+FP/OA versus OA-Plac/OA, p < 0.02).

Neither salmeterol, fluticasone, nor their combination inhibited the increase in number of neutrophils caused by 4 weeks of OA exposure (OA-Plac/OA versus OA-Plac/PBS, p < 0.0001).

Macrophage numbers were also enhanced by further OA exposure (OA-Plac/OA versus OA-Plac/PBS, p < 0.03). Only treatment with fluticasone, alone (OA-FP/OA versus OA-Plac/OA, p < 0.02) or in combination with salmeterol (OA-SLM + FP/OA versus OA-Plac/OA, p < 0.0001), significantly decreased this number.

Structural Changes
The number of goblet cells, as identified by periodic acid–Schiff staining, after 4 weeks of OA exposure did not significantly increase compared with 2 weeks of OA exposure (52.00 ± 4.42 /mm bmin the OA-Plac/OA group versus 46.79 ± 4.18 /mm bm in the OA-Plac/PBS group). Compared with placebo, monotherapy with fluticasone (OA-FP/OA, 43.85 ± 5.25 /mm bm in the OA-FP/OA group) or with salmeterol (60.59 ± 5.37 /mm bm in the OA-S/OA group) had no effect. Combining salmeterol with fluticasone, however, significantly reduced the number of goblet cells in the epithelium (34.06 ± 4.23 /mm bm in the OA-SLM+FP/OA group; p < 0.03 versus the OA-Plac/OA group and p < 0.003 versus OA-SLM/OA group). The combination of salmeterol and fluticasone in animals exposed to OA for 2 weeks, followed by 2 weeks of PBS exposure, also caused a reduction in goblet cell number compared with placebo-treated, PBS-exposed animals (OA-SLM+FP/PBS versus OA-Plac/PBS, p < 0.003) (Figure 1) .



View larger version (19K):
[in this window]
[in a new window]
  		Figure 1. The number of goblet cells in airways after 2 weeks of exposure to OA followed by 2 weeks of exposure to PBS and concomitant treatment with placebo (open bar), salmeterol (hatched bar), or the combination of salmeterol and fluticasone (vertically striped bar); and after 4 weeks of exposure to OA with treatment during the final 2 weeks with placebo (solid bar), fluticasone (shaded bar), salmeterol (checkered bar), or their combination (horizontally striped bar). Results are expressed as means ± SEM. bm = Basement membrane; FP = fluticasone propionate; GC = goblet cell; OA = ovalbumin; Plac = placebo; S = salmeterol. *p < 0.05 versus Plac/PBS; {dagger}p < 0.05 versus Plac/OA; §p < 0.05 versus S/OA.

 
OA exposure for 4 weeks also induced a significant increase in fibronectin deposition in the airway wall compared with 2 weeks of OA exposure followed by a 2-week allergen-free period (11.55 ± 0.89 µm2/µm [OA-Plac/OA] versus 8.43 ± 0.72 µm2/µm [OA-Plac/PBS]; p < 0.013). This increase was totally prevented by fluticasone treatment simultaneous with the last 2 weeks of allergen exposure (7.64 ± 1.08 µm2/µm for the OA-FP/OA group; p < 0.013 versus OA-Plac/OA). Salmeterol had no effect on the amount of fibronectin after 4 weeks of allergen exposure (11.46 ± 1.50 µm2/µm for the OA-SLM/OA group). On the other hand, in combination with fluticasone, salmeterol inhibited the reducing effect of fluticasone on fibronectin deposition (13.76 ± 1.70 µm2/µm for the OA-SLM+FP/OA group; p < 0.0001 versus OA-FP/OA). In PBS-exposed animals, salmeterol alone (7.33 ± 0.87 µm2/µm for the OA-SLM/PBS group) or in combination with fluticasone (6.62 ± 0.77 µm2/µm for the OA-SLM+FP/PBS group) did not cause an increase in fibronectin deposition versus placebo (Figures 2 and 3) .



View larger version (17K):
[in this window]
[in a new window]
  		Figure 2. The amount of fibronectin in total airway wall after 2 weeks of exposure to OA followed by 2 weeks of exposure to PBS and concomitant treatment with placebo (open bar), salmeterol (hatched bar), or a combination of salmeterol and fluticasone (vertically striped bar); and after 4 weeks of exposure to OA with treatment during the last 2 weeks with placebo (solid bar), fluticasone (shaded bar), salmeterol (checkered bar), or their combination (horizontally striped bar). Results are expressed as means ± SEM. FP = Fluticasone propionate; OA = ovalbumin; Plac = placebo; S = salmeterol. *p < 0.05 versus Plac/PBS; {dagger}p < 0.05 versus Plac/OA; §p < 0.05 versus FP/OA.

 


View larger version (92K):
[in this window]
[in a new window]
  		Figure 3. The amount of fibronectin in airway wall compared with the control group, exposed to PBS and treated with placebo during the last 2 weeks (A), after 4 weeks of exposure to OA and concomitant treatment during the final 2 weeks with placebo (B), or with a combination of salmeterol and fluticasone (C) (immunohistochemical staining for fibronectin; original magnification, x200).

 
The amount of collagen in total airway wall was significantly increased in 4 weeks versus 2 weeks in OA-exposed rats (40.60 ± 2.56 µm2/µm [OA-Plac/OA] versus 32.81 ± 1.96 µm2/µm [OA-Plac/PBS], p < 0.04). Neither fluticasone (38.38 ± 2.98 µm2/µm for the OA-FP/OA group) nor salmeterol (45.84 ± 4.80 µm2/µm for the OA-SLM/OA group) alone inhibited the OA-induced increase in collagen deposition. Treatment with a combination of salmeterol and fluticasone, on the other hand, induced a greater degree of collagen deposition compared with placebo treatment (p < 0.008) and compared with fluticasone alone (p < 0.005) in rats exposed to OA for 4 weeks. This increase was, however, not observed with treatment of PBS-exposed animals (29.15 ± 3.37 µm2/µm [OA-SLM/PBS] and 34.72 ± 3.33 µm2/µm [OA-SLM+FP/PBS]) (Figures 4 and 5) .



View larger version (19K):
[in this window]
[in a new window]
  		Figure 4. The amount of collagen in total airway wall area after 2 weeks of exposure to OA followed by 2 weeks of exposure to PBS and concomitant treatment with placebo (open bar), salmeterol (hatched bar), or the combination of salmeterol and fluticasone (vertically striped bar); and after 4 weeks of exposure to OA with treatment during the last 2 weeks with placebo (solid bar), fluticasone (shaded bar), salmeterol (checkered bar), or their combination (horizontally striped bar). Results are expressed as means ± SEM. FP = Fluticasone propionate; OA = ovalbumin; Plac = placebo; S = salmeterol. *p < 0.05 versus Plac/PBS; {dagger}p < 0.05 versus Plac/OA; §p < 0.05 versus FP/OA.

 


View larger version (92K):
[in this window]
[in a new window]
  		Figure 5. The amount of collagen in airway wall compared with the control group, exposed to PBS and treated with placebo during the last 2 weeks (A), after 4 weeks of exposure to OA and concomitant treatment during the last 2 weeks with placebo (B), or with a combination of salmeterol and fluticasone (C) (Sirius red staining; original magnification, x200).

 
Although there was a trend, the increase in the total airway wall area due to 4 weeks of OA exposure, compared with 2 weeks of OA exposure followed by a 2-week allergen-free period, was not statistically significant. Combination treatment with salmeterol and fluticasone also induced an increase in airway wall thickening compared with treatment with fluticasone alone (p < 0.04), but not compared with salmeterol alone (Table 2).

Airway Responsiveness to Carbachol
No differences were observed in PC50RL between the groups (Table 2). Neither were there significant differences between the dose–response curves of airway responsiveness to carbachol (data not shown).

DISCUSSION

The present study indicates that, in this chronic in vivo animal model, salmeterol had no effect on the progression of allergen-induced airway inflammation or structural changes. The effect of the combination with ICS on acute inflammatory changes is almost identical to monotherapy with fluticasone. Strikingly, however, adding salmeterol counteracts the effect of fluticasone on allergen-induced fibronectin and collagen deposition.

That long-acting ß-agonists might have some anti-inflammatory effect is mainly derived from in vitro experiments and animal studies (9–14). Whether this also applies in humans is less clear, although some anti-inflammatory actions have been confirmed. Salmeterol was shown to inhibit the increase in sputum eosinophils after allergen challenge (26), whereas in the study by Wallin and colleagues (27), 8 weeks of treatment with formoterol reduced eosinophils (EG2+ cells) in biopsies from patients with a high eosinophil number at the start of the study. In addition, protection against plasma exudation has been reported (28). In our model, salmeterol did not influence tissue eosinophil count, but slightly reduced BAL fluid eosinophil count. As expected, the effect of fluticasone on eosinophils was far more pronounced. It was initially feared that because of the functional antagonism of ß-agonists, airways chronically treated with long-acting ß-agonists might be exposed to doses of allergen that under nonprotected conditions would cause symptoms and thus elicit avoidance, and that this would increase the severity of airway inflammation (18). However, in our experiment the reduction in eosinophilia by fluticasone was not counteracted by the combination with salmeterol. These observations add to data from some human studies indicating that adding long-acting ß-agonists to ICS does not aggravate the underlying inflammation (3, 4).

To date, few data are available about the effect of combination therapy on long-term structural changes in asthma. One study has quantified the relative effects of adding salmeterol, compared with adding fluticasone, on the vascularity of the subepithelial lamina propria, in symptomatic subjects with asthma already receiving low to moderate doses of ICS (29). A decrease in the density of vessels to baseline was seen after treatment with salmeterol in monotherapy, which may suggest an advantageous effect of salmeterol complementary to the action of ICS. The effect of long-acting ß-agonists alone or in combination with ICS on other indices of airway remodeling in human subjects is largely unknown (15, 30). Therefore, our rat model, displaying characteristics of airway remodeling (22, 23), provides a useful tool. In the current study, sensitized and allergen-exposed rats developed goblet cell hyperplasia (GCH). GCH occurs early in the disease process (31–33) and seems to reach a plateau as prolonging allergen exposure for 2 weeks, in our experiment, did not further enhance the changes. The pathogenesis underlying allergen-induced GCH is thought to involve a variety of mediators including interleukin (IL)-4 (34), IL-13 (35), IL-9 (36), the epidermal growth factor system (37), and others, probably by upregulating the expression of MUC genes. Inhibition of cytokine expression could underlie the effect of fluticasone. Although treatment with steroids can prevent the development of allergen-induced GCH (23, 38), it has less effect once GCH has been established. Little is known about the effect of long-acting ß-agonists on mucus production. In the present study, treatment of OA-exposed animals with salmeterol in monotherapy had no influence on the number of cells positive for periodic acid–Schiff. In the study by Kamachi and coworkers, on the other hand, goblet cell number was significantly increased by treatment with the short-acting ß-agonist salbutamol (38). Interestingly, the combination of salmeterol and fluticasone significantly reduced goblet cell number versus placebo- and salmeterol-treated animals. In addition, treatment of rats exposed to PBS for the last 2 weeks with the combination of salmeterol and fluticasone reversed previously established GCH. This phenomenon could possibly be explained by a positive interaction between ß-agonist and steroid on a molecular basis (19). Glucocorticoids might modify ß-adrenergic receptor (ß-AR) function by coupling them to G proteins and hence activating adenyl cyclase, or might upregulate previously downregulated ß-AR expression (21, 39). Conversely, long-acting ß2-agonists can also influence steroid function (40). Eickelberg and coworkers (41) demonstrated that salmeterol is able to activate the glucocorticoid receptor in a ligand-dependent manner, in primary lung fibroblasts and vascular smooth muscle cells. They facilitate translocation of the glucocorticoid receptor to the nucleus, thus enhancing the glucocorticoid-mediated effect.

The overall effect of this bidirectional interaction seems to differ from cell to cell (42, 43).

Another index of airway remodeling, enhanced by prolonged allergen exposure, is increased deposition of extracellular matrix proteins. In our rat model of airway remodeling, monotherapy with salmeterol, in contrast to fluticasone, did not inhibit the increase in fibronectin. Strikingly, combined therapy significantly enhanced the deposition of fibronectin in the airway wall. Almost the same phenomenon is observed for collagen deposition. The increase in fibronectin and collagen was paralleled by an increase in airway wall thickness. Several human studies indicate that corticosteroids partly inhibit subepithelial fibrosis (7, 8, 44). Six weeks of treatment with salmeterol, on the other hand, induced no change in sub-basement membrane collagen deposition in bronchial biopsies (15). Also, Lindqvist and coworkers (30) demonstrated, in a preliminary study, that the thickness of the sub-basement membrane tenascin layer was not significantly different after treatment with salmeterol or with fluticasone. The most likely explanation for the effect of the combined therapy again resides in the molecular interaction between ICS and long-acting ß-agonists. In vitro experiments show that, depending on the experimental environment, ß-agonists and corticosteroids can induce or inhibit fibroblast function and proliferation (45–49). Silvestri and coworkers (47) demonstrated that the combination with fluticasone did not influence salmeterol-induced reduction of fibroblast proliferation. In the study by Beckett and coworkers (48), on the other hand, the increased proliferation by fluticasone was reversed by salmeterol. Although in the present model fluticasone inhibits fibronectin deposition, the interaction with salmeterol at the molecular level could tilt the balance toward an increase in fibronectin and collagen deposition. The functional consequence of the profibrotic effect of the combined therapy in our experiment is unclear. In a previous paper (22), we have shown that prolonged OA exposure resulted in increased deposition of fibronectin and collagen, and that this was accompanied by a progressive decrease in airway hyperresponsiveness (AHR). This is in line with the mathematical studies by Lambert and coworkers (50), who demonstrated that increased extracellular matrix deposition could increase airway wall stiffness, thus opposing against narrowing of the airway wall (51). Increased extracellular matrix deposition within and around airway smooth muscle bundles could also have the effect of banding, opposing against maximal shortening of airway smooth muscle and thus AHR (52). These findings raise the hypothesis that remodeling is an attempt to protect against allergen-induced AHR. The present study fits with this idea, as despite a slight increase in airway wall thickness, no increase in AHR was observed. A remarkable finding is that similar observations have not been made for the combination of ICS with short-acting ß-agonists. Wang and coworkers (53, 54) showed a significant increase in outer airway wall area of the small airways and in AHR in guinea pigs exposed to allergen, fenoterol, or their combination. Also, in a study by Kamachi and coworkers (38), OA exposure induced AHR, which was further enhanced in rats continuously treated with salbutamol. The latter was not accompanied by a thickening of the airway wall. Of note is that the beneficial effect of long-acting ß-agonists with ICS has not been described for the combination of ICS with short-acting ß-agonists (55, 56).

In conclusion, the present study indicates that in this chronic in vivo model, adding salmeterol to fluticasone resulted in a similar or even more pronounced effect on inflammatory changes, but, on the other hand, counteracted the inhibiting effect of fluticasone on allergen-induced fibronectin and collagen deposition. This had no apparent consequence for airway function. These results further illustrate that, as the precise functional role of airway remodeling remains to be established, the impact of remodeling cannot be evaluated merely by histologic analysis, but needs to be related to functional consequences.

Acknowledgments

The authors thank E. Castrique, C. Snauwaert, A. Neesen, I. De Borle, K. De Saedeleer, and M. Mouton for technical assistance. The authors also acknowledge Dr. M. Johnson, Glaxo Wellcome, for kindly providing salmeterol and fluticasone propionate.

FOOTNOTES

Supported in part by the Concerted Research Initiative of the University of Ghent (Project GOA 98-6) and by the Fund for Scientific Research, Flanders (FWO-Flanders) (Project 3G006298). N.J. Vanacker is funded by the FWO-Flanders.

Received in original form March 11, 2002; accepted in final form July 1, 2002

REFERENCES

  1. Greening AP, Ind PW, Northfield M, Shaw G. Added salmeterol versus higher-dose corticosteroid in asthma patients with symptoms on existing inhaled corticosteroid. Lancet 1994;344:219–224.[CrossRef][Medline]
  2. Woolcock A, Lundback B, Ringdal N, Jacques LA. Comparison of addition of salmeterol to inhaled steroids with doubling of the dose of inhaled steroids. Am J Respir Crit Care Med 1996;153:1481–1488.[Abstract]
  3. Kips JC, O'Connor BJ, Inman MD, Svensson K, Pauwels RA, O'Byrne PM. A long-term study of the anti-inflammatory effect of low-dose budesonide plus formoterol versus high-dose budesonide in asthma. Am J Respir Crit Care Med 2000;161:996–1001.[Abstract/Free Full Text]
  4. Li X, Ward C, Thien F, Bish R, Bamford T, Bao X, Bailey M, Wilson JW, Haydn Walters E. An antiinflammatory effect of salmeterol, a long-acting ß2 agonist, assessed in airway biopsies and bronchoalveolar lavage in asthma. Am J Respir Crit Care Med 1999;160:1493–1499.[Abstract/Free Full Text]
  5. Pauwels RA, Lofdahl CG, Postma DS, Tattersfield AE, O'Byrne P, Barnes PJ, Ullman A. Effect of inhaled formoterol and budesonide on exacerbations of asthma. Formoterol and Corticosteroids Establishing Therapy (FACET) International Study Group. N Engl J Med 1997;337:1405–1411.[Abstract/Free Full Text]
  6. Jeffery PK, Godfrey RW, Adelroth E, Nelson F, Rogers A, Johansson SA. Effects of treatment on airway inflammation and thickening of basement membrane reticular collagen in asthma: a quantitative light and electron microscopic study. Am Rev Respir Dis 1992;145:890–899.[Medline]
  7. Olivieri D, Chetta A, Del Donno M, Bertorelli G, Casalini A, Pesci A, Testi R, Foresi A. Effect of short-term treatment with low-dose inhaled fluticasone propionate on airway inflammation and remodeling in mild asthma: a placebo-controlled study. Am J Respir Crit Care Med 1997;155:1864–1871.[Abstract]
  8. Sont JK, Willems LN, Bel EH, van Krieken JH, Vandenbroucke JP, Sterk PJ. Clinical control and histopathologic outcome of asthma when using airway hyperresponsiveness as an additional guide to long-term treatment. AMPUL Study Group. Am J Respir Crit Care Med 1999;159:1043–1051.[Abstract/Free Full Text]
  9. Butchers PR, Vardey CJ, Johnson M. Salmeterol: a potent and long-acting inhibitor of inflammatory mediator release from human lung. Br J Pharmacol 1991;104:672–676.[Medline]
  10. Bissonnette EY, Befus AD. Anti-inflammatory effect of ß2-agonists: inhibition of TNF-{alpha} release from human mast cells. J Allergy Clin Immunol 1997;100:825–831.[CrossRef][Medline]
  11. Hallsworth MP, Twort CHC, Lee TH, Hirst SJ. ß2-Adrenoceptor agonists inhibit release of eosinophil-activating cytokines from human airway smooth muscle cells. Br J Pharmacol 2001;132:729–741.[CrossRef][Medline]
  12. Whelan CJ, Johnson M, Vardey CJ. Comparison of the anti-inflammatory properties of formoterol, salbutamol and salmeterol in guinea-pig skin and lung. Br J Pharmacol 1993;110:613–618.[Medline]
  13. Bolton PB, Lefevre P, McDonald DM. Salmeterol reduces early- and late-phase plasma leakage and leukocyte adhesion in rat airways. Am J Respir Crit Care Med 1997;155:1428–1435.[Abstract]
  14. Boulet LP, Chakir J, Milot J, Boutet M, Laviolette M. Effect of salmeterol on allergen-induced airway inflammation in mild allergic asthma. Clin Exp Allergy 2001;31:430–437.[CrossRef][Medline]
  15. Roberts JA, Bradding P, Britten KM, Walls AF, Wilson S, Gratziou C, Holgate ST, Howarth PH. The long-acting ß2-agonist salmeterol xinafoate: effects on airway inflammation in asthma. Eur Respir J 1999;14:275–282.[Abstract]
  16. Boulet LP, Cartier A, Milot J, Cote J, Malo JL, Laviolette M. Tolerance to the protective effects of salmeterol on methacholine-induced bronchoconstriction: influence of inhaled corticosteroids. Eur Respir J 1998;11:1091–1097.[Abstract]
  17. Yates DH, Kharitonov SA, Barnes PJ. An inhaled glucocorticoid does not prevent tolerance to the bronchoprotective effect of a long-acting inhaled ß2-agonist. Am J Respir Crit Care Med 1996;154:1603–1607.[Abstract]
  18. Mcivor RA, Pizzichini E, Turner MO, Hussack P, Hargreave FE, Sears MR. Potential masking effects of salmeterol on airway inflammation in asthma. Am J Respir Crit Care Med 1998;158:924–930.[Abstract/Free Full Text]
  19. Anderson GP. Interactions between corticosteroids and ß-adrenergic agonists in asthma disease induction, progression, and exacerbation. Am J Respir Crit Care Med 2000;161:S188–S196.[Free Full Text]
  20. Mak JC, Nishikawa M, Barnes PJ. Glucocorticosteroids increase ß2-adrenergic receptor transcription in human lung. Am J Physiol Lung Cell Mol Physiol 1995;268:L41–L46.[Abstract/Free Full Text]
  21. Giannini D, Bacci E, Dente FL, Di Franco A, Vagaggini B, Testi R, Paggiaro P. Inhaled beclomethasone dipropionate reverts tolerance to the protective effect of salmeterol on allergen challenge. Chest 1999;115:629–634.[Abstract/Free Full Text]
  22. Palmans E, Kips JC, Pauwels RA. Prolonged allergen exposure induces structural airway changes in sensitized rats. Am J Respir Crit Care Med 2000;161:627–635.[Abstract/Free Full Text]
  23. Vanacker NJ, Palmans E, Kips JC, Pauwels RA. Fluticasone inhibits but does not reverse allergen induced structural airway changes. Am J Respir Crit Care Med 2001;163:674–679.[Abstract/Free Full Text]
  24. Vanacker NJ, Palmans E, Pauwels RA, Kips JC. Fluticasone inhibits the progression of allergen-induced structural airway changes. Clin Exp Allergy 2002;32:914–920.[CrossRef][Medline]
  25. Sapienza S, Du T, Eidelman EH, Wang NS, Martin JG. Structural changes in the airways of sensitized Brown Norway rats after antigen challenge. Am Rev Respir Dis 1991;144:423–427.[Medline]
  26. Dente FL, Bancalari L, Bacci E, Bartoli ML, Carnevali S, Cianchetti S, Di Franco A, Giannini D, Vagaggini B, Testi R, et al. Effect of a single dose of salmeterol on the increase in airway eosinophils induced by allergen challenge in asthmatic subjects. Thorax 1999;54:622–624.[Abstract/Free Full Text]
  27. Wallin A, Sandstrom T, Soderberg M, Howarth P, Lundback B, Della C-G, Wilson S, Judd M, Djukanovic R, Holgate S, et al. The effects of regular inhaled formoterol, budesonide, and placebo on mucosal inflammation and clinical indices in mild asthma. Am J Respir Crit Care Med 1999;159:79–86.[Abstract/Free Full Text]
  28. Greiff L, Wollmer P, Andersson M, Svensson C, Persson CG. Effects of formoterol on histamine induced plasma exudation in induced sputum from normal subjects. Thorax 1998;53:1010–1013.[Abstract/Free Full Text]
  29. Orsida BE, Ward C, Li X, Bish R, Wilson JW, Thien F, Walters EH. Effect of a long-acting ß2-agonist over three months on airway wall vascular remodeling in asthma. Am J Respir Crit Care Med 2001;164:117–121.[Abstract/Free Full Text]
  30. Lindqvist AE, Karjalainen EM, Laitinen LA, Kava T, Altraja A, Pulkkinen M, Halme M, Laitinen A. Salmeterol (SLM), fluticasone propionate (FP) or disodium cromoglycate (DSCG) in the treatment of newly diagnosed asthma [abstract]. Am J Respir Crit Care Med 2001;163:A586.
  31. Blyth DI, Pedrick MS, Savage TJ, Hessel EM, Fattah D. Lung inflammation and epithelial changes in a murine model of atopic asthma. Am J Respir Cell Mol Biol 1996;14:425–438.[Abstract]
  32. Haile S, Lefort J, Joseph D, Gounon P, Huerre M, Vargaftig BB. Mucous-cell metaplasia and inflammatory-cell recruitment are dissociated in allergic mice after antibody- and drug-dependent cell depletion in a murine model of asthma. Am J Respir Cell Mol Biol 1999;20:891–902.[Abstract/Free Full Text]
  33. Ordonez CL, Khashayar R, Wong HH, Ferrando R, Wu R, Hyde DM, Hotchkiss JA, Zhang Y, Novikov A, Dolganov G, et al. Mild and moderate asthma is associated with airway goblet cell hyperplasia and abnormalities in mucin gene expression. Am J Respir Crit Care Med 2001;163:517–523.[Abstract/Free Full Text]
  34. Dabbagh K, Takeyama K, Lee HM, Ueki IF, Lausier JA, Nadel JA. IL-4 induces mucin gene expression and goblet cell metaplasia in vitro and in vivo. J Immunol 1999;162:6233–6237.[Abstract/Free Full Text]
  35. Shim JJ, Dabbagh K, Ueki IF, Dao-Pick T, Burgel PR, Takeyama K, Tam DCW, Nadel JA. IL-13 induces mucin production by stimulating epidermal growth factor receptors and by activating neutrophils. Am J Physiol Lung Cell Mol Physiol 2001;280:L134–L140.[Abstract/Free Full Text]
  36. Louahed J, Toda M, Jen J, Hamid Q, Renauld JC, Levitt RC, Nicolaides NC. Interleukin-9 upregulates mucus expression in the airways. Am J Respir Cell Mol Biol 2000;22:649–656.[Abstract/Free Full Text]
  37. Takeyama K, Dabbagh K, Lee HM, Agusti C, Lausier JA, Ueki IF, Grattan KM, Nadel JA. Epidermal growth factor system regulates mucin production in airways. Proc Natl Acad Sci USA 1999;96:3081–3086.[Abstract/Free Full Text]
  38. Kamachi A, Munakata M, Nasuhara Y, Nishimura M, Ohtsuka Y, Amishima M, Takahashi T, Homma Y, Kawakami Y. Enhancement of goblet cell hyperplasia and airway hyperresponsiveness by salbutamol in a rat model of atopic asthma. Thorax 2001;56:19–24.[Abstract/Free Full Text]
  39. Tan KS, Grove A, McLean A, Gnosspelius Y, Hall IP, Lipworth BJ. Systemic corticosteriod rapidly reverses bronchodilator subsensitivity induced by formoterol in asthmatic patients. Am J Respir Crit Care Med 1997;156:28–35.[Abstract/Free Full Text]
  40. Peters MJ, Adcock IM, Brown CR, Barnes PJ. ß-Adrenoceptor agonists interfere with glucocorticoid receptor DNA binding in rat lung. Eur J Pharmacol 1995;289:275–281.[CrossRef][Medline]
  41. Eickelberg O, Roth M, Lorx R, Bruce V, Rudiger J, Johnson M, Block LH. Ligand-independent activation of the glucocorticoid receptor by ß2-adrenergic receptor agonists in primary human lung fibroblasts and vascular smooth muscle cells. J Biol Chem 1999;274:1005–1010.[Abstract/Free Full Text]
  42. Kips JC, Pauwels RA. Long-acting inhaled ß2-agonist therapy in asthma. Am J Respir Crit Care Med 2001;164:923–932.[Free Full Text]
  43. Hancox RJ, Stevens DA, Adcock IM, Barnes PJ, Taylor DR. Effects of inhaled ß agonist and corticosteroid treatment on nuclear transcription factors in bronchial mucosa in asthma. Thorax 1999;54:488–492.[Abstract/Free Full Text]
  44. Laitinen A, Altraja A, Kampe M, Linden M, Virtanen I, Laitinen L. Tenascin is increased in airway basement membrane of asthmatics and decreased by an inhaled steroid. Am J Respir Crit Care Med 1997;156:951–958.[Abstract/Free Full Text]
  45. Kraft M, Lewis C, Pham D, Chu HW. IL-4, IL-13, and dexamethasone augment fibroblast proliferation in asthma. J Allergy Clin Immunol 2001;107:602–606.[CrossRef][Medline]
  46. Warshamana GS, Martinez S, Lasky JA, Corti M, Brody AR. Dexamethasone activates expression of the PDGF-{alpha} receptor and induces lung fibroblast proliferation. Am J Physiol 1998;18:L499–L507.
  47. Silvestri M, Fregonese L, Sabatini F, Dasic G, Rossi GA. Fluticasone and salmeterol downregulate in vitro, fibroblast proliferation and ICAM-1 or H-CAM expression. Eur Respir J 2001;18:139–145.[Abstract/Free Full Text]
  48. Beckett P, Shute J, Howarth P. Influence of salmeterol, fluticasone and the combination on fibroblast proliferation in vitro. Am J Respir Crit Care Med 2001;163:A473.
  49. Hershman KM, Neely CM, Ameredes BT, Calhoun WJ. Regulation of extracellular matrix components by ß agonist enantiomers: a possible role for airway remodeling. Am J Respir Crit Care Med 2001;163:A473.
  50. Lambert RK, Codd SL, Alley MR, Pack RJ. Physical determinants of bronchial mucosal folding. J Appl Physiol 1994;77:1206–1216.[Abstract/Free Full Text]
  51. Bramley AM, Roberts CR, Schellenberg RR. Collagenase increases shortening of human bronchial smooth muscle in vitro. Am J Respir Crit Care Med 1995;152:1513–1517.[Abstract]
  52. Shen X, Gunst SJ, Tepper RS. Effect of tidal volume and frequency on airway responsiveness in mechanically ventilated rabbits. J Appl Physiol 1997;83:1202–1208.[Abstract/Free Full Text]
  53. Wang ZL, Bramley AM, McNamara A, Pare PD, Bai TR. Chronic fenoterol exposure increases in vivo and in vitro airway responses in guinea pigs. Am J Respir Crit Care Med 1994;149:960–965.[Abstract]
  54. Wang ZL, Walker BA, Weir TD, Yarema MC, Roberts CR, Okazawa M, Pare PD, Bai TR. Effect of chronic antigen and ß2 agonist exposure on airway remodeling in guinea pigs. Am J Respir Crit Care Med 1995;152:2097–2104.[Abstract]
  55. Hancox RJ, Cowan JO, Flannery EM, Herbison GP, McLachlan CR, Wong CS, Taylor DR. Randomised trial of an inhaled ß2 agonist, inhaled corticosteroid and their combination in the treatment of asthma. Thorax 1999;54:482–487.[Abstract/Free Full Text]
  56. Taylor DR, Town GI, Herbison GP, Boothman-Burrell D, Flannery EM, Hancox B, Harre E, Laubscher K, Linscott V, Ramsay CM, et al. Asthma control during long-term treatment with regular inhaled salbutamol and salmeterol. Thorax 1998;53:744–752.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Mol. Pharmacol.Home page
A. M. Misior, H. Yan, R. M. Pascual, D. A. Deshpande, R. A. Panettieri, and R. B. Penn
Mitogenic Effects of Cytokines on Smooth Muscle Are Critically Dependent on Protein Kinase A and Are Unmasked by Steroids and Cyclooxygenase Inhibitors
Mol. Pharmacol., February 1, 2008; 73(2): 566 - 574.
[Abstract] [Full Text] [PDF]

Home page
 Eur Respir JHome page
E. H. Walters, D. W. Reid, D. P. Johns, and C. Ward
Nonpharmacological and pharmacological interventions to prevent or reduce airway remodelling
Eur. Respir. J., September 1, 2007; 30(3): 574 - 588.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
H. W. Chu, J. G. Rino, R. B. Wexler, K. Campbell, R. J. Harbeck, and R. J. Martin
Mycoplasma pneumoniae infection increases airway collagen deposition in a murine model of allergic airway inflammation
Am J Physiol Lung Cell Mol Physiol, July 1, 2005; 289(1): L125 - L133.
[Abstract] [Full Text] [PDF]

Home page
 ChestHome page
R. Tomic, C. C. Lassiter, J. D. Ritzenthaler, H. N. Rivera, and J. Roman
Anti-Tissue Remodeling Effects of Corticosteroids: Fluticasone Propionate Inhibits Fibronectin Expression in Fibroblasts
Chest, January 1, 2005; 127(1): 257 - 265.
[Abstract] [Full Text] [PDF]

Home page
 Eur Respir JHome page
P.E. Christie, M. Jonas, C-H. Tsai, E.Y. Chi, and W.R. Henderson Jr
Increase in laminin expression in allergic airway remodelling and decrease by dexamethasone
Eur. Respir. J., July 1, 2004; 24(1): 107 - 115.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
V. Brusasco and R. Pellegrino
Invited Review: Complexity of factors modulating airway narrowing in vivo: relevance to assessment of airway hyperresponsiveness
J Appl Physiol, September 1, 2003; 95(3): 1305 - 1313.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
M. J. Holtzman
Drug Development for Asthma
Am. J. Respir. Cell Mol. Biol., August 1, 2003; 29(2): 163 - 171.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
M. J. Tobin
Asthma, Airway Biology, and Nasal Disorders in AJRCCM 2002
Am. J. Respir. Crit. Care Med., February 1, 2003; 167(3): 319 - 332.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Vanacker, N. J.
Right arrow Articles by Kips, J. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Vanacker, N. J.
Right arrow Articles by Kips, J. C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Cell Mol. Biol.
Copyright © 2002 American Thoracic Society