Inhaled salmeterol and/or fluticasone alters structure/function in a murine model of allergic airways disease

Abstract

Background: The relationship between airway structural changes (remodeling) and airways hyperresponsiveness (AHR) is unclear. Asthma guidelines suggest treating persistent asthma with inhaled corticosteroids and long acting beta-agonists (LABA). We examined the link between physiological function and structural changes following treatment fluticasone and salmeterol separately or in combination in a mouse model of allergic asthma.

Methods: BALB/c mice were sensitized to intraperitoneal ovalbumin (OVA) followed by six daily inhalation exposures. Treatments included 9 daily nebulized administrations of fluticasone alone (6 mg/ml), salmeterol (3 mg/ml), or the combination fluticasone and salmeterol. Lung impedance was measured following methacholine inhalation challenge. Airway inflammation, epithelial injury, mucus containing cells, and collagen content were assessed 48 hours after OVA challenge. Lungs were imaged using micro-CT.

Results and discussion: Treatment of allergic airways disease with fluticasone alone or in combination with salmeterol reduced AHR to approximately naüve levels while salmeterol alone increased elastance by 39% compared to control. Fluticasone alone and fluticasone in combination with salmeterol both reduced inflammation to near naive levels. Mucin containing cells were also reduced with fluticasone and fluticasone in combination with salmeterol.

Conclusions: Fluticasone alone and in combination with salmeterol reduces airway inflammation and remodeling, but salmeterol alone worsens AHR: and these functional changes are consistent with the concomitant changes in mucus metaplasia.

Figure 1

Experimental Study Design Scheme. BALB/c mice were immunized intraperitoneally with 20 micrograms of Ovalbumin (OVA) on days 0 and 14. OVA was then nebulized daily as a challenge on days 21 to 26. Different groups of mice were treated with 20 minute nebulizations of vehicle, fluticasone 6,000 micrograms per ml, salmeterol 3,000 micrograms per ml, or the combination of fluticasone and salmeterol. These were dosed once daily from days 19 to 27 (9 doses).

Figure 2

BALF Cell Counts. Total cells (Total), macrophages (MAC), eosinophils (EOS), neutrophils (PMN), and lymphocytes (LYM). Treatment groups; Naïve mice (N), Inhaled OVA (6 doses) (O), OVA with vehicle control (V), salmeterol (S), fluticasone (F), and the combination (fluticasone and salmeterol) (FS). Mean cells per ml of BAL fluid ± SE. * in Total cells p < 0.05 for S compared to. N, FS, and F. † in Total cells p < 0.05 for N, F and FS compared to S. * in MAC p < 0.05 for S compared to V and FS. † in MAC p < 0.05 for V and FS compared to S.

Figure 3

Histology. Representative* Hematoxylin and Eosin stained tissue sections taken with 10× objective. A) Naïve, B) OVA, C) Salmeterol (arrow indicates mucus adherent to wall), D) Fluticasone, E) Fluticasone and Salmeterol, F) immersion fixed lung from OVA challenged mouse demonstrating airway obstruction with mucus in bronchial lumen. *Representative figures were chosen using criteria described in the text.

Figure 4

Tissue Scores. Peribronchial inflammation, epithelial thickening and injury. A) Semi-quantitative score for peribronchial inflammation. B) Semi-quantitative score for global epithelial damage. C) Quantitative epithelial thickness. Groups; naïve mice (N), OVA (O), and O mice with vehicle control (V), salmeterol (S), fluticasone (F), and a combination of fluticasone and salmeterol (FS). N = 4-6 mice in each group with 4 airways per mouse (averaged for each mouse/slide). Results expressed as mean ± SE. * p < 0.05.

Figure 5

Lung Mechanics. Mechanics parameters following nebulized saline and increasing concentrations of methacholine (peak response as percent of baseline). Groups; naïve mice (N) (n = 7) and OVA mice treated with vehicle (V) (n = 6), salmeterol (S) (n = 7), fluticasone (F) (n = 8), salmeterol and fluticasone (FS) (n = 8). R = R_N = Newtonian resistance, G = tissue damping, H = tissue elastance. Results expressed as mean ± SE Panel with R: NS no significant differences between the groups. Panel with G: * p < 0.05 for S compared to V, N, F, or FS. Panel with H: * p < 0.05 for S compared to V (p is also < 0.05 for S compared to N, F, or FS). † p < 0.05 for S or V compared to N, F, or FS. All comparisons in this figure are by a two way ANOVA followed by Tukey pairwise comparisons.

Figure 6

Mucus Staining. Representative* PAFS stained tissue sections imaged with a dual excitation filter (FITC/Texas Red) and the 20× objective (F imaged at 10×.). A) naïve, B) OVA, C) Salmeterol, D) Fluticasone, E) Fluticasone and Salmeterol, F) immersion fixed lung from OVA mouse demonstrating airway obstruction with mucus. *Representative figures were chosen using criteria described in the text.

Figure 7

Mucus Quantification. Mucus containing (PAFS positive) cells. Groups include naïve mice (N) and OVA sensitized and challenged mice treated with vehicle control (V), salmeterol (S), fluticasone (F), and a combination of fluticasone and salmeterol (FS). Results are expressed as means ± SE. N = 4 mice with 4 airways averaged per mouse (slide). ANOVA p < 0.0001. * p < 0.05.