Immunomodulatory Effects of Ambroxol on Airway Hyperresponsiveness and Inflammation

Abstract

Ambroxol is used in COPD and asthma to increase mucociliary clearance and regulate surfactant levels, perhaps through anti-oxidant and anti-inflammatory activities. To determine the role and effect of ambroxol in an experimental model of asthma, BALB/c mice were sensitized to ovalbumin (OVA) followed by 3 days of challenge. Airway hyperresponsiveness (AHR), lung cell composition and histology, and cytokine and protein carbonyl levels in bronchoalveolar lavage (BAL) fluid were determined. Ambroxol was administered either before the first OVA challenge or was begun after the last allergen challenge. Cytokine production levels from lung mononuclear cells (Lung MNCs) or alveolar macrophages (AM) were also determined. Administration of ambroxol prior to challenge suppressed AHR, airway eosinophilia, goblet cell metaplasia, and reduced inflammation in subepithelial regions. When given after challenge, AHR was suppressed but without effects on eosinophil numbers. Levels of IL-5 and IL-13 in BAL fluid were decreased when the drug was given prior to challenge; when given after challenge, increased levels of IL-10 and IL-12 were detected. Decreased levels of protein carbonyls were detected in BAL fluid following ambroxol treatment after challenge. In vitro, ambroxol increased levels of IL-10, IFN-γ, and IL-12 from Lung MNCs and AM, whereas IL-4, IL-5, and IL-13 production was not altered. Taken together, ambroxol was effective in preventing AHR and airway inflammation through upregulation of Th1 cytokines and protection from oxidative stress in the airways.

Keywords: Airway hyperresponsiveness; Ambroxol; Eosinophils; Neutrophils.

Figure 1. Scheme of the experimental protocol. Mice were sensitized and challenged to OVA (OVA/OVA) or sham-sensitized with PBS followed by OVA challenge (PBS/OVA). Ambroxol treatments were administered (A) beginning 2 days before through the OVA challenge days followed by 2 days of rest or (B) initiated after OVA challenges were completed. OVA, ovalbumin.

Figure 2. Effect of ambroxol on airway responses when administered prior to initiation of allergen challenge. Mice were sensitized to OVA or PBS followed by aerosolized OVA challenged (OVA/OVA and PBS/OVA, respectively). Ambroxol or vehicle treatment was initiated prior to OVA challenge. (A) Changes in lung resistance (RL). RL values were obtained in response to increasing concentrations of inhaled methacholine (MCh). (B) Cell composition in BAL fluid. (C) Cytokine levels in BAL fluid from mice which received ambroxol or saline treatment prior to OVA challenge. (D) Representative histological sections stained with H/E and PAS: (a) PBS/OVA with vehicle treatment, (b) OVA/OVA with vehicle treatment and (c) OVA/OVA with ambroxol treatment. (E) Quantitative analysis of PAS+ areas on lung histological sections. The results for each group are expressed as mean±SEM (n=8 in each group). ^*p<0.05; significant differences are shown compared to the groups of OVA/OVA with vehicle treatments. ^†p<0.05; significant differences are shown compared to the groups of PBS/OVA.

Figure 3. Effect of ambroxol on airway responses when administered after completion of the allergen challenges. Mice were sensitized and challenged to OVA (OVA/OVA) or sham-sensitized followed by OVA challenge (PBS/OVA). Ambroxol or saline were administrated following OVA challenge. (A) Changes in lung resistance (RL) and (B) Cell composition in BAL fluid. (C) Cytokine levels in BAL fluid in mice which received ambroxol or saline following OVA challenge. Mice were sensitized to OVA or PBS (OVA/OVA and PBS/OVA, respectively) followed by OVA challenge. (D) Representative histological sections after H/E and PAS staining: (a) PBS/OVA with vehicle treatment, (b) OVA/OVA with vehicle treatment and (c) OVA/OVA with ambroxol treatment. (E) Quantitative analysis of PAS+ areas. (F) The levels of protein carbonyls in BAL fluid from mice which received ambroxol or vehicle following OVA challenge were measured by ELISA and the levels are expressed as absorbance standardized to 20 µg of protein in BAL fluid. The results for each group are expressed as mean±SEM (n=8). ^*p<0.05; significant differences are shown compared to the groups of OVA/OVA with vehicle treatments. ^†p<0.05; significant differences are shown compared to the groups of PBS/OVA.

Figure 4. In vitro cytokine production from lung MNCs and alveolar macrophages. Lung MNCs were obtained from mice sensitized and challenged to OVA. Alveolar macrophages were collected from OVA sensitized mice without challenge. The levels of In vitro cytokine production from (A) lung MNCs and (B) alveolar macrophages. Cytokine levels in supernatants from cells cultured with OVA and different concentrations of ambroxol were measured by ELISA as described in Materials and Methods. The results for each group are expressed as mean±SEM (n=8). ^*p<0.05; significant differences are shown compared to controls; ^†p<0.05; significant differences are shown compared to OVA treatment alone.