Receptor for advanced glycation endproducts (RAGE) maintains pulmonary structure and regulates the response to cigarette smoke

Abstract

The receptor for advanced glycation endproducts (RAGE) is highly expressed in the lung but its physiological functions in this organ is still not completely understood. To determine the contribution of RAGE to physiological functions of the lung, we analyzed pulmonary mechanics and structure of wildtype and RAGE deficient (RAGE-/-) mice. RAGE deficiency spontaneously resulted in a loss of lung structure shown by an increased mean chord length, increased respiratory system compliance, decreased respiratory system elastance and increased concentrations of serum protein albumin in bronchoalveolar lavage fluids. Pulmonary expression of RAGE was mainly localized on alveolar epithelial cells and alveolar macrophages. Primary murine alveolar epithelial cells isolated from RAGE-/- mice revealed an altered differentiation and defective barrier formation under in vitro conditions. Stimulation of interferone-y (IFNy)-activated alveolar macrophages deficient for RAGE with Toll-like receptor (TLR) ligands resulted in significantly decreased release of proinflammatory cytokines and chemokines. Exposure to chronic cigarette smoke did not affect emphysema-like changes in lung parenchyma in RAGE-/- mice. Acute cigarette smoke exposure revealed a modified inflammatory response in RAGE-/- mice that was characterized by an influx of macrophages and a decreased keratinocyte-derived chemokine (KC) release. Our data suggest that RAGE regulates the differentiation of alveolar epithelial cells and impacts on the development and maintenance of pulmonary structure. In cigarette smoke-induced lung pathology, RAGE mediates inflammation that contributes to lung damage.

Fig 1. RAGE is expressed on alveolar epithelial cells and alveolar macrophages.

(A) Immunostaining for RAGE protein in WT and RAGE^-/- lung tissue. (B) Isolated alveolar epithelial cells (AEC) (n = 7 per group) and alveolar macrophages (AM) (n = 4 per group) were analyzed for RAGE mRNA expression using qRT-PCR. Data are shown as mean ± SEM. Scale bar: 100μm.

Fig 2. RAGE contributes to maintenance of pulmonary mechanics and structure.

Quantification of mean chord length (A) were performed by stereological analysis of alveolar parenchyma; n ≥ 4 per group. (B) Representative histology (hematoxylin and eosin staining) of ten months old WT and RAGE^-/- mice; scale bars: 200μm. Respiratory system compliance (C) and respiratory system elastance (D) were determined in two, four and ten months old WT and RAGE^-/- mice using invasive pulmonary function measurements; n ≥ 7 per group. (E) Concentration of serum protein albumin in BALF of two months old mice; n = 10. Data are shown as mean ± SEM. *p < 0.05 and **p < 0.01.

Fig 3. RAGE promotes the differentiation and formation of an intact barrier function in primary murine alveolar epithelial cells.

Alveolar epithelial cells were isolated from WT and RAGE^-/- mice and analyzed in an air-liquid interface culture system. (A) TEER of isolated alveolar epithelial cells was measured for a period of five days; n = 12 per group. Relative mRNA induction of alveolar epithelial type 2 cell marker surfactant protein C (B), alveolar epithelial type 1 cell marker aquaporin-5 (AQP5) (C) and the tight junction proteins claudin 18 (D), ZO-1 (E) and occludin (F) was determined by qRT-PCR; n = 6 per group. The proteins of cell lysate of alveolar epithelial cells at day 2 and 4 post isolation were separated by SDS-PAGE and stained with antibodies against α-Tubulin (αTub), aquaporin-5 (AQP5), pro-surfactant protein C (Sp-C), and claudin 18 (Cldn18) (G). The bands of the blot were densitometrically analyzed and the results are shown for AQP5 (H), SP-C (I), and Cldn18 (J). Data are shown as mean ± SEM. *p < 0.05; **p < 0.01 and ***p < 0.001.

Fig 4. RAGE modulates the inflammatory response in alveolar macrophages.

Alveolar macrophages were isolated from lungs of WT and RAGE^-/- mice. After activation with IFNɣ for 16 hours, alveolar macrophages were stimulated with TLR ligands LPS or Pam2CSK4 (Pam2) for 24 hours. Concentrations of TNFα (A and B), IL-6 (C and D), KC (E and F) and NO₂^- (G and H) were determined in supernatants; n ≥ 3 per group, representative for three independent experiments. Data are shown as mean ± SEM. *p < 0.05; **p < 0.01 and ***p < 0.001.

Fig 5. RAGE promotes the development of CS-induced lung damage.

WT and RAGE^-/- mice were exposed to CS or room-air for six months. Total lung capacity (A), quasi-static compliance (B) and inspiratory capacity (C) were determined by invasive pulmonary function measurements; n ≥ 18 per group. (D) Representative histology of alveolar parenchyma (hematoxylin and eosin staining); scale bars: 100μm. Quantification of mean chord length (E) were performed by stereological analysis of alveolar parenchyma; n ≥ 10 per group. Data are shown as mean ± SEM. *p < 0.05; **p < 0.01 and ***p < 0.001.

Fig 6. RAGE modulates inflammatory processes resulting from acute CS exposure.

WT and RAGE^-/- mice were exposed to acute CS exposure for four days. Numbers of total inflammatory cells (A), neutrophils (B) and macrophages (C) were determined in BALF of WT and RAGE^-/- mice; n ≥ 9 per group. Concentrations of KC in BALF were determined using ELISA (D); n ≥ 9. Data are shown as mean ± SEM. *p < 0.05; ***p < 0.001.