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.