Superoxide dismutase inactivation in pathophysiology of asthmatic airway remodeling and reactivity

Abstract

Airway hyperresponsiveness and remodeling are defining features of asthma. We hypothesized that impaired superoxide dismutase (SOD) antioxidant defense is a primary event in the pathophysiology of hyperresponsiveness and remodeling that induces apoptosis and shedding of airway epithelial cells. Mechanisms leading to apoptosis were studied in vivo and in vitro. Asthmatic lungs had increased apoptotic epithelial cells compared to controls as determined by terminal dUTP nick-end labeling-positive cells. Apoptosis was confirmed by the finding that caspase-9 and -3 and poly (ADP-ribose) polymerase were cleaved. On the basis that SOD inactivation triggers cell death and low SOD levels occur in asthma, we tested whether SOD inactivation plays a role in airway epithelial cell death. SOD inhibition increased cell death and cleavage/activation of caspases in bronchial epithelial cells in vitro. Furthermore, oxidation and nitration of MnSOD were identified in the asthmatic airway, correlating with physiological parameters of asthma severity. These findings link oxidative and nitrative stress to loss of SOD activity and downstream events that typify asthma, including apoptosis and shedding of the airway epithelium and hyperresponsiveness.

Figure 1

Immunohistochemical analysis of apoptosis in airway epithelial cells from control (A, B, G) and asthmatic patients (C–E, F, H). A to H: Increased numbers of TUNEL-positive epithelial cells in endobronchial (D, F) and brush biopsies (H) of the asthmatic airway as compared to healthy controls (B, G). In addition to routine hematoxylin (A, C) and H&E staining (E), sections or cells were subjected to TUNEL assay with no counterstaining (B, D, F), or with eosin counterstaining (G, H). Healthy control bronchial mucosa in endobronchial biopsy (B) or brush biopsy (G) was negative for TUNEL. Architecture of healthy control airway mucosa (A) is contrasted to asthmatic mucosa with thickened basement membrane (C) and marked loss of epithelium in some areas (C–E, F). D, F, and H: Red nuclei indicate TUNEL positivity in asthmatic epithelial cells, whereas only minimal positivity is found in healthy controls (B and G). I: The graph shows the mean ± SE of TUNEL-positive cells in brush biopsies from five healthy controls and four asthmatics. Endobronchial biopsies are representative of seven asthmatic and three control individuals.

Figure 2

Cell proliferation was detected by anti-human MIB-1. Brown nuclear stain indicates positive MIB-1 staining in the asthmatic epithelial cells (A) and healthy controls (B). C: The graph shows MIB-1-positive cells (mean ± SD) of three healthy controls and four asthmatics. Some fields in asthmatic airways show more than 80% MIB-1-positive cells. Arrows show positive cells.

Figure 3

Apoptosis in asthmatic epithelial cells. Immunoblots of lysates from freshly obtained human airway epithelial cells. A: Asthmatic airway epithelial cells have activation of caspase-3 as shown by the presence of the cleavage product (17 kd). B: Caspase-3 activity assay confirms significant increase in caspase activity in asthmatic airway epithelial cells as compared to controls (P < 0.05). C: Caspase-3 activity measured in asthmatic airway epithelial cells is inversely correlated with %FEV₁. Immunoblots of lysates from freshly obtained human airway epithelial cells of asthmatic individuals shows increased cleavage products of caspase-9 (35 kd) (D) and PARP (85 kd) (E), molecular evidence of increased apoptosis in asthmatic airway epithelial cells as compared to healthy control airway epithelial cells.

Figure 4

Loss of SOD leads to activation of caspase-3. A: Inhibition of SOD activity by 5 μmol/L 2-ME leads to apoptotic death of BET1A cells. Graph shows loss of SOD activity (•) and increased apoptotic cells (○) in BET1A cells after treatment with 2-ME. B: Histograms of the fluorescence of BET1A cells stained with annexin V-fluorescein isothiocyanate to demonstrate apoptosis at various times after exposure to 2-ME. Number of cells in each channel are shown plotted against a logarithmic abscissa. C: Inhibition of SOD activity by 5 μmol/L 2-ME in BET1A cells increases caspase-3 activity. D: Exposure of BET1A cells to pyrogallol and H₂O₂ leads to a decrease in SOD activity and an increase in caspase-3 activity. All data are representative of at least three experiments.

Figure 5

Silencing of MnSOD. A and C: Northern analysis and immunoblot analysis of 293T cells at 48 hours after transfection with MnSOD siRNA. γ- and β-actin were used as a control for sample loading. B: MnSOD siRNA (48 hours) increases caspase-3 activity (analysis of variance, P = 0.038). As negative control cells were transfected with 50 nmol/L siRNA control. All data are representative of three experiments.

Figure 6

A: Inhibition of SOD in BET1A cells leads to time-dependent induction of BAX levels. β-Actin was used as a control for equal loading of protein. B: Decreased levels of intracellular GSH at various times after inhibition of SOD confirms loss of reducing potential (C) whereas increased tyrosine nitration of proteins in BET1A cells after SOD inhibition indirectly indicates oxidative and nitrative stress. All data are representative of three experiments.

Figure 7

Nitration of SOD in asthmatic airway epithelial cells. A: Lysates from asthmatic or control airway epithelial cells were immunoprecipitated using anti-MnSOD Ab, run on a 4 to 20% gradient gel, and immunoblotted with anti-nitrotyrosine antibody (top panel, lanes 1 and 2). The lower band confirms equal amount of MnSOD after immunoprecipitation. Pure MnSOD and MnSOD nitrated in vitro served as controls (lanes 3 and 4). Experiments were done in triplicate. B: Protein-bound nitrotyrosine of MnSOD purified from asthmatic airway epithelium was quantified by stable isotope dilution LC-MS interfaced to an HPLX system.

Figure 8

SOD loss is related to airflow limitation in asthma. Airway epithelial cell SOD activity is inversely correlated to airway response to albuterol (% change in FEV₁) and correlate with %FEV₁/FVC.