Human alveolar epithelial cell injury induced by cigarette smoke

Abstract

Background: Cigarette smoke (CS) is a highly complex mixture and many of its components are known carcinogens, mutagens, and other toxic substances. CS induces oxidative stress and cell death, and this cell toxicity plays a key role in the pathogenesis of several pulmonary diseases.

Methodology/principal findings: We studied the effect of cigarette smoke extract (CSE) in human alveolar epithelial type I-like (ATI-like) cells. These are isolated type II cells that are differentiating toward the type I cell phenotype in vitro and have lost many type II cell markers and express type I cell markers. ATI-like cells were more sensitive to CSE than alveolar type II cells, which maintained their differentiated phenotype in vitro. We observed disruption of mitochondrial membrane potential, apoptosis and necrosis that were detected by double staining with acridine orange and ethidium bromide or Hoechst 33342 and propidium iodide and TUNEL assay after treatment with CSE. We also detected caspase 3 and caspase 7 activities and lipid peroxidation. CSE induced nuclear translocation of Nrf2 and increased expression of Nrf2, HO-1, Hsp70 and Fra1. Moreover, we found that Nrf2 knockdown sensitized ATI-like cells to CSE and Nrf2 overexpression provided protection against CSE-induced cell death. We also observed that two antioxidant compounds N-acetylcysteine and trolox protected ATI-like cells against injury by CSE.

Conclusions: Our study indicates that Nrf2 activation is a major factor in cellular defense of the human alveolar epithelium against CSE-induced toxicity and oxidative stress. Therefore, antioxidant agents that modulate Nrf2 would be expected to restore antioxidant and detoxifying enzymes and to prevent CS-related lung injury and perhaps lessen the development of emphysema.

Figure 1. CSE induces apoptosis and necrosis in ATI-like cells.

Panel I – Acridine orange and ethidium bromide double staining. A - Cells were analyzed after 4 h treatment with 5% CSE. Live cells are green, late apoptotic cells have chromatin condensation (yellow) and necrotic cells are red; B - Cells were treated with CSE for 4 h; C - Cells were exposed to CSE for 24 h. Data represent results from three independent experiments. Panel II - TUNEL assay. A - Chromatin condensation in cells exposed to 5% CSE for 24 h; B – Chromatin fragmentation after cell treatment with 5% CSE for 24 h; C - ATI-like cells were treated with CSE for 4 h and 24 h. Data represent results from three independent experiments (p<0.05). Panel III - Caspase 3 and caspase 7 activities. Cells were treated with CSE for 4 h and 24 h. Data are expressed in percentages of the control (p<0.05).

Figure 2. CSE produces 4-HNE in ATI-like cells.

A - Control; B - Cells exposed to 5% CSE for 24 h. Cells were probed by 4-HNE primary antibody, stained with Alexa Fluor 488 as secondary antibody (green) and mounted with Vectashield medium containing DAPI (blue). Shown are representative images from three independent experiments.

Figure 3. CSE induces nuclear localization of Nrf2 in ATI-like cells as detected by immunocytofluorescence.

A - Cytoplasmic localization of Nrf2 in control cells (red stain); B - Nuclear translocation of Nrf2 after 4 h treatment with 5% CSE. Representative data each from one of three experiments are shown.

Figure 4. NAC and trolox protect ATI-like cells treated with CSE.

Cells were exposed to CSE or co-treated with CSE and NAC or CSE and trolox for 24 h and toxicity was measured by propidium iodide and Hoechst 33342 double staining. A - Necrotic ATI-like cells after treatment with 5% CSE for 24 h. B – Co-treatment with CSE and 5 µM NAC or CSE with 5 µM trolox for 24 h decreases the percentage of necrotic cells in comparison with CSE alone. * - Statistically significant increase in percentage of necrotic cells induced by CSE, co-treatment with CSE and NAC or CSE and trolox in comparison with control, NAC or trolox, respectively. # - Statistically significant decrease of ATI-like necrotic cells after co-treatment with CSE and NAC or CSE and trolox in comparison with cell exposure to CSE. Data represent results from three independent experiments (p<0.05).

Figure 5. Protein expression after treatment with CSE and effect of co-treatment with NAC or Tolox.

Representative time and concentration course expression of proteins in ATI-like cells exposed to CSE and after co-treatment with CSE and 5 µM NAC or CSE and 5 µM trolox. Proteins were measured by immunoblotting. A - Cells treated for 4 h; B - Cells treated for 24 h; C - Cells were treated with CSE and NAC or trolox for 24 h. Representative data each from one of three experiments are shown.

Figure 6. Time and concentration course expression of proteins in ATII cells exposed to CSE.

A - for 4 h; B - for 24 h. Proteins were measured by immunoblotting. Representative data each from one of three experiments are shown.

Figure 7. Effect of Nrf2 knockdown and overexpression in ATI-like cells exposed to CSE.

A – Nrf2 expression (immunoblotting); Lane 1 – control; lane 2–300 nM NT siRNA; lane 3–300 nM Nrf2 siRNA; lane 4–200 MOI AdNrf2; lane 5–200 MOI AdGFP (see Materials and Methods); B - Quantitation of Nrf2 relative protein expression in ATI-like for experiments shown in A. * - Statistically significant increase in protein expression induced in comparison with negative control (p<0.05); C – Nrf2 knockdown sensitizes cells to CSE. Cells were transfected with Nrf2 siRNA for 24 h followed by exposure to CSE for 24 h and toxicity was measured by propidium iodide and Hoechst 33342 double staining. D - Nrf2 overexpression protects cells exposed to CSE. Cells were infected with AdNrf2 for 24 h, exposed to CSE for 24 h and necrotic cells were detected by Hoechst 33342 and propidium iodide double staining. * - Statistically significant increase in percentage of necrotic cells induced by CSE in comparison with control. # - Statistically significant increase for C or decrease in D in ATI-like necrotic cells after transfection with Nrf2 siRNA or infection with AdNrf2, respectively followed by CSE in comparison with CSE alone. Data represent results from three independent experiments (p<0.05).

Figure 8. NAC and trolox protect ATI-like cells with knocked down Nrf2 against injury induced by CSE.

Nrf2 was knocked down and cells were exposed for 24 h to CSE and NAC or trolox and toxicity was measured by propidium iodide and Hoechst 33342 double staining. * - Statistically significant increase in percentage of necrotic cells in comparison with control, NAC or trolox, respectively. # - Statistically significant decrease of ATI-like necrotic cells after co-treatment with CSE and NAC or CSE and trolox in comparison with cell exposure to CSE. Data represent results from three independent experiments (p<0.05).