Acute lung injury: apoptosis in effector and target cells of the upper and lower airway compartment

Abstract

Apoptotic cell death has been considered an underlying mechanism in acute lung injury. To evaluate the evidence of this process, apoptosis rate was determined in effector cells (alveolar macrophages, neutrophils) and target cells (tracheobronchial and alveolar epithelial cells) of the respiratory compartment upon exposure to hypoxia and endotoxin stimulation in vitro. Cells were exposed to 5% oxygen or incubated with lipopolysaccharide (LPS) for 4, 8 and 24 h, and activity of caspase-3, -8 and -9 was determined. Caspase-3 of alveolar macrophages was increased at all three time-points upon LPS stimulation, while hypoxia did not affect apoptosis rate at early time-points. In neutrophils, apoptosis was decreased in an early phase of hypoxia at 4 h. However, enhanced expression of caspase-3 activity was seen at 8 and 24 h. In the presence of LPS a decreased apoptosis rate was observed at 8 h compared to controls, while it was increased at 24 h. Tracheobronchial as well as alveolar epithelial cells experienced an enhanced caspase-3 activity upon LPS stimulation with no change of apoptosis rate under hypoxia. While increased apoptosis rate is triggered through an intrinsic and extrinsic pathway in alveolar macrophages, intrinsic signalling is activated in tracheobronchial epithelial cells. The exact pathway pattern in neutrophils and alveolar epithelial cells could not be determined. These data clearly demonstrate that upon injury each cell type experiences its own apoptosis pattern. Further experiments need to be performed to determine the functional role of these apoptotic processes in acute lung injury.

Fig. 1

Determination of apoptosis rate in alveolar macrophages. Caspase-3 (3), -8 (8) and -9 (9) activity was measured in control cells (co) after exposure to 5% oxygen (hyp) or exposure to 20 µg/ml lipopolysaccharide (LPS) for 4 h (a), 8 h (b) and 24 h (c). Camptothecin was used as a positive control (pos co) for apoptosis. Values are median with interquartile range from five experiments. *P < 0·05 between control and injured cells.

Fig. 2

Determination of apoptosis rate in neutrophils. Caspase-3 (3), -8 (8) and -9 (9) activity was measured in control cells (co) after exposure to 5% oxygen (hyp) or exposure to 20 µg/ml lipopolysaccharide (LPS) for 4 h (a), 8 h (b) and 24 h (c). Camptothecin was used as a positive control (pos co) for apoptosis. Values are median with interquartile range from five experiments. *P < 0·05 between control and injured cells.

Fig. 3

Determination of apoptosis rate in tracheobronchial epithelial cells. Caspase-3 (3), -8 (8) and -9 (9) activity was measured in control cells (co) after exposure to 5% oxygen (hyp) or exposure to 20 µg/ml lipopolysaccharide (LPS) for 4 h (a), 8 h (b) and 24 h (c). Camptothecin was used as a positive control (pos co) for apoptosis. Values are median with interquartile range from five experiments. *P < 0·05 between control and injured cells.

Fig. 4

Determination of apoptosis rate in alveolar epithelial cells. Caspase-3 (3), -8 (8) and -9 (9) activity was measured in control cells (co) after exposure to 5% oxygen (hyp) or exposure to 20 µg/ml lipopolysaccharide (LPS) for 4 h (a), 8 h (b) and 24 h (c). Camptothecin was used as a positive control (pos co) for apoptosis. Values are median with interquartile range from five experiments. *P < 0·05 between control and injured cells.

Fig. 5

Flow cytometric analysis of annexin V and 7-amino-actinomycin D (7-AAD) staining of neutrophils after a 4 h exposure to 5% oxygen or exposure to 20 µg/ml lipopolysaccharide (LPS). Camptothecin was used as a positive control for apoptosis. (a) Blot from a typical example (four experiments were performed). (b) Results from four experiments summarized as box-plot. *P < 0·05 between control and injured cells.