Endotoxin-induced lung alveolar cell injury causes brain cell damage

Abstract

Sepsis is the most common cause of acute respiratory distress syndrome, a severe lung inflammatory disorder with an elevated morbidity and mortality. Sepsis and acute respiratory distress syndrome involve the release of inflammatory mediators to the systemic circulation, propagating the cellular and molecular response and affecting distal organs, including the brain. Since it has been reported that sepsis and acute respiratory distress syndrome contribute to brain dysfunction, we investigated the brain-lung crosstalk using a combined experimental in vitro airway epithelial and brain cell injury model. Conditioned medium collected from an in vitro lipopolysaccharide-induced airway epithelial cell injury model using human A549 alveolar cells was subsequently added at increasing concentrations (no conditioned, 2%, 5%, 10%, 15%, 25%, and 50%) to a rat mixed brain cell culture containing both astrocytes and neurons. Samples from culture media and cells from mixed brain cultures were collected before treatment, and at 6 and 24 h for analysis. Conditioned medium at 15% significantly increased apoptosis in brain cell cultures 24 h after treatment, whereas 25% and 50% significantly increased both necrosis and apoptosis. Levels of brain damage markers S100 calcium binding protein B and neuron-specific enolase, interleukin-6, macrophage inflammatory protein-2, as well as matrix metalloproteinase-9 increased significantly after treating brain cells with ≥2% conditioned medium. Our findings demonstrated that human epithelial pulmonary cells stimulated with bacterial lipopolysaccharide release inflammatory mediators that are able to induce a translational clinically relevant and harmful response in brain cells. These results support a brain-lung crosstalk during sepsis and sepsis-induced acute respiratory distress syndrome.

Keywords: Sepsis; acute respiratory distress syndrome; apoptosis; brain injury; inflammation; lung injury.

Figure 1

Morphological changes and death in rat brain cells exposed to A549 conditioned media. Phase-contrast microscopic observation (×20) of primary rat mixed cortical cultures 24 h after treatment with 2% (b), 5% (c), 15% (d), 25% (e), or 50% (f) A539 conditioned medium as well as controls (a). Thick arrows show normal viable neuronal bodies while thin arrows show round damaged or dead cells. See text for details. (A color version of this figure is available in the online journal.)

Figure 2

Effects of A549 conditioned medium on brain cells viability at 24 h. (a) Trypan blue assay, (b) effects on lactate dehydrogenase (LDH) release, (c) effects on caspase-3 activity. *P < 0.05, **P < 0.005 compared to controls. n = 9

Figure 3

Effects of A549 conditioned medium on S100B levels in neural cells at 6 h (a) and at 24 h (b). *P < 0.05, **P < 0.005, ***P < 0.001 compared to controls. n = 9

Figure 4

Effects of A549 conditioned medium on neuron-specific enolase (NSE) levels in brain cells at 6 h (a) and at 24 h (b). *P < 0.05, **P < 0.005, ***P < 0.001 compared to controls. n = 9