Immunological responses of lung cells to brevetoxin-2 using in vitro and in vivo approaches

Naturally occurring red tides are present virtually annually in the Gulf of Mexico. These red tides are coupled with the production of brevetoxins, which are believed to have an effect on immune cells and respiratory-related functions following inhalation. Here, I show that exposure to an environmentally relevant dose of brevetoxin (PbTx)-2, the primary toxin aerosolized, results in the initiation of an acute inflammatory response in the lung. In vitro and in vivo approaches were used to determine the effects of PbTx-2 in the lung. In vitro exposure of mouse alveolar macrophages (MH-S cell line) or mouse alveolar epithelial type II cells (MLE-15 cell line) to 0.5 mug/ml PbTx-2 did not alter cell growth. Treatment of MH-S cells with 0.5 mug/ml PbTx-2 for 6 hr resulted in decreased secretion of interleukin (IL)-5, increased secretion of IL-2, IL-4, and tumor necrosis factor-alpha, and an increase in phagocytosis. Few (<0.3%) changes in MH-S cell gene expression were observed. Treatment of MLE-15 cells for 24 hr with 0.5-2 mug/ml PbTx-2 resulted in decreased secretion of surfactant protein-A in a dose-dependent manner. In vivo aspiration challenge of C57BL/6J mice with 10 or 20 mug/kg PbTx-2 resulted in the pathological representation of acute inflammation in the lung. Hematoxylin and eosin staining of lung tissue from mice challenged with 10 or 20 mug/kg PbTx-2 revealed areas of cellular infiltration and edema, as well as areas of airway and airspace collapse. Immunohistochemical staining identified an increase in infiltrating neutrophils, macrophages, and B-cells in lung tissues, while lavage fluid recovered from mice challenged with 10 mug/kg PbTx-2 contained significantly more cells than from control mice. The results presented here from the in vitro and in vivo challenges show that acute exposure to PbTx-2 results in the initiation of a non-specific innate inflammatory response in the lung that can be mediated by alveolar macrophages. This is the first study providing direct evidence that lung-only exposure of brevetoxin results in the pathological expression of inflammation.