Endocytosis of ultrafine particles by A549 cells

Alveolar epithelium's capacity to ingest inhaled ultrafine particles is not well characterized. The objectives of this study were to use an in vitro model of type II lung epithelium and evaluate the cells' ability to take up ultrafine particles (titanium dioxide [TiO2], 50 nm diameter). The human epithelial cell line A549 was grown on aclar substrates and exposed to 40 mug/ml TiO2 particles for 3, 6, and 24 h before imaging with energy-filtering transmission electron microscopy. Elemental mapping and electron energy loss spectroscopy were used to colocalize Ti/O with electron-dense particles. Particle endocytosis was compared in A549 cells with and without pretreatment with cytochalasin D (cyto a) (2 mug/ml). After 3 h of TiO2 exposure, cells internalized aggregates of the ultrafine particles which were observed in cytosolic, membrane-bound vacuoles. After 24 h of exposure there were considerably more intracellular aggregates of membrane-bound particles, and aggregated particles were also enmeshed in loosely and tightly packed lamellar bodies. Throughout 24 h of exposure a preponderance of particles remained associated with the free surface of the cells and were not internalized. The majority of membrane-bound vacuoles contained aggregates of particles and only occasionally did they contain as few as two or three particles, despite the use of several different approaches to assure the possibility for individual particles to be ingested and detected. There was morphologic evidence of microfilament disturbance, but no evidence of a decrease in internalized particles in cells pretreated with cyto D. Thus, this model of type II epithelium is able to internalize aggregates of ultrafine particles.