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Immortalization of Human Alveolar Epithelial Cells to Investigate Nanoparticle Uptake

¹ National Heart and Lung Institute, and ² Division of Medicine, Imperial College, London, United Kingdom; ³ Department of Surgery, University College London, London, United Kingdom; ⁴ Division of Clinical Chemistry and Biochemistry, University Children's Hospital Zurich, Zurich, Switzerland; and ⁵ Department of Thoracic Surgery, Royal Brompton and Harefield NHS Trust, London, United Kingdom

Correspondence and requests for reprints should be addressed to Teresa D. Tetley, BSc, PhD, Lung Cell Biology, National Heart and Lung Institute, Imperial College, London SW3 6LY, UK. E-mail: t.tetley{at}imperial.ac.uk

Primary human alveolar type 2 (AT2) cells were immortalized by transduction with the catalytic subunit of telomerase and simian virus 40 large-tumor antigen. Characterization by immunochemical and morphologic methods demonstrated an AT1-like cell phenotype. Unlike primary AT2 cells, immortalized cells no longer expressed alkaline phosphatase, pro–surfactant protein C, and thyroid transcription factor-1, but expressed increased caveolin-1 and receptor for advanced glycation end products (RAGE). Live cell imaging using scanning ion conductance microscopy showed that the cuboidal primary AT2 cells were approximately 15 µm and enriched with surface microvilli, while the immortal AT1 cells were attenuated more than 40 µm, resembling these cells in situ. Transmission electron microscopy highlighted the attenuated morphology and showed endosomal vesicles in some immortal AT1 cells (but not primary AT2 cells) as found in situ. Particulate air pollution exacerbates cardiopulmonary disease. Interaction of ultrafine, nano-sized particles with the alveolar epithelium and/or translocation into the cardiovasculature may be a contributory factor. We hypothesized differential uptake of nanoparticles by AT1 and AT2 cells, depending on particle size and surface charge. Uptake of 50-nm and 1-µm fluorescent latex particles was investigated using confocal microscopy and scanning surface confocal microscopy of live cells. Fewer than 10% of primary AT2 cells internalized particles. In contrast, 75% immortal AT1 cells internalized negatively charged particles, while less than 55% of these cells internalized positively charged particles; charge, rather than size, mattered. The process was rapid: one-third of the total cell-associated negatively charged 50-nm particle fluorescence measured at 24 hours was internalized during the first hour. AT1 cells could be important in translocation of particles from the lung into the circulation.

Key Words: alveolar • epithelial • immortalization • nanoparticle • translocation

CLINICAL RELEVANCE Immortal, human epithelial alveolar type (AT) 1 cells (not primary human AT2 cells) avidly internalized nanoparticles. These cells account for 95% of the alveolar surface area, and such interactions may contribute to the effects of ultrafine air pollution particles.