Primary human coculture model of alveolo-capillary unit to study mechanisms of injury to peripheral lung
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In order to delineate individual pathomechanisms in acute lung injury and pulmonary toxicology, we developed a primary coculture system to simulate the human alveolo-capillary barrier. Human pulmonary microvascular endothelial cells (HPMEC) were cocultivated with primary isolated human type II alveolar epithelial cells (HATII) on opposite sides of a permeable filter support, thereby constituting a bilayer. Within 7–11 days of coculture, the HATII cells partly transdifferentiated to type-I-like (HATI-like) cells, as demonstrated by morphological changes from a cuboidal to a flattened morphology, the loss of HATII-cell-specific organelles and the increase of HATI-cell-related markers (caveolin-1, aquaporin-5, receptor for advanced glycation end-products). Immunofluorescent analysis detected type-II-like and type-I-like alveolar epithelial cells mimicking the heterocellular composition of alveolar epithelium in vivo. The heterocellular epithelial monolayer showed a circumferential staining of tight-junctional (ZO-1, occludin) and adherens-junctional (E-cadherin, β-catenin) proteins. HPMEC on the opposite side also developed tight and adherens junctions (VE-cadherin, β-catenin). Under integral barrier properties, exposure to the proinflammatory cytokine tumour necrosis factor-α from either the endothelial (basolateral) or the epithelial (apical) side caused a largely compartmentalized release of the chemokines interleukin-8 and monocyte chemoattractant protein-1. Thus, the established coculture provides a suitable in vitro model to examine barrier function at the distal lung, including the interaction of microvascular endothelial cells with ATII-like and ATI-like epithelial cells. The compartmentalization of the barrier-forming bilayer also allows mechanisms of lung injury to be studied in both the epithelial (intra-alveolar) and the endothelial (intravascular) compartments.
KeywordsType II pneumocyte Microvascular endothelium Barrier Tight junctions Bilayer Lung injury Human
The authors thank Mrs A. Sartoris, L. Meyer, M. Müller and K. Molter for their excellent assistance with the cell culture and immunocytochemical and transmission electron microscopy studies.
- Kelly JJ, Moore TM, Babal P, Diwan AH, Stevens T, Thompson WJ (1998) Pulmonary microvascular and macrovascular endothelial cells: differential regulation of Ca2+ and permeability. Am J Physiol Lung Cell Mol Physiol 274:L810–L819Google Scholar
- Suter PM, Suter S, Girardin E, Roux-Lombard P, Grau GE, Dayer JM (1992) High bronchoalveolar levels of tumor necrosis factor and its inhibitors, interleukin-1, interferon, and elastase, in patients with adult respiratory distress syndrome after trauma, shock, or sepsis. Am Rev Respir Dis 145:1016–1022PubMedGoogle Scholar
- Uhal BD (1997) Cell cycle kinetics in the alveolar epithelium. Am J Physiol Lung Cell Mol Physiol 272:L1031–L1045Google Scholar