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Cell and Tissue Research

, Volume 336, Issue 1, pp 91–105 | Cite as

Primary human coculture model of alveolo-capillary unit to study mechanisms of injury to peripheral lung

  • Maria Iris HermannsEmail author
  • Sabine Fuchs
  • Michaela Bock
  • Katja Wenzel
  • Eckhard Mayer
  • Kai Kehe
  • Fernando Bittinger
  • C. James Kirkpatrick
Regular Article

Abstract

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.

Keywords

Type II pneumocyte Microvascular endothelium Barrier Tight junctions Bilayer Lung injury Human 

Notes

Acknowledgements

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.

Supplementary material

441_2008_750_Fig1_ESM.gif (152 kb)
Supplementary Fig. 1

Morphological phenotype of freshly isolated HATII cells during mono- and coculture with HPMEC and localization of markers related to alveolar epithelial cell type I and type II (caveolin-1 and TTF-1, respectively) at the single cell level. HATII cells cultivated in mono- and coculture with HPMEC were fixed after 3, 5 and 7 days and paraffin-embedded sections were stained and analysed by fluorescence microscopy. The various markers studied are arranged as separate columns: caveolin-1 (red) in monoculture (a, c, e) and coculture (g, i, k) and caveolin-1 (red) with TTF-1 (green) in monoculture (b, d, f) and coculture (h, j, l). For the cultivated HATII cells, the intensity of the caveolin-1 signal increases with ongoing culture to reach a similar intensity to that of the HPMEC on day 7 of coculture (k). The cultivated HATII cells exhibit a persistent nuclear staining for TTF-1 until day 7 in monoculture (f) and coculture (l). Bars 10 μm (GIF 156 kb)

441_2008_750_Fig1_ESM.tif (3.3 mb)
High resolution image file (EPS 3474 kb)

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Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Maria Iris Hermanns
    • 1
    Email author
  • Sabine Fuchs
    • 1
  • Michaela Bock
    • 1
  • Katja Wenzel
    • 2
  • Eckhard Mayer
    • 3
  • Kai Kehe
    • 4
  • Fernando Bittinger
    • 1
  • C. James Kirkpatrick
    • 1
  1. 1.Institute of Pathology, Mainz University Clinic, Johannes Gutenberg UniversityMainzGermany
  2. 2.Rehab Hospital, AukammtalClinic of Conservative MedicineWiesbadenGermany
  3. 3.Department for Thoracic SurgeryCatholic Hospital MainzMainzGermany
  4. 4.Bundeswehr Institute of Pharmacology and ToxicologyMunichGermany

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