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


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.


Type 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.

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)


  1. Adamson I, Bowden D (1974) The type 2 cell as progenitor of alveolar epithelial regeneration. A cytodynamic study in mice after exposure to oxygen. Lab Invest 30:35–42PubMedGoogle Scholar
  2. Adamson I, Bowden D (1975) Derivation of type 1 epithelium from type 2 cells in the developing rat lung. Lab Invest 32:736–745PubMedGoogle Scholar
  3. Beck G, Yard B, Breedijk A, Van Ackern K, Van Der Woude F (1999) Release of CXC-chemokines by human lung microvascular endothelial cells (LMVEC) compared with macrovascular umbilical vein endothelial cells. Clin Exp Immunol 118:298–303PubMedCrossRefGoogle Scholar
  4. Bhaskaran M, Kolliputi N, Wang Y, Gou D, Chintagari NR, Liu L (2007) Trans-differentiation of alveolar epithelial type II cells to type I cells involves autocrine signaling by transforming growth factor beta1 through the Smad pathway. J Biol Chem 282:3968–3976PubMedCrossRefGoogle Scholar
  5. Burg J, Krump-Konvalinkova V, Bittinger F, Kirkpatrick CJ (2002) GM-CSF expression by human lung microvascular endothelial cells: in vitro and in vivo findings. Am J Physiol Lung Cell Mol Physiol 283:L460–L467PubMedGoogle Scholar
  6. Campbell L, Hollins AJ, Al-Eid A, Newman GR, Ruhland C von, Gumbleton M (1999) Caveolin-1 expression and caveolae biogenesis during cell transdifferentiation in lung alveolar epithelial primary cultures. Biochem Biophys Res Commun 262:744–751PubMedCrossRefGoogle Scholar
  7. Clegg GR, Tyrrell C, McKechnie SR, Beers MF, Harrison D, McElroy MC (2005) Coexpression of RTI40 with alveolar epithelial type II cell proteins in lungs following injury: identification of alveolar intermediate cell types. Am J Physiol Lung Cell Mol Physiol 289:L382–L390PubMedCrossRefGoogle Scholar
  8. Crapo J, Barry B, Gehr P, Bachofen M, Weibel E (1982) Cell number and cell characteristics of the normal human lung. Am Rev Respir Dis 126:332–337PubMedGoogle Scholar
  9. Demling N, Ehrhardt C, Kasper M, Laue M, Knels L, Rieber EP (2006) Promotion of cell adherence and spreading: a novel function of RAGE, the highly selective differentiation marker of human alveolar epithelial type I cells. Cell Tissue Res 323:475–488PubMedCrossRefGoogle Scholar
  10. Dudek SM, Garcia JGN (2001) Cytoskeletal regulation of pulmonary vascular permeability. J Appl Physiol 91:1487–1500PubMedGoogle Scholar
  11. Elbert K, Schafer U, Schafers H, Kim K, Lee V, Lehr C (1999) Monolayers of human alveolar epithelial cells in primary culture for pulmonary absorption and transport studies. Pharm Res 16:601–608PubMedCrossRefGoogle Scholar
  12. Evans MJ, Cabral LJ, Stephens RJ, Freeman G (1975) Transformation of alveolar type 2 cells to type 1 cells following exposure to NO2. Exp Mol Pathol 22:142–150PubMedCrossRefGoogle Scholar
  13. Fehrenbach H, Kasper M, Tschernig T, Shearman MS, Schuh D, Muller M (1998) Receptor for advanced glycation endproducts (RAGE) exhibits highly differential cellular and subcellular localisation in rat and human lung. Cell Mol Biol 44:1147–1157PubMedGoogle Scholar
  14. Fuchs S, Hollins A, Laue M, Schaefer U, Roemer K, Gumbleton M, Lehr C (2003) Differentiation of human alveolar epithelial cells in primary culture: morphological characterization and synthesis of caveolin-1 and surfactant protein-C. Cell Tissue Res 311:31–45PubMedCrossRefGoogle Scholar
  15. Hermanns MI, Unger RE, Kehe K, Peters K, Kirkpatrick CJ (2004) Lung epithelial cell lines in coculture with human pulmonary microvascular endothelial cells: development of an alveolo-capillary barrier in vitro. Lab Invest 84:736–752PubMedCrossRefGoogle Scholar
  16. Hewett PW, Murray JC (1996) Isolation of microvascular endothelial cells using magnetic beads coated with anti-PECAM-1 antibodies. In Vitro Cell Dev Biol Anim 32:462PubMedCrossRefGoogle Scholar
  17. Hyers T, Tricomi S, Dettenmeier P, Fowler A (1991) Tumor necrosis factor levels in serum and bronchoalveolar lavage fluid of patients with the adult respiratory distress syndrome. Am Rev Respir Dis 144:268–271PubMedGoogle Scholar
  18. Isakson BE, Seedorf GJ, Lubman RL, Boitano S (2002) Heterocellular cultures of pulmonary alveolar epithelial cells grown on laminin-5 supplemented matrix. In Vitro Cell Dev Biol Anim 38:443–449PubMedCrossRefGoogle Scholar
  19. Kasper M, Reimann T, Hempel U, Wenzel K, Bierhaus A, Schuh D, Dimmer V, Haroske G, Muller M (1998) Loss of caveolin expression in type I pneumocytes as an indicator of subcellular alterations during lung fibrogenesis. Histochem Cell Biol 109:41–48PubMedCrossRefGoogle Scholar
  20. Kasper M, Seidel D, Knels L, Morishima N, Neisser A, Bramke S, Koslowski R (2004) Early signs of lung fibrosis after in vitro treatment of rat lung slices with CdCl2 and TGF-beta1. Histochem Cell Biol 121:131–140PubMedCrossRefGoogle Scholar
  21. 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
  22. Kim K-J, Malik AB (2003) Protein transport across the lung epithelial barrier. Am J Physiol Lung Cell Mol Physiol 284:L247–L259PubMedGoogle Scholar
  23. Kreda SM, Gynn MC, Fenstermacher DA, Boucher RC, Gabriel SE (2001) Expression and localization of epithelial aquaporins in the adult human lung. Am J Respir Cell Mol Biol 24:224–234PubMedGoogle Scholar
  24. Leiner KA, Newman D, Li CM, Walsh E, Khosla J, Sannes PL (2006) Heparin and fibroblast growth factors affect surfactant protein gene expression in type II cells. Am J Respir Cell Mol Biol 35:611–618PubMedCrossRefGoogle Scholar
  25. Mason RJ, Walker SR, Shields BA, Henson JE, Williams MC (1985) Identification of rat alveolar type II epithelial cells with a tannic acid and polychrome stain. Am Rev Respir Dis 131:786–788PubMedGoogle Scholar
  26. Matthay MA, Folkesson HG, Clerici C (2002) Lung epithelial fluid transport and the resolution of pulmonary edema. Physiol Rev 82:569–600PubMedGoogle Scholar
  27. Muller AM, Hermanns MI, Cronen C, Kirkpatrick CJ (2002) Comparative study of adhesion molecule expression in cultured human macro- and microvascular endothelial cells. Exp Mol Pathol 73:171–180PubMedCrossRefGoogle Scholar
  28. Murphy S, Dinsdale D, Hoet P, Nemery B, Richards R (1999) A comparative study of the isolation of type II epithelial cells from rat, hamster, pig and human lung tissue. Methods Cell Sci 21:31–38PubMedCrossRefGoogle Scholar
  29. Olsen CO, Isakson BE, Seedorf GJ, Lubman RL, Boitano S (2005) Extracellular matrix-driven alveolar epithelial cell differentiation in vitro. Exp Lung Res 31:461–482PubMedCrossRefGoogle Scholar
  30. Paine R, Rolfe M, Standiford T, Burdick M, Rollins B, Strieter R (1993) MCP-1 expression by rat type II alveolar epithelial cells in primary culture. J Immunol 150:4561–4570PubMedGoogle Scholar
  31. Planus E, Galiacy S, Matthay M, Laurent V, Gavrilovic J, Murphy G, Clerici C, Isabey D, Lafuma C, d’Ortho M (1999) Role of collagenase in mediating in vitro alveolar epithelial wound repair. J Cell Sci 112:243–252PubMedGoogle Scholar
  32. Prabhakar U, Eirikis E, Davis HM (2002) Simultaneous quantification of proinflammatory cytokines in human plasma using the LabMAP assay. J Immunol Methods 260:207–218PubMedCrossRefGoogle Scholar
  33. Ryan US (1986) Metabolic activity of pulmonary endothelium: modulations of structure and function. Annu Rev Physiol 48:263–277PubMedCrossRefGoogle Scholar
  34. Sannes PL, Khosla J, Li CM, Pagan I (1998) Sulfation of extracellular matrices modifies growth factor effects on type II cells on laminin substrata. Am J Physiol 275:L701–L708PubMedGoogle Scholar
  35. Steimer A, Laue M, Franke H, Haltner-Ukomado E, Lehr CM (2006) Porcine alveolar epithelial cells in primary culture: morphological, bioelectrical and immunocytochemical characterization. Pharm Res 23:2078–2093PubMedCrossRefGoogle Scholar
  36. Strunk R, Eidlen D, Mason R (1988) Pulmonary alveolar type II epithelial cells synthesize and secrete proteins of the classical and alternative complement pathways. J Clin Invest 81:1419–1426PubMedCrossRefGoogle Scholar
  37. 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
  38. Uhal BD (1997) Cell cycle kinetics in the alveolar epithelium. Am J Physiol Lung Cell Mol Physiol 272:L1031–L1045Google Scholar
  39. Vanderbilt JN, Mager EM, Allen L, Sawa T, Wiener-Kronish J, Gonzalez R, Dobbs LG (2003) CXC chemokines and their receptors are expressed in type II cells and upregulated following lung injury. Am J Respir Cell Mol Biol 29:661–668PubMedCrossRefGoogle Scholar
  40. Verkman AS, Matthay MA, Song Y (2000) Aquaporin water channels and lung physiology. Am J Physiol Lung Cell Mol Physiol 278:L867–L879PubMedGoogle Scholar
  41. Ware LB, Matthay MA (2000) The acute respiratory distress syndrome. N Engl J Med 342:1334–1349PubMedCrossRefGoogle Scholar
  42. Witherden IR, Vanden Bon EJ, Goldstraw P, Ratcliffe C, Pastorino U, Tetley TD (2004) Primary human alveolar type II epithelial cell chemokine release: effects of cigarette smoke and neutrophil elastase. Am J Respir Cell Mol Biol 30:500–509PubMedCrossRefGoogle Scholar
  43. Zhao X, Alexander JS, Zhang S, Zhu Y, Sieber NJ, Aw TY, Carden DL (2001) Redox regulation of endothelial barrier integrity. Am J Physiol Lung Cell Mol Physiol 281:L879–L886PubMedGoogle Scholar

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

Personalised recommendations