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

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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.

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References

  • 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 30:35–42

    PubMed  CAS  Google Scholar 

  • Adamson I, Bowden D (1975) Derivation of type 1 epithelium from type 2 cells in the developing rat lung. Lab Invest 32:736–745

    PubMed  CAS  Google Scholar 

  • 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 118:298–303

    Article  PubMed  CAS  Google Scholar 

  • 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 282:3968–3976

    Article  PubMed  CAS  Google Scholar 

  • 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–L467

    PubMed  CAS  Google Scholar 

  • 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 262:744–751

    Article  PubMed  CAS  Google Scholar 

  • 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–L390

    Article  PubMed  CAS  Google Scholar 

  • 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 126:332–337

    PubMed  CAS  Google Scholar 

  • 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 323:475–488

    Article  PubMed  CAS  Google Scholar 

  • Dudek SM, Garcia JGN (2001) Cytoskeletal regulation of pulmonary vascular permeability. J Appl Physiol 91:1487–1500

    PubMed  CAS  Google Scholar 

  • 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 16:601–608

    Article  PubMed  CAS  Google Scholar 

  • 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 22:142–150

    Article  PubMed  CAS  Google Scholar 

  • 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 44:1147–1157

    PubMed  CAS  Google Scholar 

  • 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 311:31–45

    Article  PubMed  Google Scholar 

  • 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 84:736–752

    Article  PubMed  CAS  Google Scholar 

  • 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 32:462

    Article  PubMed  CAS  Google Scholar 

  • 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 144:268–271

    PubMed  CAS  Google Scholar 

  • 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 38:443–449

    Article  PubMed  Google Scholar 

  • 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 109:41–48

    Article  PubMed  CAS  Google Scholar 

  • 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 121:131–140

    Article  PubMed  CAS  Google Scholar 

  • 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–L819

    CAS  Google Scholar 

  • Kim K-J, Malik AB (2003) Protein transport across the lung epithelial barrier. Am J Physiol Lung Cell Mol Physiol 284:L247–L259

    PubMed  CAS  Google Scholar 

  • 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 24:224–234

    PubMed  CAS  Google Scholar 

  • 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 35:611–618

    Article  PubMed  CAS  Google Scholar 

  • 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 131:786–788

    PubMed  CAS  Google Scholar 

  • Matthay MA, Folkesson HG, Clerici C (2002) Lung epithelial fluid transport and the resolution of pulmonary edema. Physiol Rev 82:569–600

    PubMed  CAS  Google Scholar 

  • 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 73:171–180

    Article  PubMed  CAS  Google Scholar 

  • 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 21:31–38

    Article  PubMed  CAS  Google Scholar 

  • Olsen CO, Isakson BE, Seedorf GJ, Lubman RL, Boitano S (2005) Extracellular matrix-driven alveolar epithelial cell differentiation in vitro. Exp Lung Res 31:461–482

    Article  PubMed  CAS  Google Scholar 

  • 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 150:4561–4570

    PubMed  CAS  Google Scholar 

  • 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 112:243–252

    PubMed  CAS  Google Scholar 

  • Prabhakar U, Eirikis E, Davis HM (2002) Simultaneous quantification of proinflammatory cytokines in human plasma using the LabMAP assay. J Immunol Methods 260:207–218

    Article  PubMed  CAS  Google Scholar 

  • Ryan US (1986) Metabolic activity of pulmonary endothelium: modulations of structure and function. Annu Rev Physiol 48:263–277

    Article  PubMed  CAS  Google Scholar 

  • 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–L708

    PubMed  CAS  Google Scholar 

  • 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 23:2078–2093

    Article  PubMed  CAS  Google Scholar 

  • 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 81:1419–1426

    Article  PubMed  CAS  Google 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 145:1016–1022

    PubMed  CAS  Google Scholar 

  • Uhal BD (1997) Cell cycle kinetics in the alveolar epithelium. Am J Physiol Lung Cell Mol Physiol 272:L1031–L1045

    CAS  Google Scholar 

  • 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 29:661–668

    Article  PubMed  CAS  Google Scholar 

  • Verkman AS, Matthay MA, Song Y (2000) Aquaporin water channels and lung physiology. Am J Physiol Lung Cell Mol Physiol 278:L867–L879

    PubMed  CAS  Google Scholar 

  • Ware LB, Matthay MA (2000) The acute respiratory distress syndrome. N Engl J Med 342:1334–1349

    Article  PubMed  CAS  Google Scholar 

  • 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 30:500–509

    Article  PubMed  CAS  Google Scholar 

  • 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–L886

    PubMed  CAS  Google Scholar 

Download references

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.

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Correspondence to Maria Iris Hermanns.

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This study was supported by the BMVg Grant E/B41G/1G302/1A402 and the 6th Framework program of the European Union, NanoBioPharmaceutics.

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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)

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Hermanns, M.I., Fuchs, S., Bock, M. et al. Primary human coculture model of alveolo-capillary unit to study mechanisms of injury to peripheral lung. Cell Tissue Res 336, 91–105 (2009). https://doi.org/10.1007/s00441-008-0750-1

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  • DOI: https://doi.org/10.1007/s00441-008-0750-1

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