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Infection and Immunity, February 2005, p. 1129-1140, Vol. 73, No. 2
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.2.1129-1140.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

A549 Lung Epithelial Cells Grown as Three-Dimensional Aggregates: Alternative Tissue Culture Model for Pseudomonas aeruginosa Pathogenesis

A. J. Carterson,1,2 K. Höner zu Bentrup,1 C. M. Ott,3 M. S. Clarke,4,{dagger} D. L. Pierson,5 C. R. Vanderburg,6 K. L. Buchanan,1 C. A. Nickerson,1 and M. J. Schurr1,2*

Department of Microbiology and Immunology, Program in Molecular Pathogenesis and Immunity, Tulane Center of Excellence in Bioengineering, Tulane University Health Sciences Center,1 Louisiana Center for Lung Biology and Immunotherapy New Orleans, Louisiana,2 EASI/Wyle Laboratories,3 Division of Space Life Sciences, Universities Space Research Association,4 Habitability and Environmental Factors Office, NASA Johnson Space Center Houston, Texas,5 Massachusetts General Hospital, Boston, Massachusetts6

Received 12 July 2004/ Returned for modification 24 August 2004/ Accepted 8 October 2004

A three-dimensional (3-D) lung aggregate model was developed from A549 human lung epithelial cells by using a rotating-wall vessel bioreactor to study the interactions between Pseudomonas aeruginosa and lung epithelial cells. The suitability of the 3-D aggregates as an infection model was examined by immunohistochemistry, adherence and invasion assays, scanning electron microscopy, and cytokine and mucoglycoprotein production. Immunohistochemical characterization of the 3-D A549 aggregates showed increased expression of epithelial cell-specific markers and decreased expression of cancer-specific markers compared to their monolayer counterparts. Immunohistochemistry of junctional markers on A549 3-D cells revealed that these cells formed tight junctions and polarity, in contrast to the cells grown as monolayers. Additionally, the 3-D aggregates stained positively for the production of mucoglycoprotein while the monolayers showed no indication of staining. Moreover, mucin-specific antibodies to MUC1 and MUC5A bound with greater affinity to 3-D aggregates than to the monolayers. P. aeruginosa attached to and penetrated A549 monolayers significantly more than the same cells grown as 3-D aggregates. Scanning electron microscopy of A549 cells grown as monolayers and 3-D aggregates infected with P. aeruginosa showed that monolayers detached from the surface of the culture plate postinfection, in contrast to the 3-D aggregates, which remained attached to the microcarrier beads. In response to infection, proinflammatory cytokine levels were elevated for the 3-D A549 aggregates compared to monolayer controls. These findings suggest that A549 lung cells grown as 3-D aggregates may represent a more physiologically relevant model to examine the interactions between P. aeruginosa and the lung epithelium during infection.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Program in Molecular Pathogenesis and Immunity, Tulane University Health Sciences Center, 1430 Tulane Ave., New Orleans, LA 70112. Phone: (504) 988-4607. Fax: (504) 588-5144. E-mail: mschurr{at}tulane.edu.

Editor: J. N. Weiser

{dagger} Present address: Laboratory of Integrated Physiology, Health and Human Performance, University of Houston, Houston, TX 77204-6015.

Infection and Immunity, February 2005, p. 1129-1140, Vol. 73, No. 2
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.2.1129-1140.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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