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Journal of tissue culture methods

, Volume 14, Issue 4, pp 209–215 | Cite as

A simple approach to measurement of electrical parameters of cultured epithelial monolayers: Use in assessing neutrophil-epithelial interactions

  • James L. Madara
  • Sean Colgan
  • Asma Nusrat
  • Charleen Delp
  • Charles Parkos
Article

Summary

Typically, electrophysiologic studies of epithelial monolayers are either performed in formalized Ussing chamber systems which yield highly accurate results or in simple setups using recording devices which have limitations in accuracy or the range of measurements which can be performed or both. Here we detail a simple method of interfacing traditionally accurate Ussing chambers systems with commercially available filter supports on which epithelial monolayers can be grown. We also detail simple methods for growing inverted monolayers, suitable for electrophysiologic assays, for use in studies where cells or particles must be layered by gravity on the undersurface (basolateral pole) of the filter. Both approaches allow experiments to be performed on large numbers of monolayers synchronously. As an example of the use of this system, we analyze the sequelae of neutrophil migration across monolayers of the intestinal cell line T84. Neutrophil migration across monolayers can occur in either direction, is dependent on neutrophil surface β2 integrins, and is paralleled by a decrease in epithelial barrier function as detected electrically. We have previously shown in formal Ussing chamber studies that neutrophil-epithelial interactions elicit a modest short-circuit current indicative of electrogenic ion transport. We show here that this short-circuit current response can be readily detected using the simple approach described.

Key words

tight junction neutrophil integrin epithelium inflammation transmigration 

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References

  1. 1.
    Arnaout, M. A. Leukocyte adhesion molecule deficiency: its structural basis, pathophysiology and implications for modulating the inflammatory response. Immunol. Rev. 14:145–180; 1990.Google Scholar
  2. 2.
    Cereijido, M.; Sabatini, D. D. Polarized monolayers formed by epithelial cells on a permeable and translucent support. J. Cell Biol. 77:853–876; 1978.PubMedGoogle Scholar
  3. 3.
    Dharmsathaphorn, K.; McRoberts, J. A.; Mandel, K. G., et al. A human colonic tumor cell line that maintains vectorial electrolyte transport. Am. J. Physiol. 246:G204-G208; 1984.PubMedGoogle Scholar
  4. 4.
    Dharmsathaphorn, K.; Madara, J. L. Established intestinal cell lines as model systems for electrolyte transport studies. Methods Enzymol. 192:354–389; 1990.Google Scholar
  5. 5.
    Gallin, J. I.; Fauci, A. S., editors, Advances in host defense mechanisms. Chronic granulomatous disease, vol. 3. New York: Raven Press; 1982.Google Scholar
  6. 6.
    Kumar, N. B.; Nostrant, T. T.; Appleman, H. D. The histologic spectrum of acute self limited colitis (acute infectious-type colitis). Am. J. Surg. Pathol. 6:523–529; 1982.PubMedGoogle Scholar
  7. 7.
    Madara, J. L.; Dharmsathaphorn, K. Occluding junctions structure-function relationships in a cultured epithelial monolayer. J. Cell Biol. 101:2124–2133; 1987.Google Scholar
  8. 8.
    Milks, L. C.; Brontoli, M. J.; Cramer, E. B. Epithelial permeability and the transepithelial migration of human neutrophils. J. Cell Biol. 96:1241–1247; 1987.Google Scholar
  9. 9.
    Murakami, H.; Masui, H. Hormonal control of human colon carcinoma cell growth in serum free medium. Proc. Natl. Acad. Sci. USA 77:3464–3468; 1980.PubMedGoogle Scholar
  10. 10.
    Nash, D.; Stafford, J.; Madara, J. L. Effects of polymorphonuclear leukocyte transmigration on the barrier function of cultured intestinal epithelial monolayers. J. Clin. Invest. 80:1104–1113; 1987.PubMedGoogle Scholar
  11. 11.
    Nash, S.; Stafford, J.; Madara, J. L. The selective and superoxide-independent disruption of intestinal epithelial tight junction during leukocyte transmigration. Lab. Invest. 59:531–537; 1988.PubMedGoogle Scholar
  12. 12.
    Nash, S.; Parkos, C. A.; Nusrat, A., et al. In vitro model of intestinal crypt abscess: a novel neutrophil-derived secretagogue activity. J. Clin. Invest. 87:1474–1477; 1991.PubMedGoogle Scholar
  13. 13.
    Omann, G. M.; Allan, R. A.; Bokoch, G. M., et al. Signal transduction and cytoskeletal activation in the neutrophil. Physiol. Rev. 67:285–322; 1991.Google Scholar
  14. 14.
    Parkos, C. A.; Delp, C.; Arnaout, A., et al. Neutrophil migration across a cultured intestinal epithelium: dependence on a CD11b/CD18 mediated event and enhanced efficiency in the physiological direction. J. Clin. Invest. 88:1605–1612; 1992.Google Scholar
  15. 15.
    Shapiro, M.; Matthews, J.; Hecht, G., et al. Stabilization of f-actin prevents cAMP-elicited Cl secretion in T84 cells. J. Clin. Invest. 87:1905–1909; 1991.Google Scholar
  16. 16.
    Yardley, J. H. Pathology of idiopathic inflammation bowel disease and relevance of specific cell findings: an overview. In: Recent developments in the therapy of inflammatory bowel disease. Proceedings of a symposium. Baltimore; Myerhoff Center for Digestive Disease at Johns Hopkins; 1986:3–9.Google Scholar

Copyright information

© Tissue Culture Association 1992

Authors and Affiliations

  • James L. Madara
    • 1
  • Sean Colgan
    • 1
  • Asma Nusrat
    • 1
  • Charleen Delp
    • 1
  • Charles Parkos
    • 1
  1. 1.Departments of Pathology, Brigham and Women's Hospital and Harvard Medical School, and the Harvard Digestive Diseases CenterBoston

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