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Infection and Immunity, October 2000, p. 5635-5644, Vol. 68, No. 10
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Transforming Growth Factor 1 Ameliorates Intestinal Epithelial Barrier Disruption by Cryptosporidium parvum In Vitro in the Absence of Mucosal T Lymphocytes

James K. Roche,^1,* Clovis A. P. Martins,¹ Rosana Cosme,¹ Ronald Fayer,² and Richard L. Guerrant¹

Divisions of Gastroenterology and of Geographic and International Medicine, Department of Medicine, University of Virginia Health System, Charlottesville, Virginia,¹ and Department of Agriculture, Beltsville, Maryland²

Received 9 February 2000/Returned for modification 13 April 2000/Accepted 30 June 2000

Exposure to oocysts of the protozoan Cryptosporidium parvum causes intestinal epithelial cell dysfunction in vivo and in vitro, but effective means by which mucosal injury might be prevented remain unclear. We examined the ability of transforming growth factor 1 (TGF-1)a cytokine synthesized and released by cells in the intestineto preserve the barrier function of human colonic epithelia when challenged with C. parvum oocysts and then studied the mechanisms involved. Epithelial barrier function was monitored electrophysiologically, receptors for TGF-1 were localized by confocal microscopy, and TGF-1-induced protein kinase C activation was detected intracellularly by translocation of its  isozyme. TGF-1 alone enhanced intestinal epithelial barrier function, while exposure to C. parvum oocysts (10⁵/monolayer) markedly reduced barrier function to 40% of that of the control. When epithelial monolayers were pretreated with TGF-1 at 5.0 ng/ml, the barrier-disrupting effect of C. parvum oocysts was almost completely abrogated for 96 h. Further investigation showed that (i) the RI and RII receptors for TGF-1 were present on 55 and 65% of human epithelial cell line cells, respectively, over a 1-log-unit range of receptor protein expression, as shown by flow cytometry and confirmed by confocal microscopy; (ii) only basolateral and not apical TGF-1 exposure of the polarized epithelial monolayer resulted in a protective effect; and (iii) TGF-1 had no direct effect on the organism in reducing its tissue-disruptive effects. In exploring mechanisms to account for the barrier-preserving effects of TGF-1 on epithelium, we found that the protein kinase C pathway was activated, as shown by translocation of its 80-kDa  isozyme within 30 s of epithelial exposure to TGF-1; the permeability of epithelial monolayers to passage of macromolecules was reduced by 42% with TGF-1, even in the face of active protozoal infection; and epithelial cell necrosis monitored by lactate dehydrogenase release was decreased by 50% 70 h after oocyst exposure. Changes in epithelial function, initiated through an established set of surface receptors, likely accounts for the remarkable barrier-sparing effect of nanogram-per-milliliter concentrations of TGF-1 when human colonic epithelium is exposed to an important human pathogen, C. parvum.

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^* Corresponding author. Mailing address: MR-4 Building, Room 1007, P.O. Box 801317, University of Virginia Health System, Charlottesville, VA 22908. Phone: (804) 243-2655. Fax: (804) 253-6169. E-mail: jkr7m{at}virginia.edu.

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Infection and Immunity, October 2000, p. 5635-5644, Vol. 68, No. 10
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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