Transforming growth factor beta 1 ameliorates intestinal epithelial barrier disruption by Cryptosporidium parvum in vitro in the absence of mucosal Tlymphocytes

Exposure to oocysts of the protozoan Cryptosporidium parvum causes intestin al epithelial cell dysfunction in vivo and in vitro, but effective means by which mucosal injury might be prevented remain unclear. We examined the ab ility of transforming growth factor beta 1 (TGF-beta 1)-a cytokine synthesi zed and released by cells in the intestine-to 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-beta 1 were localized by confoca l microscopy, and TGF-beta 1-induced protein kinase C activation was detect ed intracellularly by translocation of its a isozyme, TGF-beta 1 alone enha nced intestinal epithelial barrier function, while exposure to C,parvum ooc ysts (greater than or equal to 10(5)/monolayer) markedly reduced barrier fu nction to less than or equal to 40% of that of the control, When epithelial monolayers were pretreated with TGF-beta 1 at 5.0 ng/ml, the barrier-disru pting effect of C. parvum oocysts was almost completely abrogated for 96 h, Further investigation showed that (i) the RI and RII receptors for TGF-bet a 1 were present on 55 and 65% of human epithelial cell Line cells, respect ively, 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-beta 1 exposure of the polarized epithelial monolayer re sulted in a protective effect; and (iii) TGF-beta 1 had no direct effect on the organism in reducing its tissue-disruptive effects. In exploring mecha nisms to account for the barrier-preserving effects of TGF-beta 1 on epithe lium, we found that the protein kinase C pathway was activated, as shown by translocation of its 80-kDa alpha isozyme within 30 s of epithelial exposu re to TGF-beta 1; the permeability of epithelial monolayers to passage of m acromolecules was reduced by 42% with TGF-beta 1, even in the face of activ e protozoal infection; and epithelial cell necrosis monitored by lactate de hydrogenase release was decreased by 50% 70 h after oocyst exposure. Change s in epithelial function, initiated through an established set of surface r eceptors, likely accounts for the remarkable barrier-sparing effect of nano gram-per-milliliter concentrations of TGF-beta 1 when human colonic epithel ium is exposed to an important human pathogen, C, parvum.