Heparan Sulfate Plays a Central Role in a Dynamic in Vitro Model of Protein-losing Enteropathy

Lars Bode,Simon Murch,Hudson H Freeze

doi:10.1074/jbc.m510722200

Abstract

Protein-losing enteropathy (PLE), the loss of plasma proteins through the intestine, is a symptom in ostensibly unrelated diseases. Emerging commonalities indicate that genetic insufficiencies predispose for PLE and environmental insults, e.g. viral infections and inflammation, trigger PLE onset. The specific loss of heparan sulfate (HS) from the basolateral surface of intestinal epithelial cells only during episodes of PLE suggests a possible mechanistic link. In the first tissue culture model of PLE using a monolayer of intestinal epithelial HT29 cells, we proved that HS loss directly causes protein leakage and amplifies the effects of the proinflammatory cytokine tumor necrosis factor alpha (TNFalpha). Here, we extend our in vitro model to assess the individual and combined effects of HS loss, interferon gamma (IFNgamma), TNFalpha, and increased pressure, and find that HS plays a central role in the patho-mechanisms underlying PLE. Increased pressure, mimicking venous hypertension seen in post-Fontan PLE patients, substantially increased protein leakage, but HS loss, IFNgamma, or TNFalpha alone had only minor effects. However, IFNgamma up-regulated TNFR1 expression and amplified TNFalpha-induced protein leakage. IFNgamma and TNFalpha compromised the integrity of the HT29 monolayer and made it more susceptible to increased pressure. HS loss itself compromises the integrity of the monolayer, amplifying the effects of pressure, but also amplifies the effects of both cytokines. In the absence of HS a combination of increased pressure, IFNgamma, and TNFalpha caused maximum protein leakage. Soluble heparin fully compensated for HS loss, providing a reasonable explanation for patient favorable response to heparin therapy.

Highlights

Combination of genetic insufficiencies and environmental insults
Albumin-FITC concentrations on both sides of an empty insert reached equilibrium in less than 4 h, the HT29 monolayer grown on the insert prevented protein leakage and allowed only 1.2% albumin-FITC to pass from the basolateral to the apical side even after 8 h
The Fontan procedure vastly improves survival of patients born with univentricular hearts but elevates venous pressure [11]

Summary

Introduction

Combination of genetic insufficiencies and environmental insults. The evidence for this is that patients do not suffer from PLE continuously; rather, PLE is episodic. The most likely explanation is that genetic factors and Fontan-induced venous hypertension predispose for PLE, which precipitates upon a series of sequential or simultaneous environmental insults Consistent with this hypothesis, seven out of eight post-Fontan patients have been diagnosed with viral infections at the onset of PLE symptoms [5], indicating that this additional insult triggered PLE. Taken during episodes with PLE, revealed an increased IFN␥ concentration [7], most likely as a response to the viral infection and elevated levels of the pro-inflammatory cytokine TNF␣ [6] Both cytokines are known to impair the integrity of the intestinal epithelial barrier [13,14,15,16,17,18]. We find that HS is the central figure coordinating their synergistic effects

Methods

Results

Conclusion