Coordinated epithelial NHE3 inhibition and barrier dysfunction are required for TNF-mediated diarrhea in vivo
Daniel R. Clayburgh, … , Yang-Xin Fu, Jerrold R. Turner
Daniel R. Clayburgh, … , Yang-Xin Fu, Jerrold R. Turner
Published October 2, 2006
Citation Information: J Clin Invest. 2006;116(10):2682-2694. https://doi.org/10.1172/JCI29218.
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Research Article Gastroenterology

Coordinated epithelial NHE3 inhibition and barrier dysfunction are required for TNF-mediated diarrhea in vivo

Abstract

Acute T cell–mediated diarrhea is associated with increased mucosal expression of proinflammatory cytokines, including the TNF superfamily members TNF and LIGHT. While we have previously shown that epithelial barrier dysfunction induced by myosin light chain kinase (MLCK) is required for the development of diarrhea, MLCK inhibition does not completely restore water absorption. In contrast, although TNF-neutralizing antibodies completely restore water absorption after systemic T cell activation, barrier function is only partially corrected. This suggests that, while barrier dysfunction is critical, other processes must be involved in T cell–mediated diarrhea. To define these processes in vivo, we asked whether individual cytokines might regulate different events in T cell–mediated diarrhea. Both TNF and LIGHT caused MLCK-dependent barrier dysfunction. However, while TNF caused diarrhea, LIGHT enhanced intestinal water absorption. Moreover, TNF, but not LIGHT, inhibited Na+ absorption due to TNF-induced internalization of the brush border Na+/H+ exchanger NHE3. LIGHT did not cause NHE3 internalization. PKCα activation by TNF was responsible for NHE3 internalization, and pharmacological or genetic PKCα inhibition prevented NHE3 internalization, Na+ malabsorption, and diarrhea despite continued barrier dysfunction. These data demonstrate the necessity of coordinated Na+ malabsorption and barrier dysfunction in TNF-induced diarrhea and provide insight into mechanisms of intestinal water transport.

Authors

Daniel R. Clayburgh, Mark W. Musch, Michael Leitges, Yang-Xin Fu, Jerrold R. Turner

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Figure 1

Administration of TNF and LIGHT induces barrier dysfunction similar to that caused by anti-CD3 injection, but only TNF causes net water secretion.

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Administration of TNF and LIGHT induces barrier dysfunction similar to t...
(A) In vivo perfusion assays show that anti-CD3 causes a large increase in BSA flux (P < 0.0001 versus control). The MLCK inhibitor PIK completely prevented this increased BSA flux, which was also attenuated by anti-TNF or use of LTβR–/– mice. (B) Anti-CD3 injection reverses water movement, from net water absorption in control animals to net water secretion (P < 0.0001). PIK restored water flow to net absorption, although absorption was still significantly less than in control animals. Anti-TNF completely restored water absorption after anti-CD3 injection. No significant water absorption or secretion was observed in LTβR–/– animals treated with anti-CD3 (P < 0.001). (C) Two hours following anti-CD3 injection, TNF, LIGHT, and IFN-γ mRNA were assessed in intestinal mucosa using quantitative real-time PCR. Anti-CD3 caused significant increases in transcripts for all 3 cytokines (P < 0.001). (D) Either TNF or LIGHT, but not IFN-γ, causes significant increases in BSA flux, though not as large as that caused by anti-CD3. Simultaneous injection of TNF and LIGHT led to a larger increase in BSA flux that resembled the increase following anti-CD3 injection. (E) While IFN-γ does not alter water movement, TNF reverses water movement from net water absorption to net water secretion in a manner similar to anti-CD3. In contrast, LIGHT caused an increase in water absorption (P = 0.03). Simultaneous TNF and LIGHT treatment caused water secretion similar to that caused by treatment with TNF alone.