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P069. Glycine and glutamine down-regulate IL-6-induced claudin-2 expression in human colonic epithelial cell monolayer

T. Nakagawa1, Y. Takahashi1, K. Saito1, S. Sazuka1, T. Katsuno1, O. Yokosuka1, 1Chiba University Hospital, Chiba, Japan

Background

It is well-known that tight junction contributes epithelial barrier function of intestinal mucosa, and claudins which are major component proteins of tight junction are identified twenty four subtypes in human. However some previous reports show claudin-2 decrease epithelial barrier function in increasing paracellular permeability, and claudin-2 over-expression was implicated in exacerbation of active Crohn's disease. On the other hand, it is becoming apparent that amino acids are not only a material of protein synthesis but also own some pharmacological activity by itself. Thus we sought to determine whether any amino acids modulate barrier function and claudin expressions in colonic epithelial cell monolayers.

Methods

T84 cells which are human colonic epithelial cells were cultured as confluent monolayer models for a week in insert well of transwell assay. The epithelial barrier function of the cells was determined by monitoring trans-epithelial electrical resistance (TER) with administration of pro-inflammatory cytokines and amino acids into basal surface and apical surface of the well respectively. Furthermore protein assay was performed by Western blotting assay.

Results

TER of the monolayers was significantly reduced when incubated with 10 ng/ml of IL-6 (533±42 Ω·cm2) compared with that of untreated monolayers (1,074±54 Ω·cm2). Further, the monolayers were incubated with 2 mM of 20 kinds of each amino acid (pH = 7.4) with or without IL-6. Notably, treatment with glycine and glutamine significantly increased TER of the IL-6-untreated monolayers (1,205±118, 1,151±113 Ω·cm2, respectively), whereas other amino acids had minimum effects. Moreover, glycine and glutamine neutralized IL-6-induced decrease in TER (1,054±103, 924±91 Ω·cm2, respectively). The protein level of claudin-2 that deteriorates barrier function was selectively and significantly increased when the cells were incubated with IL-6 (829±161%) compared with that of the untreated monolayers (100%). Intriguingly, treatment with glycine and glutamine selectively and remarkably down-regulated IL-6-induced claudin-2 protein expressions (83±8.8%, 60±1.4%, respectively).

Conclusion

This study demonstrated a novel physiological effect of glycine and glutamine, both of which enhanced barrier function of human colonic epithelial cell monolayers by a selective down-regulation of claudin-2 proteins.