Induction of claudin-1 in IEC-6 cells by IL-4

• Published in: The Journal of surgical research 2004-10, Vol.121 (2), p.311-312
• Main Authors: Poritz, L.S., Garver, K.I., Thompson, J., Boyer, M., Koltun, W.A.
• Format: Article
• Language: English
• ISSN: 0022-4804; 1095-8673
• DOI: 10.1016/j.jss.2004.07.146

The purpose of this study was to evaluate the effect of IL-4 on the tight junction complex (TJ) in IEC-6 cells. Background. IL-4, an anti-inflammatory cytokine, is thought to have a protective effect in inflammatory bowel disease (IBD), although the exact mechanism is unknown. Patients with IBD have increased intestinal permeability, suggesting an alteration in the TJ. One of the protective roles of IL-4 may be to decrease permeability by altering the TJ. Under normal conditions IEC-6 cells do not express transmembrane TJ proteins such as occludin and claudin-1. Methods. Confluent IEC-6 cells were incubated with recombinant rat IL-4 (0, 5, 50, 100, 200, and 250 ng/ml) for 8, 24, 48, 72, or 96 h. The cells were then lysed and a Western blot was performed with polyclonal rabbit anti-claudin-1 antibody and detected with ECL. Additional IEC-6 cells were grown to confluence in 12-well transwell plates for 21 days. Permeability was then assayed using fluorescent-labeled dextran-4000MW. IL-4 was added (0, 5, or 50 ng/ml) basally 48 h prior to assay. Fluorescence was measured at 5, 12, and 24 h at 515 nm and compared by ANOVA. Results. Claudin-1 expression was detectable in IEC-6 cells after 8 h of IL-4 incubation. Expression increased and peaked at 48 h. Maximal claudin-1 expression was seen with 50 ng/ml of IL-4. The figure shows induction of claudin-1 (22Kda) in IEC-6 cells after 48 h of IL-4. MDCK cells were a positive control. There was an increase in permeability of the IEC-6 monolayer to F-dextran with time up to 24 h. There was no difference in permeability in IL-4-treated cells (at either 5 or 50 ng/ml IL-4) compared to untreated cells. Conclusions. (1) Incubation of IEC-6 cells with IL-4 leads to the induction of claudin-1 expression in this cell line. (2) Expression of claudin-1 is optimal at a dose of 50 ng/ml of IL-4 and 48 h of incubation. (3) Despite the induction of claudin-1, permeability of the monolayer did not change. This suggests a complex and multifactorial regulation of the permeability of the TJ and potentially a more limited role for claudin-1.