Gastroenterology

Gastroenterology

Volume 132, Issue 7, June 2007, Pages 2395-2411
Gastroenterology

Basic–alimentary tract
Interferon-γ Inhibits Intestinal Restitution by Preventing Gap Junction Communication Between Enterocytes

https://doi.org/10.1053/j.gastro.2007.03.029Get rights and content

Background & Aims: Necrotizing enterocolitis (NEC) is characterized by interferon-gamma (IFN-γ) release and inadequate intestinal restitution. Because enterocytes migrate together, mucosal healing may require interenterocyte communication via connexin 43-mediated gap junctions. We hypothesize that enterocyte migration requires interenterocyte communication, that IFN impairs migration by impairing connexin 43, and that impaired healing during NEC is associated with reduced gap junctions. Methods: NEC was induced in Swiss-Webster or IFN−/− mice, and restitution was determined in the presence of the gap junction inhibitor oleamide, or via time-lapse microscopy of IEC-6 cells. Connexin 43 expression, trafficking, and localization were detected in cultured or primary enterocytes or mouse or human intestine by confocal microscopy and 35S-labeling, and gap junction communication was assessed using live microscopy with oleamide or connexin 43 siRNA. Results: Enterocytes expressed connexin 43 in vitro and in vivo, and exchanged fluorescent dye via gap junctions. Gap junction inhibition significantly reduced enterocyte migration in vitro and in vivo. NEC was associated with IFN release and loss of enterocyte connexin 43 expression. IFN inhibited enterocyte migration by reducing gap junction communication through the dephosphorylation and internalization of connexin 43. Gap junction inhibition significantly increased NEC severity, whereas reversal of the inhibitory effects of IFN on gap junction communication restored enterocyte migration after IFN exposure. Strikingly, IFN−/− mice were protected from the development of NEC, and showed restored connexin 43 expression and intestinal restitution. Conclusions: IFN inhibits enterocyte migration by preventing interenterocyte gap junction communication. Connexin 43 loss may provide insights into the development of NEC, in which restitution is impaired.

Section snippets

Cell Culture, Transfection, and Treatment

Cultured intestinal cell lines (small intestine: IEC-6 and large intestine: HT-29, T84, and CaCO-2) were obtained from the American Type Culture Collection (Manassas, VA) and maintained as described.19, 20, 21 Where indicated, cells were treated with IFN-gamma (IFN, Sigma-Aldrich Corp., St. Louis, MO, 1000 IU/mL for 1–14 hours, 37°C), or the following phosphatase inhibitors 20 minutes to 1 hour prior to IFN treatment: calyculin A (2 μmol/L), okadaic acid (100 nmol/L, Calbiochem, San Diego, CA),

Enterocytes Express Functional Gap Junctions

We first sought to determine whether enterocytes express the gap junction protein Cx43 (Cx43), and whether gap junctions are capable of exchanging molecules between adjacent enterocytes. As shown in Figure 1, Cx43, which is the most widely expressed connexin isoform, was detected in several cultured enterocyte cell lines, including the small intestinal epithelial cell line IEC-6, the colonic epithelial lines CaCO-2, HT-29, and T84, and in mucosal scrapings from the terminal ilea of

Discussion

NEC is characterized by damage to the mucosal barrier and a profound inhibition in the rate and extent of enterocyte migration.9 Repair of the damaged intestine occurs through the process of intestinal restitution, in which healthy enterocytes adjacent to the site of injury migrate to areas of mucosal disruption to reconstitute an intact barrier.37 Despite excellent work from a variety of authors studying the regulation of enterocyte migration,38, 39 few studies have distinguished the

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    D. J. H. was supported by 1R01-GM078238-01 from the National Institutes of Health and the State of Pennsylvania Tobacco Settlement Fund. C. L. L. was supported in part by the Loan Repayment Program for Pediatric Research of the National Institutes of Health.

    There are no conflicts of interest to disclose with respect to this manuscript.

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