Phosphodiesterase inhibition attenuates alterations to the tight junction proteins occludin and ZO-1 in immunostimulated Caco-2 intestinal monolayers Todd W. Costantini, Jessica Deree, William Loomis, James G. Putnam, Sunghyuk Choi, Andrew Baird, Brian P. Eliceiri, Vishal Bansal, Raul Coimbra ⁎ Division of Trauma, Surgical Critical Care, and Burns, Department of Surgery, University of California-San Diego School of Medicine, San Diego, California, United States abstract article info Article history: Received 13 May 2008 Accepted 10 October 2008 Keywords: Tight junction ZO-1 Occludin Phosphodiesterase inhibition Pentoxifylline Caco-2 cells Intestinal permeability Shock Cytokines Inflammation Tight junction Aims: Under normal conditions, the intestinal mucosa acts as a local barrier to prevent the influx of luminal contents. The intestinal epithelial tight junction is comprised of several membrane associated proteins, including zonula occludens-1 (ZO-1) and occludin. Disruption of this barrier can lead to the production of pro-inflammatory mediators and ultimately multiple organ failure. We have previously shown that Pentoxifylline (PTX) decreases histologic gut injury and pro-inflammatory mediator synthesis. We hypothesize that PTX prevents the breakdown of ZO-1 and occludin in an in vitro model of immuno- stimulated intestinal cell monolayers. Main methods: Caco-2 human enterocytes were grown as confluent monolayers and incubated under control conditions, or with PTX (2 mM), Cytomix (TNF-α, IFN-γ, IL-1), or Cytomix + PTX for 24 h. Occludin and ZO-1 protein levels were analyzed by Western blot. Confocal microscopy was used to assess the cytoplasmic localization of ZO-1 and occludin. Key findings: Cytomix stimulation of Caco-2 cells resulted in a 50% decrease in both occludin and ZO-1 protein. Treatment with Cytomix+PTX restored both occludin and ZO-1 protein to control levels. Confocal microscopy images show that Cytomix caused an irregular, undulating appearance of ZO-1 and occludin at the cell junctions. Treatment with PTX prevented the Cytomix-induced changes in ZO-1 and occludin localization. Significance: Treatment with PTX decreases the pro-inflammatory cytokine induced changes in the intestinal tight junction proteins occludin and ZO-1. Pentoxifylline may be a useful adjunct in the treatment of sepsis and shock by attenuating intestinal barrier breakdown. © 2008 Elsevier Inc. All rights reserved. Introduction Multiple organ failure (MOF) remains a significant cause of morbidity and mortality after shock despite numerous advances in critical care. Exaggeration of the immune response can result in neutrophil activation, generation of pro-inflammatory cytokines, and distant organ injury. It is clear that the gut plays a key role in this process. Recent research has suggested the “gut-lymph” hypothesis for MOF, with studies indicating that gut derived factors carried in the intestinal lymph are key mediators of systemic inflammation (Senthil et al., 2006; Deitch et al., 2004; Magnotti et al., 1998). Intestinal epithelial barrier integrity, maintained by the tight junctions, is important in preventing the egress of luminal bacteria and endotoxin. Therefore, preventing intestinal inflammation and tight junction breakdown may limit systemic inflammation and decrease MOF after shock. The tight junction is the rate limiting step for paracellular transit and defines the overall barrier function of the intact intestinal epithelium (Turner, 2006). Disruption of the tight junction can result in increased intestinal permeability. Tight junction function is regulated by both structural and functional elements. Actin and myosin form a ring encircling the cell at the level of the tight junction. Activation of myosin light chain kinase (MLCK) regulates contraction of this actomyosin ring, and has been shown to increase tight junction permeability in response to pro-inflammatory stimuli (Ma et al., 2005; Blair et al., 2006). The structural proteins occludin and zonula occludens protein-1 (ZO-1) have also been shown to play an important role in the maintenance of epithelial barrier integrity. Previous studies have shown decreased expression of occludin and ZO-1 in response to pro- inflammatory cytokines (Bruewer et al., 2003; Poritz et al., 2007). In addition, these proteins are redistributed away from the tight junction during times of increased permeability (Musch et al., 2006). These inflammatory changes to the epithelial tight junction have been shown to be mediated, in part, by nuclear factor kappa-B (NF-κB) (Ma et al., 2004; Al-Sadi and Ma, 2007). Life Sciences 84 (2009) 18–22 ⁎ Corresponding author. 200 W. Arbor Drive, #8896, San Diego, CA 92103-8896, United States. Tel.: +1 619 543 7100; fax: +1 619 543 7202. E-mail address: rcoimbra@ucsd.edu (R. Coimbra). 0024-3205/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2008.10.007 Contents lists available at ScienceDirect Life Sciences journal homepage: www.elsevier.com/locate/lifescie