Phosphodiesterase inhibition attenuates alterations to the tight junction proteins occludin and ZO-1 in immunostimulated Caco-2 intestinal monolayers.
OBJECTIVE
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 immunostimulated intestinal cell monolayers.


METHODS
Caco-2 human enterocytes were grown as confluent monolayers and incubated under control conditions, or with PTX (2 mM), Cytomix (TNF-alpha, IFN-gamma, 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.


RESULTS
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.


CONCLUSIONS
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.
Costantini
Todd W
TW
Division of Trauma, Surgical Critical Care, and Burns, Department of Surgery, University of California-San Diego School of Medicine, San Diego, California, United States.
Deree
Jessica
J
Loomis
William
W
Putnam
James G
JG
Choi
Sunghyuk
S
Baird
Andrew
A
Eliceiri
Brian P
BP
Bansal
Vishal
V
Coimbra
Raul
R
D018938:Caco-2 Cells; D016207:Cytokines / Q000494:pharmacology*; D006801:Humans; D007413:Intestinal Mucosa / Q000737:chemistry / Q000187:drug effects*; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008565:Membrane Proteins / Q000032:analysis*; D018613:Microscopy, Confocal; D016328:NF-kappa B / Q000037:antagonists & inhibitors; D062793:Occludin; D010431:Pentoxifylline / Q000494:pharmacology* / Q000627:therapeutic use; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D010750:Phosphoproteins / Q000032:analysis*; D018805:Sepsis / Q000188:drug therapy; D062826:Zonula Occludens-1 Protein
D016207:Cytokines; D008565:Membrane Proteins; D016328:NF-kappa B; C568985:OCLN protein, human; D062793:Occludin; D010726:Phosphodiesterase Inhibitors; D010750:Phosphoproteins; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; D010431:Pentoxifylline