Tumor necrosis factor alpha disrupts tight junction assembly.
BACKGROUND
We have previously shown an increase in intestinal permeability and a corresponding decrease in the expression of tight junction (TJ) proteins in the in testines of patients with Crohn's disease (CD). Tumor necrosis factor-alpha (TNFalpha) has been implicated in the inflammatory process of CD and its suppression has therapeutic benefit. ZO-1, occludin, and the claudins are key proteins in the TJ.


OBJECTIVE
TNFalpha disrupts the TJ.


METHODS
MDCK cells were incubated with TNFalpha (0-100 ng/ml) for 5 days. Qualitative evaluation of the TJ was done with monoclonal antibody to ZO-1 detected by an immunofluorescence. Duplicate cells were lysed and ZO-1, occludin, and claudin-1 amount determined by western blot.


RESULTS
Immunofluorescent staining of MDCK cells for ZO-1 showed TJ structural disruption with increasing amount of TNFalpha characterized by fragmented staining of ZO-1. There were no significant differences in quantitation of ZO-1 or occludin in the MDCK cells for all TNFalpha concentrations. There was a significant decrease in the amount of claudin-1 with increasing concentration of TNFalpha.


CONCLUSIONS
(1) MDCK TJs are qualitatively disrupted by TNFalpha. (2) This disruption is not because of a decrease in cell number, lack of cell layer confluency, or a decrease in the amount of ZO-1 or occludin. (3) The amount of claudin-1 present in the cell is decreased with increasing amounts of TNFalpha suggesting that the lack of claudin-1 may cause a relocation of ZO-1 away from the TJ. (4) This rearrangement may play a role in the increased intestinal permeability seen in CD and other diseases.
Poritz
Lisa S
LS
Department of Surgery, The Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033, USA. lporitz@psu.edu
Garver
Kristian I
KI
Tilberg
Anna F
AF
Koltun
Walter A
WA
D000818:Animals; D002452:Cell Count; D002460:Cell Line; D062445:Claudin-1; D004285:Dogs; D005455:Fluorescent Antibody Technique; D006801:Humans; D015151:Immunoblotting; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D062793:Occludin; D010750:Phosphoproteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D013194:Staining and Labeling; D019108:Tight Junctions / Q000187:drug effects*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D062826:Zonula Occludens-1 Protein
C568108:CLDN1 protein, human; D062445:Claudin-1; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; D010750:Phosphoproteins; D011994:Recombinant Proteins; C569042:TJP1 protein, human; D014409:Tumor Necrosis Factor-alpha; D062826:Zonula Occludens-1 Protein