Corneal epithelial tight junctions and their response to lipopolysaccharide challenge.
OBJECTIVE
To investigate the expression and cellular distribution of putative tight junction (TJ) proteins occludin, ZO-1, ZO-2, and claudin-1 in rat corneal epithelium and alterations of TJs in cultured human corneal epithelial cells in response to lipopolysaccharide (LPS) challenge.


METHODS
Immunohistochemistry was used to determine tissue distribution of occludin, ZO-1, ZO-2, and claudin-1 in the rat cornea. Reverse transcription-polymerase chain reaction was used to reveal the expression of mRNAs for claudins in simian virus (SV)40-immortalized human corneal epithelial (THCE) cells. To assess epithelial response to LPS challenge, THCE cells were cultured on the upper chamber of Transwell filters (Costar, Cambridge, MA), transepithelial electrical resistance (TER) was measured using a voltohmmeter. Immunocytochemistry and immunoblotting were used to assess alteration in the levels and localization of TJ-associated proteins occludin, ZO-1, and ZO-2 in LPS-treated THCE cells.


RESULTS
Occludin, ZO-1, and ZO-2 were found at the cell borders of the superficial layer, whereas claudin-1 was localized mainly in the basal and wing cell layers of rat corneal epithelium. In addition to claudin-1, the transcripts for several other isotypes of claudins-2, -3, -7, -9, -14, and -15 were identified in THCE cells. Treatment of cultured THCE cells with LPS caused a dose- and time-dependent increase in monolayer permeability as assessed by TER measurements. The maximal decrease of TER was observed at approximately 6 to 9 hours after LPS challenge. The TER was then recovered gradually and returned to baseline after 24 hours. Examination of specific proteins associated with TJs by immunoblot analysis and immunomicroscopy revealed changes in the expression levels and localization of some of these proteins after their exposure to LPS. Specifically, LPS challenge resulted in a decrease in the levels of ZO-1 and ZO-2 compared with untreated cells. Reduction of the ZO-2 level was associated with the disappearance of ZO-2 staining from cell borders in 6-hour LPS-treated cells.


CONCLUSIONS
Occludin, ZO-1, and ZO-2, but not claudin-1, are components of corneal epithelial TJs. LPS induces breakdown of the epithelial barrier through disruption of TJs, and ZO-1 and ZO-2 are targets for the induction.
Yi
X
X
Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts, USA.
Wang
Y
Y
Yu
F S
FS
D000818:Animals; D002478:Cells, Cultured; D062445:Claudin-1; D017931:DNA Primers / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D017097:Electric Impedance; D019573:Epithelium, Corneal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D015151:Immunoblotting; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D062793:Occludin; D010539:Permeability; D010750:Phosphoproteins / Q000378:metabolism; D011550:Pseudomonas aeruginosa; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019108:Tight Junctions / Q000187:drug effects* / Q000378:metabolism; D013997:Time Factors; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein
C568108:CLDN1 protein, human; D062445:Claudin-1; C568114:Cldn1 protein, rat; D017931:DNA Primers; D008070:Lipopolysaccharides; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; C568987:Ocln protein, rat; D010750:Phosphoproteins; D012333:RNA, Messenger; C569042:TJP1 protein, human; C569046:TJP2 protein, human; C569044:Tjp1 protein, rat; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein; D012313:RNA