Corneal epithelial tight junctions and their response to lipopolysaccharide challenge

PURPOSE. To investigate the expression and cellular distribution of putativ e tight junction (TJ) proteins occludin, ZO-1, ZO-2, and claudin-1 in rat c orneal epithelium and alterations of TJs in cultured human corneal epitheli al 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 transcriptio n-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 cu ltured on the upper chamber of Transwell filters (Costar, Cambridge, MA), t ransepithelial electrical resistance (TER) was measured using a voltohmmete r. 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 sup erficial layer, whereas claudin-1 was localized mainly in the basal and win g cell layers of rat corneal epithelium. In addition to claudin-1, the tran scripts 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 ca used a dose- and time-dependent increase in monolayer permeability as asses sed by TER measurements. The maximal decrease of TER was observed at approx imately 6 to 9 hours after LPS challenge. The TER was then recovered gradua lly and returned to baseline alter 24 hours. Examination of specific protei ns associated with TJs by immunoblot analysis and immunomicroscopy revealed changes in the expression levels and localization of some of these protein s after their exposure to LPS. Specifically, LPS challenge resulted in a de crease in the levels of ZO-1 and ZO-2 compared with untreated cells. Reduct ion of the ZO-2 level was associated with the disappearance of ZO-2 stainin g 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 th rough disruption of TJs, and ZO-1 and ZO-2 are targets for the induction.