December 2000
Volume 41, Issue 13
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Cornea  |   December 2000
Corneal Epithelial Tight Junctions and Their Response to Lipopolysaccharide Challenge
Author Affiliations
  • Xian-jin Yi
    From the Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts; and the
  • Yuan Wang
    Laboratory of Molecular Biology and Department of Biochemistry, Anhui Medical University, Peoples Republic of China.
  • Fu-Shin X. Yu
    From the Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts; and the
Investigative Ophthalmology & Visual Science December 2000, Vol.41, 4093-4100. doi:
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      Xian-jin Yi, Yuan Wang, Fu-Shin X. Yu; Corneal Epithelial Tight Junctions and Their Response to Lipopolysaccharide Challenge. Invest. Ophthalmol. Vis. Sci. 2000;41(13):4093-4100.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. 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.

Corneal epithelium functions as a barrier that separates the eye from the outside environment. 1 Zonula occludens or tight junctions (TJs) encircle the cells just below the apical surface and constitute the principal barrier to passive movement of fluid, electrolytes, macromolecules, and cells through the paracellular pathway, in a regulated manner. 2 3 4 Recent studies have revealed that the TJ complex includes integral transmembrane proteins claudins; occludin; membrane-associated proteins ZO-1, ZO-2, and ZO-3; and actin filaments. 5 6 7 8 9 In the cornea, ZO-1 is found in the superficial layer of epithelium where TJs are located, as well as between wing cells and basal epithelial cells that have no TJ structure. 2 3 The expression and localization of other TJ proteins in stratified corneal epithelium has not been reported. 
As described in recent review articles, epithelial TJs are under the control of a wide variety of agents and signal transduction pathways. 4 8 Different TJ components are known to be targeted for barrier modulation by extracellular stimuli. Occludin was the first transmembrane protein identified and has been the subject of extensive studies. 7 10 Occludin contains four transmembrane domains with both N and C termini oriented into the cytoplasm. 10 Current data indicate that occludin plays a regulatory rather than a structural role in TJs. 8 10 11 Recent in vitro studies suggested that the members of the claudin family form TJ strands that are associated laterally with those of adjacent cells to form paired strands to eliminate the extracellular space. 12 13 To date, 16 claudins have been identified and are referred to as the claudin family. 8 At the molecular level, distinct species of claudins interact with themselves and with each other, within and between TJ strands. 14 ZO-1, ZO-2, and ZO-3 are members of the membrane-associated guanylate kinase homologue (MAGUK) family. 8 They are peripheral membrane proteins located at the points of TJ-membrane contact in epithelial and endothelial cells. 6 15 16 17 The MAGUK proteins are able to interact with each other, with occludin and claudins, and with the actin cytoskeleton to form TJ complexes. 17 18 19 20 21 Thus, the cell content and other physiological states, such as phosphorylation and cytoskeletal association, of MAGUK proteins and occludin serve as relevant indicators for TJ integrity and barrier function in epithelial cells. 
Because of the corneal epithelial barrier, the eye is relatively impermeable to micro-organisms and other environmental elements. However, if corneal integrity is breached by trauma or routine contact lens wear, a sight-threatening bacterial infection may occur. In the human cornea, Pseudomonas aeruginosa is the organism most commonly involved in bacterial keratitis associated with contact lens wear. 22 23 This corneal infection is rapidly progressive, difficult to treat, and can cause severe visual impairment. 24 An increase in permeability and breakdown of epithelial barrier function is associated with corneal bacterial infections. 25 26 27 Although it remains unclear whether barrier breakdown is an initiating event or a consequence of inflammation, it is readily apparent that loss of the barrier contributes to propagation and exacerbation of infection and inflammation. It is therefore of interest to study epithelial barrier function and regulation. To date, the mechanism underlying TJ disruption and barrier breakdown remain to be determined. Cell culture studies have facilitated analysis of TJ structure and epithelial barrier functions, adding to our knowledge of the molecular and biochemical properties of these cellular structures and of their regulation in response to extracellular stimuli. 
Recently, a number of corneal epithelial cell lines with an extended life span have been established. 28 29 One example is a simian virus (SV)40-transformed human corneal epithelial cell line (THCE) developed by Araki–Sasaki et al. 30 This cell line continues to grow and exhibits a cobblestone-like appearance similar to normal corneal epithelial cells in culture. 30 Long-term stable expression of N-terminal mutants of ZO-1 in THCE cells results in disruption of endogenous ZO-1 and ZO-2 localization and a dramatic cell shape change from characteristic epithelial cobblestone morphology to an elongated fibroblast-like shape. 31 Thus, this cell line can be used as an in vitro model that enables the study of corneal epithelial barrier regulation. 
In this report, we examined the distribution of TJ proteins ZO-1, ZO-2, occludin, and claudin-1 in rat corneal epithelium. We also investigated the effects of lipopolysaccharide (LPS) isolated from P. aeruginosa on barrier function and regulation of corneal epithelial cells, using THCE cells grown on permeable supports (Transwell membrane; Costar, Cambridge, MA) as a model epithelium. The results show that LPS challenge disrupts corneal epithelial barrier function by altering the levels and/or distribution of ZO-1 and ZO-2. 
Materials and Methods
Immunohistochemistry and Immunocytochemistry of TJ Proteins in Corneal Epithelial Cells
Procedures involving experimental animals were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Two Sprague–Dawley rats (weighing 150–175 g) were killed by CO2 inhalation. The corneas were excised, embedded in optimal cutting temperature (OCT) medium, and frozen immediately in liquid nitrogen. Eight-μm-thick cryostat sections of corneas and corneal epithelial cells cultured in the filter membrane were both fixed in absolute methanol for 15 minutes and blocked by 5% nonfat milk for 3 hours. The polyclonal rabbit IgG against occludin, ZO-1 and ZO-2 (Zymed Laboratory, South San Francisco, CA; 1:500 dilution each), and claudin-1 (Zymed, 1:1000 dilution) were then applied overnight at 4°C in a moist chamber. After three washings with Tris-buffered saline with 0.01% Tween-20 (TBST), fluorescein isothiocyanate (FITC)–conjugated goat anti-rabbit IgG was applied to the sections for 1 hour at room temperature in a moist chamber. Negative controls included incubation of tissue sections with preimmune rabbit serum instead of primary or secondary antibodies alone. After mounting, sections were photographed under a microscope (Eclipse E-800; Nikon, Tokyo, Japan) equipped with a digital camera. 
Cell Culture
THCE cells were kindly provided by Kaoru Araki–Sasaki, Ehime University, Japan. The cells were grown in the keratinocyte growth medium (Clonetics, San Diego, CA) with penicillin and streptomycin. Cells were routinely passaged when they reached 80% confluence. To assess whether LPS caused cell damage, THCE cells were treated with or without LPS (1 and 5 μg/ml) for 6 or 24 hours and examined under a light microscope for morphologic changes and with a nonradioactive cell proliferation assay (CellTiter 96 Aqueous; Promega, Madison, WI) for cytotoxicity determination. 
RNA Isolation and RT-PCR Amplification of Claudin Species
For reverse transcription–polymerase chain reaction (RT-PCR), THCE cells cultured on 150-mm culture dishes were directly lysed with Tri Reagents (Molecular Research Center, Cincinnati, OH), and total cellular RNA was isolated according to the manufacturer’s instructions. Because many claudin genes contain no introns, we treated isolated RNA with RNase-free DNase I (1 U DNase I/5 μg RNA; Promega). First-strand cDNA was generated in the presence of 0.5 μg oligo(dT) from 5 μg total RNA with reverse transcriptase (SuperScript II; Life Technologies, Rockville, MD). The paired primers for amplifying claudin species are listed in Table 1 . The polymerase chain reaction (PCR) was performed with the cDNA working mixture in a 50-μl reaction volume containing 20 mM Tris (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 200 μM dNTP, specific 5′ and 3′ primers (10 picomoles each), and 1 U Taq DNA polymerase (Promega). Amplification was performed in a Personal Cycler (Biometra, Göttingen, Germany) programmed for 35 cycles of 94°C for 45 seconds, 58°C for 2 minutes, and 72°C for 1 minute. The PCR products were examined by 0.7% agarose gel and ethidium bromide staining. The observed PCR products corresponded to their expected molecular weights. 
Measurement of Transepithelial Electrical Resistance (TER)
THCE cells were seeded in the upper chamber of a Transwell tissue culture plate (Costar; 12-mm diameter, 0.4-μm pore size) and allowed to reach confluence. The TER of cells grown on filters was measured with an epithelial voltohmmeter (World Precision Instruments, Sarasota, FL). Cells were used only if their TER was more than 100Ω /cm2. Cells with stable TER were treated with LPS isolated from P. aeruginosa (Serotype 10; Sigma, St. Louis, MO) that was added to both chambers for the indicated times. TER was calculated from the measured resistance and normalized by the area of the monolayer (ohms per square centimeter). The background TER of blank Transwell filters was subtracted from the TER of cell monolayers. 
Immunoblot Analysis
Whole-cell extracts were prepared from both rat corneal epithelium and cultured THCE cells by lysing tissues and cells in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate [SDS], 50 mM Tris, [pH 8.0]) with protease inhibitors leupeptin, aprotinin, pepstatin A, soybean trypsin inhibitor (5 μg/ml each), and 1 mM phenylmethylsulfonyl fluoride. The protein concentrations were determined by a protein assay reagent kit (Micro BCA; Pierce, Rockford, IL). Equal amounts of protein were mixed with SDS-sample buffer and boiled for 5 minutes before loading. Proteins were separated by 5% to 15% gradient SDS-PAGE and transferred electronically to the nitrocellulose membranes. The quality of the transfer was monitored by ponceau S staining. After blocking with 5% nonfat milk, the membranes were incubated with polyclonal antibodies against occludin, ZO-1, ZO-2, and claudin-1 (Zymed, 1:1000 dilution in 5% nonfat milk) in TBST. Horseradish peroxidase–conjugated goat anti-rabbit IgG (Bio-Rad, Hercules, CA) was applied for 1 hour. Immune complexes were visualized with an enhanced chemiluminescence reagent (Pierce). Results were quantified by capturing the exposed x-ray film image with BDS Image System (Biological Detection System, Pittsburgh, PA), and using area measurements from image analysis software (NIH Image, ver. 1.60; National Institutes of Health, Bethesda, MD). Experimental values were within the linear range of the assay. 
Results
Subcellular Distribution of TJ Components in Rat Corneal Epithelium
To assess the potential role of TJ-associated proteins in barrier function of the cornea, the distribution of occludin, ZO-1, ZO-2, and claudin-1 in corneal epithelium was examined by immunohistochemistry. We first used human eye bank corneas that were no longer usable for transplantation and found that, in addition to edema, most of the superficial layer(s) of epithelium had disappeared. Because we were interested in TJs that are located only in the superficial layer of stratified epithelium, these corneas were not suitable for our studies. We then used the rat corneas and antibodies known to cross-react with rat antigens (Fig. 1) . As reported by Sugrue and Zieske, 2 ZO-1 immunoreactivity was found at the lateral margins of cells comprising the superficial layer of the epithelium, characteristic of TJs in epithelium. The staining patterns of occludin and ZO-2 were very similar to that of ZO-1; both were concentrated at cell–cell borders only in the superficial layer of the corneal epithelium. Surprisingly, claudin-1 was primarily found in the basal cell and wing cell layers, and its staining intensity gradually faded toward the superficial layer. To confirm the cross-species reactivity and specificity of claudin-1 antibody, we performed immunoblotting of proteins extracted from rat corneal epithelia. A single band with molecular weight of approximately 20 kDa was observed (inset in claudin-1 panel, Fig. 1 ), indicating that the observed immunostaining in epithelial layers in the rat cornea was claudin-1–specific. Thus, these data suggest that, in the stratified epithelium of the rat cornea, claudin-1 immunoreactivity is not associated with TJs. 
Claudins Expressed in Cultured Corneal Epithelial Cells Detected by RT-PCR
To date, 16 species of claudins have been identified. 13 32 33 At the present time, except for that of claudin-1, the expression of claudin isotypes in corneal epithelial cells are not known. Absence of claudin-1 immunoreactivity in the superficial layer of rat corneal epithelium implies involvement of other members of the claudin family in the formation of TJ strands. We used RT-PCR to determine which types of claudins are expressed in THCE cells. Three claudin species (claudins 6, 8, and 13) have no human expressed sequence tags (ESTs), and most, if not all, murine ESTs for these three claudins are from embryonic DNA libraries, which suggests that these genes may not be expressed in adult tissues. Claudin-11 has been found only in oligodendrocytes and in Sertoli cells in the testis. 34 No information was available for claudin-12. We therefore designed PCR primers for human claudins-1 to -5, -7, -9, -10, and -14 to -16 (Table 1) and determined the presence of transcripts of these 11 species in THCE cells. Using RT-PCR (Fig. 2) , seven species (claudins-1, -2, -3, -7, -9, -14, and -15) were amplified from THCE cells, which suggests that the transcripts of these claudin species were expressed in corneal epithelial cells. 
Effects of LPS on the TER of Corneal Epithelial Cell Monolayers
To study TJ-based corneal epithelial barrier function, we cultured THCE cells on porous filters. Epithelial cells cultured on the membrane formed a polarized, impermeable monolayer that possessed relatively high TER (∼100 Ω/cm2). We also measured the TER of primary cultured human corneal epithelial cells and observed a slightly higher TER, approximately 120 Ω/cm2 (data not shown). 
Treatment of THCE cells with different doses of LPS isolated from P. aeruginosa (0.3, 1, and 3 μg/ml) added to both chambers of the Transwell resulted in a time- and dose-dependent decrease in TER (Fig. 3) . Monolayers treated with a low dose of LPS (0.3 μg/ml) showed a 20% decrease in TER at 9 hours; higher concentrations of LPS (1 and 3μ g/ml) showed a 40% decrease in TER after 9 hours of treatment in comparison with control cells. The TER of THCE cells gradually recovered after 9 hours and returned to baseline after 24-hour incubation of LPS. No significant cell damages was observed during the 24-hour LPS-incubation period, as assessed using morphology examination and cytotoxicity determination (data not shown). 
Reduction of ZO-1 and ZO-2 Levels by LPS Challenge
To examine the effects of LPS on proteins associated with TJs, ZO-1, ZO-2, and occludin were analyzed by immunoblot. Cells were lysed with RIPA buffer containing NP-40 and SDS, which solubilizes membrane and cytoskeleton associated TJ components. Cells treated for 6 hours with different doses of LPS were processed for immunoblot analysis. The same blot was cut into three strips according to molecular weight, and the strips were then probed with ZO-1, ZO-2, and occludin, respectively. As shown in Figure 4 , a number of changes were apparent in the various proteins after LPS treatment. In normal cornea, single bands with molecular weights of approximately 220 kDa, 160 kDa, and 68 kDa were detected with ZO-1, ZO-2, and occludin antibodies, respectively (Fig. 4) . The intensity of TJ proteins after gel electrophoresis was quantitatively assessed by image capture and analysis. Treatment of the cells with LPS (1 μg/ml, 6-hour incubation) reduced ZO-1 and ZO-2 content to 40% and 35% of control levels, respectively, correlating with the decrease in TER (Fig. 3) . The changes in occludin immunoreactivity, however, were different from those in ZO-1 and ZO-2. At low concentrations of LPS (up to 2 μg/ml), the occludin band (∼68 kDa) was slightly decreased, whereas a smaller band (∼65 kDa) appeared. The 65-kDa may have been the dephosphorylation product of occludin. 35 At 4 μg/ml of LPS, the total immunoreactivity of occludin also decreased. After 24 hours, the immunoreactivities of ZO-1, ZO-2, and occludin in LPS-challenged cells were similar to that of control (data not shown). 
Alteration of the Subcellular Distribution of ZO-2 by LPS Challenge
To determine whether LPS affects the localization of TJ-associated proteins, we performed immunocytochemistry of ZO-1, ZO-2, occludin, and claudin-1 (Fig. 5) . In untreated cells, ZO-1, ZO-2, and occludin were all localized to the cell boundaries (control), although the immunostaining intensities for those proteins were different, with occludin staining the most defused. The immunostaining pattern for these proteins is characteristic of TJs in monolayer culture of epithelial cells. Claudin-1 was also localized to the cell boundaries of THCE cells. Because many cell–cell adhesion proteins also exhibit similar cell boundary localization, it is not clear whether claudin-1 immunoreactivity is associated with TJ structure in THCE cells. The cell boundary staining of ZO-1 and occludin in LPS-treated THEC cells remained apparently unchanged, whereas staining intensity was somewhat reduced. Analysis of LPS-treated THCE cells showed that immunostaining for ZO-2 was faint at tricellular corners, where the borders of three epithelial cells meet, and was undetectable in the remainder of the epithelial borders. When TER returned to a level comparable with that in untreated cells after 24 hours of LPS incubation, ZO-2 staining also recovered to a pattern similar to the control (data not shown). 
Discussion
In this study, we investigated the molecular composition of corneal epithelial TJs and found that occludin and two MAGUK proteins, ZO-1 and ZO-2, were localized at the lateral aspect of the superficial cell layer and exhibited the characteristic pattern of TJ staining. We were surprised to detect claudin-1 in basal and wing, but not in superficial, cell layers of the epithelium. As a first step toward understanding regulation of the epithelial barrier in the cornea, we have examined the effects of LPS on THCE cells. We observed that LPS induces disruption of the corneal epithelial barrier as assessed by TER, a parameter of paracellular permeability, and causes a decrease in the levels of ZO-1 and ZO-2, two key components of TJs, as well as loss of cell staining of ZO-2. 
Stratified corneal epithelium consists of five to seven cell layers, and TJs are found at the superficial layer. 1 2 Our data showing that claudin is localized in the cell layers of stratified corneal epithelium that does not possess TJs or perform barrier functions raise the question of claudin-1’s role in corneal epithelial cells. Recent studies provide evidence of a pivotal role for claudins in TJ structure. Deletion of claudin-11 (found only in myelin sheaths in the brain and in Sertoli cells in the testis) resulted in both neurologic and male reproductive deficits. 36 Mutations in claudin-16, a kidney-specific gene, caused hereditary human renal Mg2+ wasting and renal hypomagnesemia. 33 To date, it is not clear whether claudins perform other functions than forming TJ strands. Localization at apical–lateral, but not basal, sides of the basal cells, and at suprabasal and wing cell boundaries, suggests a possible role for claudin-1 in cell–cell interaction and adhesion. That at least seven isotypes of claudin transcripts are present in the corneal epithelial cells suggests that other members of the claudin family may be involved in the formation of TJ strands in the cornea. 
A major function of the corneal epithelial barrier is to defend ocular tissues from infection. In the normal cornea, experimentally induced devitalization of overlying cells induces rapid de novo generation of a paracellular barrier between the newly exposed intrastratal cells, 3 whereas the epithelial barrier function is compromised in infected corneas. 25 26 27 Thus, there must be a mechanism(s) operating during infection that first triggers the breakdown and then prevents the de novo generation of epithelial TJs. LPS is a major virulence factor for P. aeruginosa and elicits many of the clinical manifestations of bacterial keratitis. 37 We proposed that LPS may be one of the factors that influence epithelial TJs in vivo and investigated whether P. aeruginosa–isolated LPS induces TJ disruption in vitro by determining the effect of LPS on corneal epithelial paracellular permeability. We observed that LPS challenge indeed decreased the TER of cultured corneal epithelial cells. The effects of LPS on TER could be seen at 3 hours and reached a peak at 9 hours. The response of corneal epithelial cells to LPS was transient; epithelial monolayers regained baseline resistance values by 24 hours. This pattern of response to an endotoxin is similar to that observed in cultured T84 cells—a model for intestinal epithelium—with Helicobacter pylori 26 or epithelial cell-small intestinal lamina propria fibroblast coculture with LPS. 38 Thus, LPS may contribute to the alteration of epithelial barrier properties observed in vivo during corneal infection. It should be noted that, in addition to LPS, protease, exotoxin A, and elastase have also been implicated in P. aeruginosa keratitis 39 40 41 42 and are known to affect TJs in epithelial cells. 27 43  
Recently, toll-like receptor (TLR)-4 has been identified as a receptor for LPS. 44 TLR4 is a member of the TLR family, and to date six members (TLRs 1–6) have been identified in mammals. 45 TLR family members are transmembrane proteins containing repeated leucine-rich motifs in their extracellular portion and a cytoplasmic domain that is homologous to the signaling domain of the interleukin (IL)-1 receptor. 45 46 47 Using RT-PCR, we detected transcripts for TLR2 and 4, but not TLR1, 3, and 5 in THCE cells; the presence of TLR2 and 4 in rat and human corneal epithelial cells was confirmed by Western blot analysis (F.-S.Yu et al., unpublished results, 2000). The involvement of TLR-initiated signal transduction pathway activated by LPS and/or P. aeruginosa in modulation of epithelial permeability remains to be determined. The same signal transduction pathway may also modulate the production of proinflammatory cytokines such as IL-1, IL-6, and tumor necrosis factor-α, 48 as well as defensin-2 49 and possibly mucins, 50 as observed in lung epithelial cells. 51 52 Of note, many of these cytokines are known to mediate TJ structure and are involved in the development of barrier dysfunction in vitro and in vivo. 53 54 55 56 Thus, the released proinflammatory cytokines may contribute to the induction of epithelial barrier disruption in LPS-challenged corneal epithelial cells. 
Our data also showed that LPS had diverse effects on TJ components, including reduction of the levels of TJ proteins ZO-1 and ZO-2 and alteration of ZO-2 localization. Although ZO-1 has been reported to be a target of extracellular stimuli, 56 57 this was the first time that the level and the distribution of ZO-2 have been shown to change in response to an extracellular stimulus in cells. The biochemical properties of MAGUK proteins suggest that they may serve to organize the TJ complex by recruiting membrane proteins such as occludin and/or claudins to the cell–cell contact sites and also by connecting it to the actin cytoskeleton. Thus, loss of ZO-1 and/or ZO-2 would result in a disorganization of the TJ. 
In summary, we showed that the localization of occludin, ZO-1, and ZO-2, but not claudin-1, in corneal epithelium is consistent with where TJs are formed. We showed that LPS isolated from P. aeruginosa induced disruption of TJs, an increase in paracellular permeability, and alteration of ZO-1 and ZO-2 expression and/or distribution in cultured corneal epithelial cells. Because loosening of TJs may be an early change in the epithelial barrier that contributes to destructive events caused by P. aeruginosa infection in the cornea, studies designed to determine the mechanisms of LPS-induced changes in TJ structure and function should increase our understanding of pathogenesis of infection. 
 
Table 1.
 
Primers for Detecting mRNA Expression of Claudins in THCE Cells by RT-PCR
Table 1.
 
Primers for Detecting mRNA Expression of Claudins in THCE Cells by RT-PCR
Claudin Sequence (5′–3′) PCR Size Reference
Claudin-1 s TCA GCA CTG CCC TGC CCC AGT 506 bp 41
as TGG TGT TGG GTA AGA GGT TGT
Claudin-2 s ACA CAC AGC ACA GGC ATC AC 319 bp 12
as TCT CCA ATC TCA AAT TTC ATG C
Claudin-3 s AAG GCC AAG ATC ACC ATC GTG 304 bp 13
as AGA CGT AGT CCT TGC GGT CGT
Claudin-4 s TGG ATG AAC TGC GTG GTG CAG 361 bp 42
as GAG GCG GCC CAG CCG ACG TA
Claudin-5 s ATG TCG TGC GTG GTG CAG AG 413 bp 43
as GGT GCA GAC CCA GGC GCC GCA
Claudin-7 s AGT GGC AGA TGA GCT CCT ATG 364 bp
as GTT ATA AAA GTC TGT GAC AAT CT
Claudin-9 s TTC ATC GGC AAC AGC ATC GT 403 bp
as GCC CAG CCC AGG TAG AGG GA
Claudin-10 s TGT ACC AAA GTC GGA GGC TC 370 bp
as GCA TTT TTA TCA AAC TGT TTT GAA GG
Claudin-14 s CTC ATG GTC ATC TCC TGC CTG 469 bp
as ACG TAG TCG TTC AGC CTG TAC
Claudin-15 s CAT CAC CAC CAA CAC CAT CTT 540 bp
as GCT GCT GTC GCC TTC TTG GTC
Claudin-16 s TTT GGA TTT CTC ACC CTG CTC 398 bp 30
as TGT GCG AGG GGC TGA GTA TGA
Figure 1.
 
Distribution of ZO-1, ZO-2, occludin, and claudin-1 in rat corneal epithelium. Cryostat sections (8 μm) of the normal rat corneas were immunofluorescence stained with antibodies against ZO-1, ZO-2, occludin, and claudin-1. Like control, in which nonspecific rabbit serum was used instead of first antibody, all four specific antibodies exhibited evenly distributed, weak nonspecific staining in entire epithelial layer. However, strong immunoreactivity of ZO-1, ZO-2, and occludin was present along the borders of the superficial epithelial cells, whereas claudin-1 was mainly located in the wing and basal cell layers. M: Stained with hematoxylin to reveal corneal morphology. Bar, 50 μm.
Figure 1.
 
Distribution of ZO-1, ZO-2, occludin, and claudin-1 in rat corneal epithelium. Cryostat sections (8 μm) of the normal rat corneas were immunofluorescence stained with antibodies against ZO-1, ZO-2, occludin, and claudin-1. Like control, in which nonspecific rabbit serum was used instead of first antibody, all four specific antibodies exhibited evenly distributed, weak nonspecific staining in entire epithelial layer. However, strong immunoreactivity of ZO-1, ZO-2, and occludin was present along the borders of the superficial epithelial cells, whereas claudin-1 was mainly located in the wing and basal cell layers. M: Stained with hematoxylin to reveal corneal morphology. Bar, 50 μm.
Figure 2.
 
RT-PCR detecting claudin species in THCE cells. Primers derived from claudin-1 to -5, -7, -9, -10, and -14 to -16 were used in RT-PCR amplification. Seven species (claudins -1, - 2, -3, -7, -9, -14, and -15) were detected in the human corneal cell line. The observed PCR products corresponded to their expected molecular weights. Right: Molecular size standards in base pairs.
Figure 2.
 
RT-PCR detecting claudin species in THCE cells. Primers derived from claudin-1 to -5, -7, -9, -10, and -14 to -16 were used in RT-PCR amplification. Seven species (claudins -1, - 2, -3, -7, -9, -14, and -15) were detected in the human corneal cell line. The observed PCR products corresponded to their expected molecular weights. Right: Molecular size standards in base pairs.
Figure 3.
 
LPS treatment of THCE cells decreases in TER in a dose- and time-dependent manner. THCE cells grown on tissue culture–treated filters were treated with LPS. At the times indicated, TER measurements were taken. Results represent an average of three filters per time point. (♦), Control; (▪), 0.3 μg/ml LPS; (▴), 1.0 μg/ml LPS; and (•), 3.0 μg/ml LPS.
Figure 3.
 
LPS treatment of THCE cells decreases in TER in a dose- and time-dependent manner. THCE cells grown on tissue culture–treated filters were treated with LPS. At the times indicated, TER measurements were taken. Results represent an average of three filters per time point. (♦), Control; (▪), 0.3 μg/ml LPS; (▴), 1.0 μg/ml LPS; and (•), 3.0 μg/ml LPS.
Figure 4.
 
Influence of LPS on the levels of TJ-associated proteins ZO-1, ZO-2, and occludin. Confluent THCE cells on six-well tissue culture plates were incubated with LPS (concentrations marked in micrograms per milliliter) for 6 hours. Total cell extracts were separated on a 5% to 15% gradient gel and subsequently transferred to nitrocellulose membrane. The strips that were divided into three pieces were then probed with ZO-1 (220 kDa), ZO-2 (160 kDa), and occludin (65 kDa), respectively. A dose-dependent decline in ZO-2 content was apparent, and an extra, smaller band of occludin appeared in LPS-treated cells.
Figure 4.
 
Influence of LPS on the levels of TJ-associated proteins ZO-1, ZO-2, and occludin. Confluent THCE cells on six-well tissue culture plates were incubated with LPS (concentrations marked in micrograms per milliliter) for 6 hours. Total cell extracts were separated on a 5% to 15% gradient gel and subsequently transferred to nitrocellulose membrane. The strips that were divided into three pieces were then probed with ZO-1 (220 kDa), ZO-2 (160 kDa), and occludin (65 kDa), respectively. A dose-dependent decline in ZO-2 content was apparent, and an extra, smaller band of occludin appeared in LPS-treated cells.
Figure 5.
 
Indirect immunofluorescence staining of occludin, ZO-1, ZO-2, and claudin-1 in cultured THCE cells. THCE cells were cultured in Transwell filters and allowed to form a monolayer. After incubation with 3.0μ g/ml LPS for 6 hours, cells were fixed and immunofluorescence staining was performed with antibodies against occludin, ZO-1, and ZO-2. The immunostaining of ZO-2 disappeared from cell borders after LPS treatment, whereas ZO-1, occludin, and claudin-1 staining apparently did not change. Bar, 50 μm.
Figure 5.
 
Indirect immunofluorescence staining of occludin, ZO-1, ZO-2, and claudin-1 in cultured THCE cells. THCE cells were cultured in Transwell filters and allowed to form a monolayer. After incubation with 3.0μ g/ml LPS for 6 hours, cells were fixed and immunofluorescence staining was performed with antibodies against occludin, ZO-1, and ZO-2. The immunostaining of ZO-2 disappeared from cell borders after LPS treatment, whereas ZO-1, occludin, and claudin-1 staining apparently did not change. Bar, 50 μm.
The authors thank James Zieske, Schepens Eye Research Institute, for critical reading and comments and other members of Fu-Shin Yu’s laboratory for useful discussion of the manuscript. 
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Figure 1.
 
Distribution of ZO-1, ZO-2, occludin, and claudin-1 in rat corneal epithelium. Cryostat sections (8 μm) of the normal rat corneas were immunofluorescence stained with antibodies against ZO-1, ZO-2, occludin, and claudin-1. Like control, in which nonspecific rabbit serum was used instead of first antibody, all four specific antibodies exhibited evenly distributed, weak nonspecific staining in entire epithelial layer. However, strong immunoreactivity of ZO-1, ZO-2, and occludin was present along the borders of the superficial epithelial cells, whereas claudin-1 was mainly located in the wing and basal cell layers. M: Stained with hematoxylin to reveal corneal morphology. Bar, 50 μm.
Figure 1.
 
Distribution of ZO-1, ZO-2, occludin, and claudin-1 in rat corneal epithelium. Cryostat sections (8 μm) of the normal rat corneas were immunofluorescence stained with antibodies against ZO-1, ZO-2, occludin, and claudin-1. Like control, in which nonspecific rabbit serum was used instead of first antibody, all four specific antibodies exhibited evenly distributed, weak nonspecific staining in entire epithelial layer. However, strong immunoreactivity of ZO-1, ZO-2, and occludin was present along the borders of the superficial epithelial cells, whereas claudin-1 was mainly located in the wing and basal cell layers. M: Stained with hematoxylin to reveal corneal morphology. Bar, 50 μm.
Figure 2.
 
RT-PCR detecting claudin species in THCE cells. Primers derived from claudin-1 to -5, -7, -9, -10, and -14 to -16 were used in RT-PCR amplification. Seven species (claudins -1, - 2, -3, -7, -9, -14, and -15) were detected in the human corneal cell line. The observed PCR products corresponded to their expected molecular weights. Right: Molecular size standards in base pairs.
Figure 2.
 
RT-PCR detecting claudin species in THCE cells. Primers derived from claudin-1 to -5, -7, -9, -10, and -14 to -16 were used in RT-PCR amplification. Seven species (claudins -1, - 2, -3, -7, -9, -14, and -15) were detected in the human corneal cell line. The observed PCR products corresponded to their expected molecular weights. Right: Molecular size standards in base pairs.
Figure 3.
 
LPS treatment of THCE cells decreases in TER in a dose- and time-dependent manner. THCE cells grown on tissue culture–treated filters were treated with LPS. At the times indicated, TER measurements were taken. Results represent an average of three filters per time point. (♦), Control; (▪), 0.3 μg/ml LPS; (▴), 1.0 μg/ml LPS; and (•), 3.0 μg/ml LPS.
Figure 3.
 
LPS treatment of THCE cells decreases in TER in a dose- and time-dependent manner. THCE cells grown on tissue culture–treated filters were treated with LPS. At the times indicated, TER measurements were taken. Results represent an average of three filters per time point. (♦), Control; (▪), 0.3 μg/ml LPS; (▴), 1.0 μg/ml LPS; and (•), 3.0 μg/ml LPS.
Figure 4.
 
Influence of LPS on the levels of TJ-associated proteins ZO-1, ZO-2, and occludin. Confluent THCE cells on six-well tissue culture plates were incubated with LPS (concentrations marked in micrograms per milliliter) for 6 hours. Total cell extracts were separated on a 5% to 15% gradient gel and subsequently transferred to nitrocellulose membrane. The strips that were divided into three pieces were then probed with ZO-1 (220 kDa), ZO-2 (160 kDa), and occludin (65 kDa), respectively. A dose-dependent decline in ZO-2 content was apparent, and an extra, smaller band of occludin appeared in LPS-treated cells.
Figure 4.
 
Influence of LPS on the levels of TJ-associated proteins ZO-1, ZO-2, and occludin. Confluent THCE cells on six-well tissue culture plates were incubated with LPS (concentrations marked in micrograms per milliliter) for 6 hours. Total cell extracts were separated on a 5% to 15% gradient gel and subsequently transferred to nitrocellulose membrane. The strips that were divided into three pieces were then probed with ZO-1 (220 kDa), ZO-2 (160 kDa), and occludin (65 kDa), respectively. A dose-dependent decline in ZO-2 content was apparent, and an extra, smaller band of occludin appeared in LPS-treated cells.
Figure 5.
 
Indirect immunofluorescence staining of occludin, ZO-1, ZO-2, and claudin-1 in cultured THCE cells. THCE cells were cultured in Transwell filters and allowed to form a monolayer. After incubation with 3.0μ g/ml LPS for 6 hours, cells were fixed and immunofluorescence staining was performed with antibodies against occludin, ZO-1, and ZO-2. The immunostaining of ZO-2 disappeared from cell borders after LPS treatment, whereas ZO-1, occludin, and claudin-1 staining apparently did not change. Bar, 50 μm.
Figure 5.
 
Indirect immunofluorescence staining of occludin, ZO-1, ZO-2, and claudin-1 in cultured THCE cells. THCE cells were cultured in Transwell filters and allowed to form a monolayer. After incubation with 3.0μ g/ml LPS for 6 hours, cells were fixed and immunofluorescence staining was performed with antibodies against occludin, ZO-1, and ZO-2. The immunostaining of ZO-2 disappeared from cell borders after LPS treatment, whereas ZO-1, occludin, and claudin-1 staining apparently did not change. Bar, 50 μm.
Table 1.
 
Primers for Detecting mRNA Expression of Claudins in THCE Cells by RT-PCR
Table 1.
 
Primers for Detecting mRNA Expression of Claudins in THCE Cells by RT-PCR
Claudin Sequence (5′–3′) PCR Size Reference
Claudin-1 s TCA GCA CTG CCC TGC CCC AGT 506 bp 41
as TGG TGT TGG GTA AGA GGT TGT
Claudin-2 s ACA CAC AGC ACA GGC ATC AC 319 bp 12
as TCT CCA ATC TCA AAT TTC ATG C
Claudin-3 s AAG GCC AAG ATC ACC ATC GTG 304 bp 13
as AGA CGT AGT CCT TGC GGT CGT
Claudin-4 s TGG ATG AAC TGC GTG GTG CAG 361 bp 42
as GAG GCG GCC CAG CCG ACG TA
Claudin-5 s ATG TCG TGC GTG GTG CAG AG 413 bp 43
as GGT GCA GAC CCA GGC GCC GCA
Claudin-7 s AGT GGC AGA TGA GCT CCT ATG 364 bp
as GTT ATA AAA GTC TGT GAC AAT CT
Claudin-9 s TTC ATC GGC AAC AGC ATC GT 403 bp
as GCC CAG CCC AGG TAG AGG GA
Claudin-10 s TGT ACC AAA GTC GGA GGC TC 370 bp
as GCA TTT TTA TCA AAC TGT TTT GAA GG
Claudin-14 s CTC ATG GTC ATC TCC TGC CTG 469 bp
as ACG TAG TCG TTC AGC CTG TAC
Claudin-15 s CAT CAC CAC CAA CAC CAT CTT 540 bp
as GCT GCT GTC GCC TTC TTG GTC
Claudin-16 s TTT GGA TTT CTC ACC CTG CTC 398 bp 30
as TGT GCG AGG GGC TGA GTA TGA
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