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Published ahead of print on January 23, 2003, doi:10.1165/rcmb.2002-0180OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 62-70, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0180OC

Heterogeneity of Claudin Expression by Alveolar Epithelial Cells

Fushan Wang*, Brandy Daugherty*, Lisa L. Keise, Zhangyong Wei, Joseph P. Foley, Rashmin C. Savani and Michael Koval

Departments of Physiology and Pediatrics, and Institute for Environmental Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania

Address correspondence to: Michael Koval, Department of Physiology, University of Pennsylvania School of Medicine, B 400 Richards Building/6085, 3700 Hamilton Walk, Philadelphia, PA 19104-6085. E-mail: mkoval{at}mail.med.upenn.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Claudins are proteins that participate in epithelial barrier function and regulate paracellular permeability. By immunohistochemistry of adult rat lung sections, claudin-3, claudin-4, and claudin-5 were found to be co-expressed by type II alveolar epithelial cells. Claudin-3 and claudin-4 were also co-expressed by some alveolar epithelial cells adjacent to type II cells. In contrast, claudin-5 was expressed throughout the alveolus. Isolated primary rat alveolar epithelial cells in culture also expressed claudin-3, claudin-4, and claudin-5, but showed little claudin-1 and claudin-2 expression. Claudin expression by isolated cells at both the mRNA and protein level varied with time in culture. In particular, claudin-3 and claudin-5 co-localized and were distributed around the alveolar cell periphery, but claudin-4 expression was heterogeneous. We also found that paracellular permeability was increased when cultured alveolar epithelial cells were treated with a fatty acid amide, methanandamide. Methanandamide did not alter cell viability. Claudin-3, claudin-4, claudin-5, occludin, and zona occludens 1 remained localized to cell–cell contact sites at the plasma membrane in methanandamide-treated cells, suggesting that plasma membrane localization of these junction proteins is not sufficient for maintaining barrier function. However, methanandamide-treated cells showed a 12-fold increase in claudin-5 expression and a 2- to 3-fold increase in claudin-3, consistent with the notion that specific changes in claudin expression levels may correlate with changes in alveolar epithelial barrier function.

Abbreviations: fetal bovine serum, FBS • minimum essential medium, MEM • phosphate-buffered saline, PBS • transepithelial resistance, TER • zona occludens 1, ZO-1


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Disruption of the paracellular alveolar permeability barrier is a significant pathologic consequence of acute lung injury (1). The permeability barrier in terminal airspaces of the lung is due in large part to tight junctions between alveolar epithelial cells, which regulate the flow of molecules between extracellular apical and basolateral compartments (2, 3). Transmembrane proteins in the occludin and claudin families are the major transmembrane structural elements of tight junctions (4–6). It has previously been shown that alveolar epithelial cells express occludin and zona occludens 1 (ZO-1) as part of the tight junction complex (7, 8). In addition to these components, the importance of claudins in pulmonary barrier function is underscored by the viability of occludin-deficient mice (9). However, to date, the claudins expressed by alveolar epithelial cells have not been defined.

Using primary type II alveolar epithelial cells cultured on permeable supports, it is routinely possible to obtain transepithelial resistances greater than 500 {Omega} x cm2, consistent with the formation of tight junctions (10–12). This system was used to examine the expression pattern of five different claudins by alveolar epithelial cells at the level of protein and mRNA expression as a function of time in culture. Claudin expression by alveolar epithelial cells in situ was also examined by immunohistochemistry. In the course of unrelated studies examining the effect of methanandamide (13) on intercellular communication between alveolar epithelial cells, methanandamide was fortuitously found to increase paracellular permeability between alveolar epithelial cells, without decreasing cell viability. Agents which cause controlled increases in alveolar paracellular permeability are of interest because they might be useful for delivery of aerosolized drugs to the vasculature (14) or for gene therapy (15), where access of vectors to the epithelial basolateral surface is desired. Given this, the effect of methanandamide on alveolar epithelial cells in culture was characterized to determine whether methanandamide-induced increases in paracellular permeability resulted from changes in claudin expression and/or localization.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
Sprague-Dawley rat alveolar type II cells were isolated from lavaged, perfused lungs by elastase digestion using the method of Dobbs and colleagues (16), with modifications (17). Cells were biopanned using IgG-coated culture dishes to remove alveolar macrophages and other Fc-receptor–expressing cells. To obtain type II cells with high purity, we used a second round of immunodepletion in which cells were incubated with Biomag beads coated with rabbit IgG (Polysciences, Warrington, PA). Using this approach, preparations routinely contained 90–95% type II cells (17). To allow cells to progress to a type I–like phenotype (11, 18), cells were cultured on standard tissue culture–treated plastic dishes (seeding density: 6 x 10 6 cells/35 mm dish) or 0.4 µm pore size, Transwell permeable membrane supports (Corning Life Sciences, Acton, MA) (seeding density: 1.5 x 10 6 cells/1 cm2) in Earle's Minimal Essential Medium (Life Technologies, Rockville, MD) containing 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (MEM) and 10% fetal bovine serum (FBS).

A549 cells were routinely cultured in MEM + 10% FBS. Human and mouse claudin-5 cDNAs were obtained from the American Type Culture Collection (Manassas, VA). The mouse claudin-5 cDNA was in the pCMV-SPORT6 mammalian expression vector and human claudin-5 was excised from the pOTB7 vector with Eco RI and Xho I and ligated into pcDNA3. A549 cells at 80% confluence were transiently transfected with claudin-5 cDNA using FuGENE-6 reagent (Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturer's instructions 2 d before use for experiments.

Measurement of Cell Barrier Function
Cells were cultured in MEM + 1% FBS 1 d before measurements of barrier function. Transepithelial resistance (TER) was measured using an Ohmmeter (World Precision Instruments, Sarasota, FL). Alternatively, epithelial barrier function was assessed by simultaneously measuring the diffusion of fluorescent dyes across the cell monolayer. In these experiments, the medium in the bottom chamber was replaced with phosphate-buffered saline (PBS) + 0.675 mM CaCl2 + 0.2 mM MgCl2 and medium in the top chamber was replaced with PBS + Ca +Mg containing 0.1 mg/ml carboxyfluorescein (0.37 kD) and 1.0 mg/ml Texas Red Dextran (10 kD). The cells were then further incubated at 37°C for 2 h. The cell chamber was removed, and the amount of fluorophore that diffused into the lower chamber was measured using a PE Biosystems (Foster City, CA) multiwell plate fluorimeter.

Viability Assay
A variation of the Live/Dead fluorometric assay (Molecular Probes, Eugene, OR) was used to measure cell viability, because both esterase cleavage of calcein-AM and retention of free calcein are properties of living cells (19). Alveolar epithelial cells plated in 24-well culture plates were incubated with either vehicle control or methanandamide (Sigma, St. Louis, MO) for 16 h. The cells were then washed and incubated with MEM containing 1 µM calcein-AM for 30 min, washed, and the amount of calcein-AM hydrolysis (cell-associated calcein) was determined using a PE Biosystems microplate fluorometer. Cells treated with either agent did not show significant differences in cell-associated calcein (see RESULTS). Comparable results were also routinely obtained by measuring trypan blue exclusion.

Immunofluorescence
Anti-occludin, anti–ZO-1, anti-connexin32, and anti-claudin antibodies were from Zymed (S. San Francisco, CA); anti-RTI40 antibodies and anti-ABCA3 antibodies were kind gifts of Drs. Leland Dobbs (UCSF) (20) and Henry Shuman (U. Penn School of Medicine) (21). Anti-connexin43 antibodies were developed as previously described (22). Cultured cells were plated onto permeable supports, washed with PBS, fixed and permeabilized with methanol/acetone 1:1 for 2 min, washed with PBS + 0.5% Triton X-100 (PBS/TX), followed by PBS/TX + 2% goat serum (PBS/TX/GS). The cells were then incubated with primary antiserum diluted into PBS + 2% goat serum (PBS/GS) for 1 h at room temperature, washed, and then incubated with Cy2-conjugated Goat anti-rabbit IgG (Jackson Immunoresearch, Malvern, PA, catalog #711–225–152) and Cy3-conjugated Goat anti-mouse IgG (Jackson Immunoresearch, catalog #715–165–151) for 2 h at room temperature. Note that these secondary antibodies are designed for multiple labeling experiments and are specially purified to avoid cross species reactivity with primary IgG. Single label experiments were done to confirm the lack of cross-reactivity and signal spillover in immunofluorescence experiments (not shown). Also, immunofluorescence experiments using HeLa cells transfected to express claudin-1, -2, -3, -4 or -5 were done to confirm that anti-claudin antibodies did not recognize other claudin species (not shown).

For immunohistochemistry, rat lungs were inflated, fixed by perfusion with 1% paraformaldehyde, and then sequentially incubated in 5 mM NH4Cl, 10% sucrose, 20% sucrose, and 30% sucrose in PBS. The samples were chopped into 3 mm3 pieces and then frozen into embedding medium (Tissue-Tek). The samples were sliced with a microtome to 5 µm thickness and mounted onto slides. Tissue sections were incubated for 30 min at room temperature with 1 M glycine in PBS to reduce nonspecific cross-linking and autofluorescence. The samples were then washed with PBS, incubated for 10 min at room temperature with 1 mg/ml of NaBH4 in PBS to further reduce autofluorescence, and washed with PBS/TX and PBS/TX/GS. The sections were incubated overnight at 4°C with primary antisera diluted with PBS/TX/GS, washed, and then incubated with Cy2-conjugated Goat anti-rabbit IgG and Cy3-conjugated Goat anti-mouse IgG for 2 h at room temperature. Cells and tissues were imaged using either a Nikon inverted fluorescence microscope (Nikon, Melville, NY) outfitted with a BioRad confocal fluorescence system (BioRad, Hercules, CA) or an Olympus IX70 conventional fluorescence microscope (Olympus, Melville, NY) with a Hamamatsu Orca CCD camera (Hamamatsu Orca, Hamamatsu City, Japan) and ImagePro image analysis software (Media Cyternetics, Carlsbad, CA).

Immunoblot
Cultured cells were harvested in PBS containing 1.0% Triton X-100, 0.2% SDS, 1 mM sodium vanadate, 10 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml of leupeptin, and 1 µg/ml of pepstatin. Protein concentration was measured with the bicinchoninic acid reagent (BioRad). Samples normalized for total amount of protein were resolved using a 12% NuPAGE BIS-TRIS polyacrylamide gel run with the MES SDS running buffer system (Invitrogen, Carlsbad, CA), transferred to polyvinylidene difluoride membrane, and then blocked overnight with 40 mM TRIS, pH 7.4, 5% Carnation powdered milk, and 0.1% Tween 20 (Blotto). The blots were incubated with Blotto containing specific antisera, washed, and then further incubated with goat anti-rabbit IgG or goat anti-mouse IgG conjugated to horseradish peroxidase (Roche Molecular Biochemicals). Specific signals corresponding to a given protein were detected with an enhanced chemiluminescence reagent (ECL; Amersham, Piscataway, NJ) and quantified with the Kodak EDAS 1D analysis system (Kodak, Rochester, NY). Data were from triplicate determinations ± SE, and significance was determined using Student's t test.

RT-PCR
PCR primers corresponding to mouse claudin-1, -2, -3, -4, and -5 are shown in Table 1 and were obtained from Invitrogen/Life Technologies. Semiquantitative RT-PCR was performed as previously described (23). RNA was isolated from untreated or treated cells with TRIzol reagent (Life Technologies). RNA was treated with DNase (Promega, Madison, WI) to remove contaminating genomic DNA and then converted to cDNA with reverse transcriptase and random hexamer primers (Invitrogen). The resulting cDNAs were used as source material for PCR reactions using primers specific for a given claudin construct. QuantumRNA 18S internal standards (Ambion, Austin, TX) were used to normalize for input cDNA. The identity of the PCR products amplified from rat alveolar epithelial cells was confirmed by DNA sequencing (not shown). The size and amount of the PCR products generated were determined by agarose gel electrophoresis in the presence of ethidium bromide and analyzed with the Kodak EDAS 1D analysis package. Different starting conditions and ratios of cDNA to 18S standards were examined to ensure that measurements were in a linear range for product formation.


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  		TABLE 1 Primers used for RT-PCR

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Claudin Expression by Alveolar Epithelial Cells
Because claudins play an important role in epithelial barrier function in other cell systems, we wanted to define the expression pattern of claudins by alveolar epithelial cells. The analysis was restricted to claudin-1, -2, -3, -4, and -5 using commercially available antibodies. Claudin-1 and claudin-2 showed extremely low levels of immunostaining for lung sections (not shown); however, there was prominent expression of claudin-3, claudin-4, and claudin-5 by adult rat lung (Figure 1). Cells expressing ABCA3 also expressed claudin-3, suggesting expression of claudin-3 by type II cells (24). However, claudin-3 was not restricted to type II cells, because there were also areas of the alveolus where claudin-3 was expressed, but ABCA3 was not. Claudin-3 and claudin-4 showed considerable overlap, indicating that nearly all of the cells expressing claudin-3 also expressed claudin-4. There were also some cells that showed claudin-3, but not claudin-4 expression (Figures 1D–1F, arrow). A typical field from a normal adult rat lung at this magnification shows one or two cells expressing claudin-3, but not claudin-4. The expression pattern for claudin-5 was much more extensive than claudin-3 or claudin-4, suggesting that most alveolar epithelial cells expressed claudin-5. In fact, both claudin-3 (not shown) and claudin-4 (Figure 1) showed partial overlap with cells expressing claudin-5 in the alveolus.



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  		Figure 1. Claudin expression by alveolar epithelial cells in situ. Adult rat lung sections were immunostained for claudin-3 (A, D), claudin-4 (E, H) claudin-5 (G), and ABCA3 (B) as a type II cell marker. Sections incubated with Cy2 Goat anti-rabbit IgG (J) and Cy3 Goat anti-mouse IgG (K) are also shown to indicate the level of nonspecific labeling. Merged images are shown in (C, F, I, L). Claudin-3 and ABCA3 showed significant overlap (A–C; arrowhead), although there were also areas where cells stained for claudin-3 expression alone (A–C; arrows). Claudin-3 and claudin-4 showed very similar immunolabeling patterns, and cells frequently expressed both claudins (D–F; arrowheads) or claudin-3 alone (arrow). Claudin-5 expression was prominent, with areas showing cells co-expressing claudin-5 and claudin-4 (G–I; arrowheads) or mainly claudin-5 alone (G–I; arrows).

 
Given previous reports suggesting that claudin-5 was not expressed by alveolar epithelial cells (25), claudin expression by isolated primary rat alveolar epithelial cells was examined by confocal imumunofluorescence microscopy (Figure 2). Alveolar epithelial cells cultured for 6 d on permeable supports showed virtually complete co-localization of claudin-3 and claudin-5 at the plasma membrane, with smooth, continuous labeling surrounding each cell. In contrast, claudin-4 was heterogeneously expressed, where 38.7 ± 8.1% of the cells showed claudin-4 labeling by immunofluorescence (n = 10 fields, two independent experiments). Thus, claudin-3 and claudin-4 were not coordinately regulated by cultured alveolar epithelial cells. Claudin-3 and claudin-4 showed partial co-localization at the cell surface; however, there were areas where claudin-4 was distinct from claudin-3. Spatial separation of claudin-3 and claudin-4 was particularly noticeable in the x-z plane (Figures 2D–2F), suggesting that some of the apparent overlap in x-y images may have been due to expression of claudin-3 and claudin-4 in different planes of the junctional membrane.



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  		Figure 2. Claudin localization by cultured alveolar epithelial cells. Rat alveolar epithelial cells were cultured for 6 d on permeable supports, fixed, permeabilized, and then immunolabeled with rabbit anti-claudin-3 (A, D) and mouse anti-claudin-5 (B) or mouse anti-claudin-4 (E). The cells were subsequently labeled with Cy2-goat anti-rabbit IgG (A, D) and Cy3-goat anti-mouse IgG (B, E) and then imaged by confocal immunofluorescence microscopy. Merged images are shown in C, F. The main images show the X-Y plane of the sample, and black arrowheads denote the location of the X-Z scan depicted below each image. White arrowheads in D–F show areas where claudin-3 and claudin-4 co-localized. (G–I) Day 6 alveolar epithelial cells were immunostained for RTI40 (G), ABCA3 (H, arrowheads) or Connexin43 (I) as markers for cell phenotype. Bars: 20 µ.

 
Considering the heterogeneous expression of claudin-4 by these cells, we decided to examine the expression of well characterized markers of cell phenotype by cultured alveolar epithelial cells (Figures 2G–2J). Nearly all of the cells cultured for 6 d on permeable supports expressed RTI40 (also referred to as T1{alpha}), a distinctive marker of type I alveolar epithelial cells (20, 26, 27). In contrast, there were very few cells expressing the type II cell marker ABCA3, and those that did, did not have ABCA3-positive lamellar bodies with the typical ring labeling pattern (24). We also examined expression of gap junction proteins (connexins) which are differentially expressed by alveolar epithelial cells (28). Connexin32, which is highly expressed by type II cells, was not present in Day 6 cells cultured on permeable supports. In contrast, nearly all of the cells expressed connexin43 (Figure 2I) . At the level of immunofluorescence, cells cultured on tissue culture plastic showed expression of RTI40, ABCA3, connexin32, and connexin43 comparable to that observed for alveolar epithelial cells cultured on permeable supports (not shown). Thus, Day 6 alveolar epithelial cells shared some characteristics of type I alveolar epithelial cells. Progression of these cells toward a type I–like phenotype was expected, given that the medium contained 10% FBS, which as been shown to promote loss of type II cell markers and acquisition of type I cell markers (11, 29, 30). However, FBS may have unanticipated effects on alveolar epithelial cells. Also, given the heterogeneous expression of claudin-4 in the monolayer, these results suggest that regulation of claudin expression by these cells was not necessarily tightly linked to the loss of type II cell markers and gain of type I cell markers.

Claudin expression was also examined biochemically. As shown in Figure 3, alveolar epithelial cells show varying levels of claudin expression as a function of days in culture. There was little, if any, expression of claudin-1 or claudin-2 by cells either on a solid substratum or on permeable supports. Claudin-3 showed the highest level of expression after overnight culture (Day 1) and decreased with increasing time in culture. For cells cultured on solid substrata, the total level of claudin-4 protein expression slightly decreased with time in culture. In contrast, cells cultured on permeable supports showed a slight increase in total claudin-4 protein expression with time in culture. Note that in either case, the percentage of claudin-4 expressing cells at Day 6 was roughly equivalent for alveolar epithelial cells cultured on solid substrata (47.3 ± 6.6%, n = 6 fields) and cells plated on permeable supports (38.7 ± 8.1%). Because the total levels of claudin-4 protein expression were largely unchanged with time in culture, but the number of cells expressing claudin-4 greatly decreased, this would suggest that Day 6 cells, which retained claudin-4 expression, did so at a higher level, on a per cell basis, as compared with Day 1 cells. However, given that indirect immunofluorescence is not quantitative, this cannot be definitively determined.



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  		Figure 3. Claudin expression by cultured alveolar epithelial cells. (A) Alveolar epithelial cells were cultured on permeable supports (lanes 1–3, 7–9, 13–15, 19–21) or tissue culture plastic (lanes 4–6, 10–12, 16–18, 22–24) for 1 (lanes 1, 4, 7, 10, 13, 16, 19, 22), 3 (lanes 2, 5, 8, 11, 14, 17, 20, 23) or 6 d (lanes 3, 6, 9, 12, 15, 18, 21, 24), then harvested and analyzed by immunoblot for claudin-1 (lanes 1–6), claudin-3 (lanes 7–12), claudin-4 (lanes 13–18) or claudin-5 (lanes 19–24). Claudin-2 immunoblots showed no protein expression (not shown). Densitometric analysis of claudin-3 (B), claudin-4 (C), and claudin-5 (D) expression, normalized to the level of expression by Day 1 cells plated on tissue culture plastic. In the case of claudin-5, the 29-kD band was normalized to the 27 kD band for Day 1 cells cultured on plastic (*P < 0.05 compared with Day 1 cells). (E) A549 cells transfected with mouse claudin-5 cDNA (lanes 1 and 6), human claudin-5 cDNA (lanes 2 and 7), nontransfected controls (lanes 5 and 10) or Day 3 rat alveolar epithelial cells (lanes 4 and 9) were harvested and analyzed by immunoblot using monoclonal (lanes 1–5) or polyclonal (lanes 6–10) anti–claudin-5 IgG from Zymed (see MATERIALS AND METHODS). Lanes 3 and 8 were not loaded with samples.

 
To further confirm claudin expression by alveolar epithelial cells, claudin mRNA expression was analyzed by semi-quantitative RT-PCR. As shown in Figure 4, mRNA expression of claudin-3, claudin-4, and claudin-5 partially agreed with total protein expression levels, confirming our immunoblot results and suggesting partial regulation of claudin expression at the transcriptional level. However, there were also some differences between levels of mRNA and protein expression, particularly for claudin-4 expression by cells cultured on plastic and, to a lesser extent, for claudin-5 expression, suggesting post-translational regulation of claudin expression levels as well.



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  		Figure 4. Claudin mRNA expression by cultured alveolar epithelial cells. (A) Alveolar epithelial cells were cultured on permeable supports (lanes 1–3, 7–9, 13–15) or tissue culture plastic (lanes 4–6, 10–12, 16–18) for 1 (lanes 1, 4, 7, 10, 13, 16), 3 (lanes 2, 5, 8, 11, 14, 17) or 6 (lanes 3, 6, 9, 12, 15, 18) d, then harvested and analyzed by semiquantitative RT-PCR for claudin-3 (lanes 1–6), claudin-4 (lanes 7–12) or claudin-5 (lanes 13–18). The upper band corresponds to 18 S RNA, and the lower band (arrowhead) corresponds to the specific claudin mRNA. Claudin-3 (B), claudin-4 (C), and claudin-5 (D) expression were analyzed by densitometry and normalized to 18 S RNA expression. Results were the average ± SE of independent triplicate measurements. (*P < 0.05, compared with Day 1 cells)

 
In contrast to the other claudins, claudin-5 resolved on 12% NuPAGE gels with MES SDS running buffer showed an apparent 27 kD + 29 kD doublet. To determine the identity of the two bands recognized by anti–claudin-5 antibodies, A549 cells transfected with either mouse or human claudin-5 cDNA were examined (Figure 3). Recombinant claudin-5 showed only a single band by immunoblot, which migrated with a molecular mass comparable to that of the lower, 27-kD form. Similar results were obtained for claudin-5–transfected HeLa cells (not shown). Interestingly, different commercially available anti–claudin-5 antibodies showed different affinities for the two bands expressed by rat alveolar epithelial cells. Monoclonal anti–claudin-5 (Zymed #35–2500) recognized both bands equivalently, however, polyclonal anti–claudin-5 (Zymed #34–1600) preferentially labeled the upper band. Both antibodies recognized recombinant mouse and human claudin-5. Given that Day 1 alveolar epithelial cells expressed claudin-5 mRNA and showed only the 27-kD band, it seems likely that this band corresponds to claudin-5. Whether the upper band is a claudin isoform or post-translational modification of claudin-5 is unclear. Preliminary studies indicate that both bands were resistant to alkaline phosphatase, although phosphorylation or some other potential modification cannot be ruled out at this time. Given these caveats, the monoclonal antibody, which equivalently recognized both bands, was used for immunoblot analysis and each band was separately quantified.

Methanandamide Decreases Alveolar Epithelial Barrier Function
While screening pharmacologic agents for other effects on alveolar epithelial cells, it was found that methanandamide caused a decrease in the barrier function of alveolar epithelial cells. Primary type II alveolar epithelial cells were isolated and cultured on permeable supports for 5 d. Cells cultured in this manner routinely formed monolayers with TER greater than 500 {Omega} x cm2, consistent with formation of a paracellular barrier. The cells were then incubated with either ethanol or increasing concentrations of methanandamide. As shown in Figure 5B, alveolar epithelial cells incubated for 16 h with methanandamide showed significant increases in paracellular permeability (decreased TER). Overnight incubation was required for this effect, because cells treated for 4 h with methanandamide showed little effect on TER (Figure 5A). Also, the decrease in barrier function was not due to cell death. Using a calcein-AM cleavage assay for assessing cell viability, we found no significant difference in viability of control cells (RFU = 386 ± 27 [n = 4]) and cells treated with 20 µM methanandamide for 16 h (RFU = 377 ± 46 [n = 4]). Comparable results were also routinely obtained by measuring trypan blue exclusion.



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  		Figure 5. Decreased barrier function induced by methanandamide. (A, B) Alveolar epithelial cells were cultured on permeable supports for 5 d and transepithelial resistance (TER) was measured to obtain baseline values. The cells were then treated with methanandamide and TER was measured 4 h (A) or 16 h (B) following treatment and normalized to baseline values. After 16 h treatment, cells treated with methanandamide showed significant decreases in TER as compared with vehicle-treated controls (*P < 0.05). (C, D) The cells were treated with methanandamide for 16 h, and then transepithelial barrier permeability was determined by measuring the relative fluorescence units (RFU) of Texas Red (C) or carboxyfluorescein (D) transferred from the upper chamber to the lower one. The dashed line corresponds to RFU values obtained from cells treated with vehicle alone. Cells treated with methanandamide showed significant increases in paracellular dye transfer in all cases as compared with controls (P < 0.05).

 
The transcellular permeability of two different sized fluorescent dyes, carboxyfluorescein and Texas Red dextran, was also used to measure paracellular permeability (Figures 5C and 5D). Consistent with the decrease in TER induced by methanandamide, a 16-h incubation with methanandmide caused significant increases in the paracellular permeability of carboxyfluorescein and Texas Red dextran across the cell monolayer. Both dyes showed enhanced paracellular diffusion, suggesting that the alveolar epithelial barrier permeability to both macromolecules and small molecules was enhanced by treatment with methanandamide.

The effect of methanandamide on tight junction protein localization was also examined. As shown in Figure 6, alveolar epithelial cells treated for 16 h with 20 µM methanandamide showed little, if any, alteration in claudin localization. Both claudin-3 and -5 retained their plasma membrane localization. Claudin-4 expression also remained heterogeneously expressed by methanandamide-treated cells (40.7 ± 10.0%, n = 10 fields, two independent experiments) and was similar to levels of expression by untreated cells (38.7 ± 8.1%). However, claudin-4 appeared to be more stringently localized to the plasma membrane in methanandamide-treated cells as compared with untreated controls, which might contribute to changes in paracellular permeability. Two other markers for tight junction morphology, occludin and ZO-1, remained localized to the plasma membrane in the presence of methanandamide (Figure 6). However, there were some subtle changes in the appearance of these makers, notably an increase in linear inclusions originating at the plasma membrane and oriented toward the nucleus, suggesting potential reorganization of the plasma membrane in response to methanandamide treatment.



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  		Figure 6. Junction-associated proteins remain plasma membrane localized in the presence of methanandamide. Alveolar epithelial cells were cultured for 5 d and then treated with 20 µM methanandamide for 16 h. The cells were then fixed, permeabilized and immunostained for claudin-3 (A, D), claudin-4 (E) or claudin-5 (B). Merged images are shown in C, F. The main images show the X-Y plane of the sample, and black arrowheads denote the location of the X-Z scan depicted below each image. The localization of these proteins in methanandamide treated alveolar epithelial cells was comparable to controls (Figure 2). (G–J) Alveolar epithelial cells were cultured for 5 d and then treated with control vehicle (control; G, I) or 20 µM methanandamide (MA; H, J) for 16 h. The cells were then fixed, permeabilized, and immunostained for either occludin (occ; G, H) or ZO-1 (I, J). Bar: 20 µ.

 
Exposure to methanandamide might also alter the level of claudin expression, which, in turn, could have downstream effects on barrier function. To determine whether this was the case, the expression of claudins and other junction-associated proteins by control and methanandamide-treated cells was examined. By immunoblot, there was little, if any, effect of methanandamide on expression of claudin-4, occludin or ZO-1 (Figure 7). Claudin-3 protein expression was modestly enhanced by methanandamide; however, at the mRNA level neither claudin-3 nor claudin-4 showed a significant change in expression (not shown).



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  		Figure 7. Claudin expression is upregulated by methanandamide treatment. (A) Alveolar epithelial cells were cultured for 5 d and then treated with control vehicle (con; lanes 1, 3, 5, 7, 9) or 20 µM methanandamide (MA; lanes 2, 4, 6, 8, 10) for 16 h. The cells were then harvested and analyzed by immunoblot for claudin-3 (lanes 1 and 2), claudin-4 (lanes 3 and 4), claudin-5 (lanes 5 and 6), occludin (lanes 7 and 8) and ZO-1 (lanes 9 and 10; *). (B) Densitometric analysis of protein expression. Shown is the ratio of protein expression for methanandamide-treated cells versus control cells for triplicate independent determinations. For claudin-5, the gray bar denotes the upper band in the immunoblot and the black bar denotes the lower band. (*P < 0.05 versus control).

 
In contrast, claudin-5 expression showed a dramatic increase in protein level in response to methanandamide treatment, where the intensity of the lower claudin-5 band increased greater than 12-fold in response to methanandamide. The magnitude of the upper band did not change. Interestingly, the elevated claudin-5 protein level was reflected by only a modest increase in claudin-5 mRNA, which increased 1.4 ± 0.2 -fold (n = 3) for methanandamide treated-cells as compared with control cells. This suggests that methanandamide treatment altered claudin-5 protein levels largely by a post-translational mechanism.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Claudins form and regulate the paracellular permeability barrier between epithelial cells. Claudin-3, claudin-4, and claudin-5 were found to be three of the major claudins expressed by type II alveolar epithelial cells. In addition, claudin-5 was also present throughout the alveolus, consistent with expression by type I cells and endothelial cells (25), as well as type II cells. In contrast, claudin-3 and claudin-4 expression was more limited, showing mainly expression by type II cells and, to a lesser extent, other cells immediately adjacent to type II cells. Taken together, these results suggest that there are likely to be multiple classes of tight junction interfaces between alveolar epithelial cells, which might reflect different states of paracellular permeability in different locations in the alveolus.

Differences in claudin expression suggest differences in paracellular permeability. This has largely been examined in model cell systems. For instance, MDCK cells transfected to overexpress claudin-3 (31) or claudin-4 (32) show increased transepithelial resistance, suggesting a function for claudins in forming the paracellular permeability barrier. However, claudins not only form the barrier to prevent diffusion of macromolecules across a polarized cell monolayer, but also form specific paracellular ion channels (33). For instance, a role for claudin-4 in regulating paracellular sodium transport can be inferred from studies showing that overexpression of claudin-4 by MDCK cells results in increased TER, due to a specific decrease in paracellular sodium permeability (34). Because regulation of airspace sodium content is critical for maintaining proper lung fluid levels (1, 34), it seems feasible that one role for claudin-4 in lung might be to help regulate the vectorial flow of sodium from the airspace to the interstitium, in concert with apical membrane sodium channels (10, 35, 36) and aquaporins (37–39).

In contrast to claudin-3 and claudin-4, there is no direct evidence implicating a role for claudin-5 in increasing epithelial barrier function, although it is associated with tight junction strands in endothelial cells and transfected fibroblasts (25, 40). In fact, previous studies indicated that claudin-5 was expressed exclusively by endothelial cells (25). However, the data presented above and published reports of claudin-5 expression by retinal pigment epithelium (41) support the argument that epithelial cells also have the capacity to express claudin-5.

There are a total of 20 different mammalian claudins, and it is likely that alveolar epithelial cells express some of these other claudin species as well, in addition to the claudins examined in this study. For instance, the expression of claudin-7 and claudin-8 at the mRNA level by cultured primary alveolar epithelial cells was recently reported (42) and claudin-18 is also expressed by alveolar epithelium (43). In addition, claudin-10, claudin-12, claudin-13, and claudin-15 are all present in total lung at the mRNA level (5, 43, 44). However, this study was restricted to claudins which could be examined at the protein level using commercially available anti-claudin antibodies.

Claudin protein expression was largely equivalent for alveolar epithelial cells cultured on either permeable supports or tissue culture plastic, although claudin mRNA expression showed some differences. For instance, claudin-4 mRNA expression by cells on permeable supports was largely unchanged, although it decreased with time in culture when alveolar epithelial cells were cultured on tissue culture plastic. Because differences between cells cultured in the two different systems showed greater differences in claudin mRNA expression as opposed to claudin protein expression, this suggests that post-translational mechanisms, such as claudin trafficking and turnover, may play a significant role in regulating claudin expression. Also, despite these differences, alveolar epithelial cells on permeable supports and tissue culture plastic at Day 6 showed comparable high levels of RTI40 expression, a type I cell marker (20, 26, 27) and low levels of ABCA3 expression, a type II cell marker (24). Thus, variability in claudin expression by cultured cells suggests that claudin expression may not be tightly linked to determinants of alveolar epithelial cell phenotype. Also, claudin expression by alveolar epithelial cells is likely to reflect culture conditions and may be influenced by medium components, such as serum. Despite these caveats, alveolar epithelial cells cultured on permeable supports enabled us to measure transepithelial barrier function of these cells in vitro.

Methanandamide was found to increase paracellular permeability between cultured alveolar epithelial cells, without decreasing cell viability. Methanandamide is a nonhydrolyzable analog of the fatty acid amide anandamide, which binds to the cannabinoid receptors CB1 and CB2 (45). Type II alveolar epithelial cells express the CB1 cannabinoid receptor, although type I cells do not (46). Also, CB1 expression is rapidly lost by alveolar epithelial cells in culture (46). Methanandamide, and the naturally occurring compounds anandamide and oleamide, can also interact with another unknown class of receptors to inhibit gap junctional communication through a signaling cascade involving Gi/o activation (47, 48) or MAP kinases (49). Whether these signaling pathways are stimulated by methanandamide to increase paracellular permeability remains to be determined. Consistent with these possibilities, trimeric G protein activity tends to enhance tight junction assembly and transepithelial barrier function (50), and inhibiting MAP kinases enables ras-transformed MDCK cells to form a tight paracellular barrier (51).

Although methanandamide increased paracellular permeability, claudins, occludin, and ZO-1 remained localized to the plasma membrane. However, methanandamide-treated cells showed some subtle changes in plasma membrane morphology, consistent with the notion that a gross perturbation of tight junction morphology is not necessary for increased paracellular permeability (52). In addition, methanandamide altered the level of claudin expression, with a fairly dramatic increase in claudin-5 expression (Figure 7). One possibility is that expression of claudin-5 and claudin-3 is upregulated by the cells in an attempt to compensate for the methanandamide-induced decrease in barrier function. A more provocative possibility is that claudin-5 might induce a leaky tight junction phenotype. Consistent with this possibility, studies of retinal pigment epithelial development showed that increased claudin-5 expression is associated with decreased retinal TER in E7 stage chick embryos (41). The notion of leaky claudins is also suggested by studies where overexpression of claudin-2 by MDCK cells caused a dramatic decrease in TER (31). Whether a comparable effect is induced by claudin-5 expression remains to be determined. Also, it will be of interest to determine whether stages of lung injury where the alveolar epithelial barrier is compromised also show corresponding changes in claudin expression.


   Acknowledgments
 
The authors thank Drs. Michael Beers and Susan Margulies for their advice and comments regarding this manuscript. They thank Dr. Leland Dobbs for anti-RTI40 and advice regarding alveolar cell phenotype, Dr. Henry Shuman for anti-ABCA3, and Philip George for technical assistance. Confocal microscopy was performed at the shared equipment core maintained by the Institute for Environmental Medicine. This work was supported by grants from the American Heart Association, NIH R01-GM61012 and P01-HL019737, Program 3 (M.K.) and NIH R01-HL62472 (R.C.S.). B.D. is supported by an NRSA Postdoctoral Training Grant from NHLBI.


   Footnotes
 
* These authors contributed equally to the work presented in this article. Back

Received in original form September 3, 2002

Received in final form January 8, 2003


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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