Expression and Function of Tight Junctions in the Crypt Epithelium of Human Palatine Tonsils
Departments of Otolaryngology (MG,KT,HT,TH) and Pathology (MG,TK,KT,MM,SI,NS), Sapporo Medical University School of Medicine, Sapporo, Japan
Correspondence to: Takashi Kojima, PhD, Dept. of Pathology, Sapporo Medical University School of Medicine, S1, W17, Sapporo 060-8556, Japan. E-mail: ktakashi{at}sapmed.ac.jp
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Summary |
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Key Words: tight junctions human palatine tonsil epithelial barrier crypt tonsillitis
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Introduction |
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Tight junctions have been well characterized in simple epithelium and endothelium but have been not fully described in stratified epithelium. Recently, continuous claudin-based tight junctions have been shown to occur in stratified epithelium such as that of the epidermis, and these tight junctions are crucial for the barrier function of the mammalian skin (Tsukita and Furuse 2002). Furthermore, bacterialepithelial tight junction "crosstalk" can be mediated by many virulent factors, mainly secreted toxins, or can be induced by direct contact of the pathogen with the epithelial membrane (Hofman 2003
). In simple epithelium, it has been demonstrated that bacteria or their toxins cause the tight junctions to open, leading to a breakdown of barrier function (Katahira et al. 1997
; Sonoda et al. 1999
; Fasano 2000
; Fullner and Mekalanos 2000
; Nusrat et al. 2001
). Claudin-3 and -4 are also known as receptors for enterotoxin of Clostridium perfringens (CPE), which is a common cause of food poisoning (Katahira et al. 1997
; Fujita et al. 2000
). Furthermore, other tight junction-associated proteins, JAM and CAR, are suggested to be receptors for reovirus (Barton et al. 2001
) or coxsackievirus and adenovirus (Cohen et al. 2001
), respectively. Recently, Rescigno et al. (2001)
discovered a route by which some pathogenic microorganisms might invade through the epithelial cells via intraepithelial dendritic cells, because the dendritic cells expressed tight junction proteins such as occludin, claudin-1, and ZO-1 between the epithelial cells.
The adenoids and tonsils are lymphoid tissues in the pharynx, where they play a crucially important role in host defense against invading antigens of the upper respiratory tract. The palatine tonsils are believed to belong to the family of nasal-associated lymphoreticular tissues (NALTs) and are critical in the priming of antigen-specific T-cells and IgA-committed B-cells, with dissemination of the primed lymphocytes to distant mucosal sites for generation of antigen-specific IgA immune responses (Debertin et al. 2003; Yuki and Kiyono 2003
). Immunological processes are initiated in the different specialized compartments of the palatine tonsils, such as the surface epithelium, the crypt epithelium, lymphoid follicles, and extrafollicular region. The epithelium of the palatine tonsils is stratified and is characterized as lymphoepithelium, which consists not only of epithelial cells but also of non-epithelial cells, lymphocytes, macrophages, and dendritic cells (Graeme-Cook et al. 1993
). The palatine tonsils have surface and crypt stratified epithelium and are considered to be initiated via the epithelium to mount immune responses to various presenting antigens. In addition, we hypothesized that there might be a specific barrier system of the crypt epithelium, although expression and function of the tight junctions in the palatine tonsillar epithelium remain unclear.
To analyze the specific barrier system in the crypt epithelium of human palatine tonsils, we investigated the expression, distribution, and function of tight junctions in the epithelium of human palatine tonsils from patients with tonsillar hypertrophy or recurrent tonsillitis.
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Materials and Methods |
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RNA Isolation and RT-PCR Analysis
Total RNA was extracted from the palatine tonsils from patients with hypertrophy or recurrent tonsillitis after homogenization in TRIzol reagent (Gibco BRL; Gaithersburg, MD). For RT-PCR, 1 µg of total RNA was reverse-transcribed into cDNA using the manufacturer's recommended conditions (Invitrogen; Carlsbad, CA). Each cDNA synthesis was performed in a total volume of 20 µl for 50 min at 42C and was terminated by incubation for 15 min at 70C. RT-PCR was performed using 2 µl of the 20 µl total RT product, PCR buffer, dNTPs and Premix Taq DNA polymerase under the manufacturer's recommended conditions (Takara; Siga, Japan). Conditions applied for PCR were 96C for 30 sec, 30 cycles of 96C for 15 sec, 55C (except for claudin-3 and claudin-14; 60C) for 30 sec, 72C for 1 min, and 72C for 7 min using a Perkin Elmer/Cetus Thermocycler Model 2400. Ten µl of 20-µl total PCR reaction was analyzed by electrophoresis in ethidium bromide-impregnated 1% agarose gels. Primers used to detect occludin, ZO-1, JAM-1, claudin-1, claudin-2, claudin-3, claudin-4, claudin-5, claudin-6, claudin-7, claudin-8, claudin-9, and claudin-14 had the following sequences: occludin (sense 5'-TCAGGGAATATCCACCTATCACTTCAG-3' and antisense 5'-CATCAGCAGCAGCCATGTACTCTTCAC-3', amplicon length 136 bp); ZO-1 (5'-CGG TCCTCTGAGCCTGTAAG-3' and antisense 5'-GGATCTACATGCGACGACAA-3', amplicon length 435 bp); JAM-1 (5'-GGTCAAGGTCAAGCTCAT-3' and antisense 5'-CTGAGTAAGGCAAATGCAG-3', amplicon length 765 bp); claudin-1 (5'-GCTGCTGGGTTTCATCCTG-3' and antisense 5'-CACATAGTCTTTCCCACTAGAAG-3', amplicon length 619 bp); claudin-2 (5'-GCAAACAGGCTCCGAAGATACT-3' and antisense 5'-CTCTGTACTTGGGCATCATCTC-3'); claudin-3 (5'-TGCTGTTCCTTCTCGCCGCC-3' and antisense 5'-CTTAGACGAAGTCCATGCGG-3', amplicon length 247 bp), claudin-4 (5'-AGCCTTCCAGGTCCTCAACT-3' and antisense 5'-AGCAGCGAGTAGAAG-3', amplicon length 249 bp); claudin-5 (5'-GACTCGGTGCTGGCTCTGAG-3' and antisense 5'-CGTAGTTCTTCTTGTCGTAG-3'); claudin-6 (5'-TGAGGCCCAAAAGCGGGAGC-3' and antisense 5'-CGTAATTCTTGGTAGGGTAC-3'); claudin-7 (5'-AGGCATAATTTTCATCGTGG-3' and antisense 5'-GAGTTGGACTTAGGGTAAGAGCG-3', amplicon length 210 bp); claudin-8 (5'-TCATCCCTGTGAGCTGGGTT-3' and antisense 5'-TGGAGTAGACGCTCGGTGAC-3', amplicon length 215 bp); claudin-9 (5'-AGGCCCGTAT CGTGCTCACC-3' and antisense 5'-ACGTAGTCCC TCTTGTCCAG-3'); and claudin-14 (5'-CGCGCCCTCATGGTCATCT-3' and antisense 5'-CCCCCTCTGTCCCTGTGCT-3', amplicon length 627 bp). To provide a qualitative control for reaction efficiency, PCR reactions were performed with primers coding for the housekeeping gene G3PDH (sense 5'-ACCACAGTCCATGCCATCAC-3' and antisense 5'-TCCACCACCCTGTTGCTGTA-3', amplicon length 452 bp). Signals were quantified by the Scion-Image Densimetric analysis program (Scion; Frederick, MA).
Immunohistochemistry
For IHC of palatine tonsil slices, 10 µm-thick frozen sections were made with a cryostat. The sections were fixed with cold acetone and ethanol (1:1) for 10 min. After rinsing in PBS, the sections were incubated with monoclonal anti-occludin (33-1500; concentration 0.25 mg/ml; 1:100), polyclonal anti-ZO-1 (61-7300; 0.25 mg/ml; 1:100), polyclonal anti-JAM-1 (36-1700; 0.25 mg/ml; 1:100), polyclonal anti-claudin-1 (71-7800; 0.25 mg/ml; 1:100), polyclonal anti-claudin-3 (34-1700; 0.25 mg/ml; 1:100), monoclonal anti-claudin-4 (32-9400; 0.25 mg/ml; 1:100), polyclonal anti-claudin-7 (34-9100; 0.25 mg/ml; 1:100), polyclonal and anti-claudin-14 (36-4200; 0.25 mg/ml; 1:100) antibodies at RT for 1 hr and then were incubated with Alexa 488 (green)-conjugated anti-mouse IgG or anti-rabbit IgG (Molecular Probes; Eugene, OR) at RT for 1 hr. All primary antibodies were obtained from Zymed Laboratories (San Francisco, CA). Some sections were used for double staining of occludin and claudin-1, occludin and claudin-4, occludin and claudin-7, occludin and claudin-14, claudin-1 and claudin-4. The specimens were examined with an epifluorescence microscope (Oympus; Tokyo, Japan) and a laser-scanning confocal microscope (MRC 1024; Bio-Rad, Hercules, CA).
Freeze-fracture Analysis
For freeze-fracture experiments, tonsil tissues were immersed in 40% glycerin solution after fixation in 2.5% glutaraldehyde in 0.1 M PBS (pH 7.3). The specimens were mounted on a copper stage, frozen in liquid nitrogen, fractured at 150C to 160C, replicated by platinum/carbon from an electron beam gun positioned at a 45° angle followed by carbon applied from overhead in a JFD-7000 freeze-fracture device (JEOL; Tokyo, Japan). After the replicas were thawed, they were floated on filtered 10% sodium hypochlorite solution for 10 min in a Teflon dish. Replicas were washed in distilled water for 30 min, mounted on copper grids, and examined at 100 kV on a JEOL 1200EX transmission electron microscope.
Barrier Function Assay
To evaluate the barrier function of tight junctions, the palatine tonsils from patients with recurrent tonsillitis were incubated with rhodaminedextran (molecular weight 1000 Da; Molecular Probes) dissolved in PBS for 15 min on ice. The specimens were immediately frozen in liquid nitrogen, sectioned at 10 µm with a cryostat, and mounted on glass slides. Some sections were double stained with occludin and claudin-4 visualized by Alexa 488 (green)-conjugated anti-mouse IgG. The specimens were examined with an epifluorescence microscope (Olympus).
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Results |
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Discussion |
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Constitutive tight junction proteins and tight junction-related structures are identified in squamous stratified epithelia, including the epidermis, where they occur in special positions, most prominently in the uppermost living epidermal cell layer, the stratum granulosum (Langbein et al. 2003). Using RT-PCR, mRNAs encoding protein ZO-1, occludin, and claudin-1, -4, -7, -8, -11, -12, and -17 are detected in both skin and cultured keratinocytes (Brandner et al. 2002
). Whereas claudin-1 occurs in plasma membranes of all living cell layers, protein ZO-1 is concentrated in or even restricted to the uppermost layers, and occludin is often detected only in the stratum granulosum (Brandner et al. 2002
).
Recently, Tsukita and Furuse (2002) found a unique expression and a crucially important function of tight junctions in the stratified epithelium. Occludin is known to be highly concentrated at tight junction strands in most simple epithelial cells and is the best general marker for tight junction strands, whereas occludin is not indispensable for the formation of tight junction strands (Tsukita and Furuse 1999
). In mouse epidermis, the diffusion of subcutaneously injected tracer was sharply halted at occludin-positive tight junctions (Furuse et al. 2002
). In the present study, occludin was localized in the uppermost layer of human palatine tonsillar surface epithelium, whereas ZO-1, JAM-1, and claudin-1, -4, and -7 were found throughout the stratified epithelium (Figure 4B). The diffusion of tracer was stopped at an occludin-positive layer in the surface epithelium. These results suggest that occludin may also play a crucial role in tight junctions of human palatine tonsillar epithelium.
Palatine tonsils are believed to belong to the family of nasal-associated lymphoreticular tissues (NALTs) (Debertin et al. 2003, Yuki and Kiyono 2003
). In the follicle-associated epithelium (FAE) of Peyer's patches, which is a gut-associated lymphoreticular tissue, occludin and claudin-2, -3, and -4 expression were detected. Claudin-4 was preferentially expressed in the apical region; claudin-2 was only weakly expressed on the crypt side of the FAE compared with stronger expression on the crypt side of villous epithelial cells; and claudin-3 and occludin were found throughout the dome (Tamagawa et al. 2003
). In an experiment using MDCK cells, expression of exogenous claudin-4 specifically affected the permeability to sodium (Van Itallie et al. 2001
). Claudin-4 is also known as a receptor for the enterotoxin of Clostridium perfringens (CPE), which is a common cause of food poisoning (Katahira et al. 1997
; Fujita et al. 2000
). In the crypt epithelium of human tonsils, claudin-4 was preferentially expressed in the upper layers, whereas it was found throughout the surface epithelium. In the crypt of human tonsils, claudin-4 expressed in the upper layers may be important in acting as selective cation channels or receptors for bacteria, similar to the follicle-associated epithelium of Peyer's patches. This study suggests that claudin-4 expression may be associated with loosening of intercellular junctions to allow passage of various pathogens from the epithelium of lymphoreticular tissues, including the crypt epithelium of human tonsils.
Claudin-14 has been investigated as a gene responsible for human hereditary deafness (Wilcox et al. 2001). Mutations in this gene were believed to downregulate the barrier function of tight junctions in the organ of Corti and thus to result in hereditary deafness with degeneration of hair cells and supporting cells, which act as a cation-restrictive barrier to maintain the proper ionic composition (Ben-Yosef et al. 2003
). In the present study, claudin-14 was co-localized with occludin in the uppermost layer of human palatine tonsillar surface epithelium, whereas claudin-1 was found throughout the epithelium (Figure 4). Therefore, claudin-14 may have a specific function in human palatine tonsillar surface epithelium, although its role remains largely unclear in the stratified epithelium.
It is well known that inflammatory conditions of the intestinal mucosa result in compromised barrier function, which is regulated by the epithelial apical junctional complex consisting of the tight junctions and the adherens junctions (Berkes et al. 2003). The proinflammatory cytokines, such as interferon-
(IFN-
) and tumor necrosis factor-
(TNF-
), have been reported to influence epithelial barrier function (Madara and Stafford 1989
; Fish et al. 1999
; Youakim and Ahdieh 1999
; Sugi et al. 2001
; Bruewer et al. 2003
). Moreover, downregulation of occludin but not claudin-1 by treatment with IFN-
and TNF-
was observed at the transcriptional level (Mankertz et al. 2000
). In the present study, in the crypt epithelium of the palatine tonsils from patients of recurrent tonsillitis, discontinuous immunoreactivity of occludin and claudin-4 was observed at the upper layer, whereas claudin-1 expressed throughout the epithelium was not affected. The diffusion of tracer was observed to pass through the epithelium where occludin and claudin-4 disappeared. Expression of mRNAs of occludin, JAM-1, ZO-1, and claudin-1, -3, -4, -14, but not claudin-7, was markedly decreased in recurrent tonsillitis compared to that of tonsillar hypertrophy. The epithelium of human palatine tonsils is characterized as lymphoepithelium. Cytokines, such as IL-6, IFN-
and TNF-
, are predominantly produced at sites of local antigen stimulation by intraepithelial lymphocytes (Andersson et al. 1994
; Harabuchi et al. 1996
; Wakashima et al. 1999
). Accordingly, downregulation of the expression and function of tight junctions in recurrent tonsillitis may be modulated by the proinflammatory cytokines.
Bacterialepithelial tight junction "crosstalk" can be mediated by many virulent factors, mainly secreted toxins, or can be induced by direct contact of the pathogen with the epithelial membrane (Hofman 2003). Recently, Rescigno et al. (2001)
discovered a route by which some pathogenic microorganisms might invade through the epithelial cells via intraepithelial dendritic cells, because the dendritic cells expressed tight junction proteins such as occludin, claudin-1, and ZO-1 between the epithelial cells. Although this in itself is not enough to characterize the dendritic cells in the epithelium of human palatine tonsils, in the crypts of the palatine tonsils bacteria or pathogens may in part invade through the epithelium via the dendritic cells.
The adenoids and tonsils are lymphoid tissues located in the pharynx that play an important role in host defense against invading antigens of the upper respiratory tract. In the present study, in freeze-fracture replicas of human palatine tonsils, short fragments of continuous tight junction strands were observed but never formed networks. However, preliminary freeze-fracture experiments in the epithelium of human adenoid revealed well-developed networks of continuous tight junction strands (data not shown). These results indicate that the level of barrier function of the epithelium differs between the adenoid and palatine tonsils that they can play specific roles in host defense against invading antigens, and that the palatine tonsils may be easily initiated through the epithelium to mount immune responses against various presenting antigens.
In summary, unique expression of tight junctions in the surface epithelium and the crypt epithelium of human palatine tonsils was observed, and the crypt epithelium may have an epithelial barrier different from that of the surface epithelium. In the crypt epithelium from patients with recurrent tonsillitis, partial disruption of tight junctions was observed.
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Acknowledgments |
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We are grateful to Dr T. Kita (Kyoto University) for the JAM-1 antibody. We thank Ms E. Suzuki (Sapporo Medical University) for technical support.
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Footnotes |
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