Copyright ©The Histochemical Society, Inc.

Expression and Function of Tight Junctions in the Crypt Epithelium of Human Palatine Tonsils

Mitsuru Go, Takashi Kojima, Ken-ichi Takano, Masaki Murata, Shingo Ichimiya, Hiroshi Tsubota, Tetsuo Himi and Norimasa Sawada

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


    Summary
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
The human palatine tonsils have surface and crypt stratified epithelium and may be initiated via the epithelium to mount immune responses to various presenting antigens. Here we investigated the expression and function of tight junctions in the epithelium of human palatine tonsils from patients with tonsillar hypertrophy or recurrent tonsillitis. Occludin, ZO-1, JAM-1, and claudin-1, -3, -4, -7, -8, and -14 mRNAs were detected in tonsillar hypertrophy. Occludin and claudin-14 were expressed in the uppermost layer of the tonsil surface epithelium, whereas ZO-1, JAM-1, and claudin-1, -4, and -7 were found throughout the epithelium. In the crypt epithelium, claudin-4 was preferentially expressed in the upper layers. In freeze-fracture replicas, short fragments of continuous tight junction strands were observed but never formed networks. In the crypt epithelium of recurrent tonsillitis, the tracer was leaked from the surface regions where occludin and claudin-4 disappeared. Occludin, ZO-1, JAM-1, and claudin-1, -3, -4, and –14, but not claudin-7, mRNAs were decreased in recurrent tonsillitis compared with those of tonsillar hypertrophy. These studies suggest unique expression of tight junctions in human palatine tonsillar epithelium, and the crypt epithelium may possess an epithelial barrier different from that of the surface epithelium. (J Histochem Cytochem 52:1627–1638, 2004)

Key Words: tight junctions • human palatine tonsil • epithelial barrier • crypt • tonsillitis


    Introduction
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 Introduction
 Materials and Methods
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 Literature Cited
 
TIGHT JUNCTIONS, the most apical component of intercellular junctional complexes, separate the apical from the basolateral cell surface domains to establish cell polarity (performing the function of a fence). Tight junctions also possess a barrier function, inhibiting the flow of solutes and water through the paracellular space (Schneeberger and Lynch 1992Go; Gumbiner 1993Go). They demonstrate a particular net-like meshwork of fibrils formed by the integral membrane proteins occludin, the claudin family, and JAM (Tsukita et al. 2001Go; Sawada et al. 2003Go). Several peripheral membrane proteins, ZO-1, ZO-2, ZO-3, 7H6 antigen, cingulin, symplekin, Rab3B, Ras target AF-6, and ASIP, an atypical protein kinase C-interacting protein, have been reported (Tsukita et al. 2001Go; Sawada et al. 2003Go). Occludin was the first discovered integral membrane protein of tight junctions and is the most ubiquitously expressed at the apicalmost basolateral membranes, being the most reliable immunohistochemical (IHC) marker for tight junctions (Tsukita and Furuse 2002Go). The claudin family, consisting of more than 20 members, is solely responsible for forming tight junction strands (Tsukita et al. 2001Go). Two or more different claudin species are generally expressed in single cells in various tissues. Recently, it has become clear that the integral membrane proteins of tight junctions claudin-2, claudin-4, and claudin-16 form selective channels through the tight junction barrier (Schneeberger and Lynch 2004Go). The cytoplasmic domains of occludin, claudins, and JAM-1 are reported to bind to ZO-1, forming a macromolecular complex at cell membranes (Schneeberger and Lynch 2004Go).

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 2002Go). Furthermore, bacterial–epithelial 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 2003Go). 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. 1997Go; Sonoda et al. 1999Go; Fasano 2000Go; Fullner and Mekalanos 2000Go; Nusrat et al. 2001Go). 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. 1997Go; Fujita et al. 2000Go). Furthermore, other tight junction-associated proteins, JAM and CAR, are suggested to be receptors for reovirus (Barton et al. 2001Go) or coxsackievirus and adenovirus (Cohen et al. 2001Go), respectively. Recently, Rescigno et al. (2001)Go 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. 2003Go; Yuki and Kiyono 2003Go). 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. 1993Go). 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.


    Materials and Methods
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Human Tissue Samples
Nine palatine tonsils were obtained from four patients with tonsillar hypertrophy and five patients with recurrent tonsillitis (donors ranged in age from 2 to 35 years) who underwent tonsillectomy at Sapporo Medical University. Informed consent was obtained from all patients and the study was approved by the Ethics Committee of Sapporo Medical University. The palatine tonsils from the patients were processed within 2 hr after surgery and the epithelial layers were sectioned by mechanical dissociation without affecting the structure. They were immediately frozen in liquid nitrogen and stored at –70C for IHC and RNA extraction. Some tissue samples were fixed in 10% formaldehyde in PBS for hematoxylin–eosin (HE) staining.

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 rhodamine–dextran (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).


    Results
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Expression in mRNAs of Tight Junctions in Human Palatine Tonsils
To investigate the expression in mRNAs of tight junctions in human palatine tonsillar epithelium, RT-PCR was carried out in palatine tonsils from patients with tonsillar hypertrophy. As shown in Figure 1, expression in mRNAs of occludin, ZO-1, JAM-1, and claudin-1, -3, -4, -7, -8, and -14 were detected.



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Figure 1

Expression in mRNAs of tight junctions in human palatine tonsils from patients of hypertrophy using semiquantative RT-PCR. M, 100-bp ladder DNA marker; Oc, occludin; CL, claudin. Expression in mRNAs of occludin, JAM-1, ZO-1 and claudin-1, -3, -4, -7, -8, -14 was detected.

 
Distribution of Tight Junction Proteins in Human Palatine Tonsils
Human palatine tonsillar epithelium consists of two different compartments: surface epithelium and crypt epithelium (Perry and Whyte 1998Go). To investigate the distribution of tight junction proteins in human palatine tonsillar epithelium, IHC was carried out using palatine tonsils from patients with tonsillar hypertrophy. Occludin and claudin-14 were localized in the upper layers of the tonsillar surface epithelium, whereas ZO-1, JAM-1, and claudin-1, -4, and -7 were found throughout the epithelium, and claudin-3 was only faintly stained (Figure 2). Crypts of human palatine tonsils contained bacteria and cell debris (Figure 3). In the crypt epithelium, claudin-4 was preferentially expressed in the upper layers where occludin was observed, whereas claudin-1 was found throughout the epithelium (Figure 3). By using various double stainings (see Materials and Methods), occludin and claudin-14 were co-localized in the uppermost layer of the crypt epithelium, unlike claudin-1, -4, and -7 (Figure 4A). Furthermore, occludin was localized in a more upper layer of the epithelium than were JAM-1 and ZO-1 (data not shown). A schematic diagram of the distribution of tight junction proteins in the surface and the crypt epithelium of human platine tonsils is shown in Figure 4B.



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Figure 2

Immunohistochemistry for occludin, JAM-1, ZO-1, and claudin-1, -3, -4, -7, -14 in the surface epithelium of human palatine tonsils from patients with tonsillar hypertrophy. F, follicular area; Oc, occludin; CL, claudin. Bar = 400 µm.

 


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Figure 3

HE staining and single IHC staining for occludin and claudin-1, -4 in the surface and crypt epithelium of human palatine tonsils from patients with hypertrophy. Arrowheads, bacteria; arrows, cell debris; Oc, occludin; CL, claudin. Bar = 200 µm.

 


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Figure 4

(A) Double IHC staining for occludin and claudin-1, -4, -7, -14, and claudin-4 and claudin-1 in the crypt epithelium of human palatine tonsils from patients with hypertrophy. Oc, occludin; CL, claudin. Bar = 100 µm. (B) Schematic diagram of the distribution of tight junction proteins in human palatine tonsils.

 
Freeze-fracture Analysis of Human Palatine Tonsils
To investigate whether tight junction structures in human palatine tonsillar epithelium express several claudins that can form tight junction strands, we performed freeze-fracture of palatine tonsils from patients with tonsillar hypertrophy. In the freeze-fracture replicas, short fragments of continuous tight junction strands were observed on the subapical membranes, but the strands never formed networks (Figures 5A and 5B).



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Figure 5

Freeze-fracture replicas of human palatine tonsils from patients with hypertrophy. AP, apical membrane; BL, basolateral membrane. Short fragments of continuous tight junction strands were observed (arrows in B) but the strands never formed networks. Bars: A = 50 nm; B = 100 nm.

 
Distribution and Barrier Function of Tight Junctions in Crypt Epithelium of Recurrent Tonsillitis
To investigate changes in distribution and barrier function of tight junctions in palatine tonsils from patients with recurrent tonsillitis, we performed IHC for occludin, claudin-1 and -4, and a barrier function assay using exudation of rhodamine–dextran. HE-stained slides were carefully reviewed and the diagnosis of recurrent tonsillitis was confirmed according to the established criteria: formation of deep tubular crypts, degenerated cells and cell debris outside the epithelium, and infiltration of many lymphoid cells into the epithelium (Figure 6A). In the crypt epithelium, discontinuous immunoreactivity for occludin and claudin-4 was observed at the upper layer, whereas claudin-1 expressed throughout the epithelium was not affected (Figures 6B–6D). However, in the surface epithelium no changes in distribution of the tight junction proteins were observed (data not shown). In double staining for occludin and rhodamine-dextran of the crypt epithelium, the dye was leaked from the surface where occludin disappeared, while in the surface epithelium the dye was not leaked from the surface regions in which occludin was highly expressed (Figures 6E and 6F).



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Figure 6

HE staining (A) and single IHC staining for occludin (B), claudin-1 (D), -4 (C) in the crypt epithelium of human palatine tonsils from patients with recurrent tonsillitis. Double staining for occludin and rhodamine–dextran in the surface epithelium (E) and in the crypt epithelium (F) of human palatine tonsil from the same patient with recurrent tonsillitis. Oc, occludin; CL, claudin. Bars: A–C,D = 200 µm; E,F = 100 µm.

 
Changes in mRNAs of Tight Junctions in Recurrent Tonsillitis
To investigate changes of expression in mRNAs of tight junctions in palatine tonsils from patients with recurrent tonsillitis, we performed semiquantitive RT-PCR encoding the tight junction-associated proteins occludin, JAM-1, ZO-1, and claudin-1, -3, -4, -7, and -14 in comparison with palatine tonsils from patients with tonsillar hypertrophy. Expression of mRNAs of occludin, JAM-1, ZO-1, and claudin-1, -3, -4, -14, but not claudin-7, was significantly decreased in recurrent tonsillitis involving less than 50% tonsillar hypertrophy (Figures 7A and 7B).



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Figure 7

(A) Expression of mRNAs in tight junctions in human palatine tonsils from patients with hypertrophy (1) and recurrent tonsillitis (2) using semiquantative RT-PCR. M, 100-bp ladder DNA marker. The signals are shown in the bar graph (B). Oc, occludin; CL, claudin. The results were obtained from three independent sets of tonsillar hypertrophy and recurrent tonsillitis. Expression of mRNAs for occludin, JAM-1, ZO-1, and claudin-1, -3, -4, -7, -14 but not claudin-7 in recurrent tonsillitis was significantly decreased compared with tonsillar hypertrophy.

 

    Discussion
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 Summary
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 Materials and Methods
 Results
 Discussion
 Literature Cited
 
In the present study we demonstrated the expression and distribution of tight junctions in human palatine tonsillar stratified epithelium of tonsillar hypertrophy and downregulation of expression and function of tight junctions in the crypt epithelium of recurrent tonsillitis.

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. 2003Go). 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. 2002Go). 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. 2002Go).

Recently, Tsukita and Furuse (2002)Go 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 1999Go). In mouse epidermis, the diffusion of subcutaneously injected tracer was sharply halted at occludin-positive tight junctions (Furuse et al. 2002Go). 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. 2003Go, Yuki and Kiyono 2003Go). 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. 2003Go). In an experiment using MDCK cells, expression of exogenous claudin-4 specifically affected the permeability to sodium (Van Itallie et al. 2001Go). 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. 1997Go; Fujita et al. 2000Go). 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. 2001Go). 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. 2003Go). 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. 2003Go). The proinflammatory cytokines, such as interferon-{gamma} (IFN-{gamma}) and tumor necrosis factor-{alpha} (TNF-{alpha}), have been reported to influence epithelial barrier function (Madara and Stafford 1989Go; Fish et al. 1999Go; Youakim and Ahdieh 1999Go; Sugi et al. 2001Go; Bruewer et al. 2003Go). Moreover, downregulation of occludin but not claudin-1 by treatment with IFN-{gamma} and TNF-{alpha} was observed at the transcriptional level (Mankertz et al. 2000Go). 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-{gamma} and TNF-{alpha}, are predominantly produced at sites of local antigen stimulation by intraepithelial lymphocytes (Andersson et al. 1994Go; Harabuchi et al. 1996Go; Wakashima et al. 1999Go). Accordingly, downregulation of the expression and function of tight junctions in recurrent tonsillitis may be modulated by the proinflammatory cytokines.

Bacterial–epithelial 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 2003Go). Recently, Rescigno et al. (2001)Go 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.


    Acknowledgments
 
Supported by Grants-in-Aid from the Ministry of Education, Culture, Sports and Science, and the Ministry of Health, Labor and Welfare of Japan and by the Kato Memorial Bioscience Foundation, the Uehara Memorial Foundation, the Suhara Memorial Foundation, the Smoking Research Foundation, and the Long-Range Research Initiative Project of Japan Chemical Industry Association.

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.


    Footnotes
 
Received for publication April 6, 2004; accepted August 13, 2004


    Literature Cited
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 

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