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(The FASEB Journal. 1998;12:1349-1357.)
© 1998 FASEB

RESEARCH COMMUNICATION

Targeting of gliadin peptides, CD8, {alpha}/ß-TCR, and {gamma}/{delta}-TCR to Golgi complexes and vacuoles within celiac disease enterocytes

Klaus-peter Zimmera,1, Hassan Naimb, Peter Webera, H. Julia Ellisc, and Paul J. Ciclitirac

a Universitätskinderklinik, D-48149 Münster, Germany
b Institut für physiologische Chemie, Tierärztliche Hochschule, D-30559 Hannover, Germany
c Gastroenterology Unit (UMDS),St. Thomas' Hospital, London SEI 7EH, U.K.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Celiac disease (CD) is characterized by autodestruction of enterocytes after exposure of genetically susceptible individuals to dietary gluten. To define the transport pathways of proteins involved in the celiac immune response, we wished to determine the subcellular compartments of the intestinal mucosa where wheat gliadin peptides colocalize with receptors of T lymphocytes, including {alpha}/ß-TCR, {gamma}/{delta}-TCR, and CD8. Semithin and ultrathin frozen section of jejunal biopsies from CD patients and controls were used to perform immunofluorescence and immunogold labeling as well as in situ hybridization experiments. In patients with active CD, we detected gliadin peptides in vacuoles and Golgi complexes of enterocytes. CD8, {alpha}/ß-TCR, and {gamma}/{delta}-TCR were found in vacuoles and Golgi complexes within these gliadin-containing enterocytes in addition to the surface of intraepithelial and mucosal T lymphocytes. In contrast, we observed that the localization of CD4, CD3, T cell-restricted intracellular antigen (TIA), and leukocyte common antigen (LCA) was restricted to lymphocytes in CD patients. We further detected labeling signals for gliadin peptides, CD8, {alpha}/ß-TCR, and {gamma}/{delta}-TCR at the basal membrane of enterocytes that were interdigitated by extensions of lymphocytes. In situ hybridization experiments revealed that CD8 and {gamma}/{delta}-TCR were not expressed by CD enterocytes. We conclude that CD8, {alpha}/ß-TCR, and {gamma}/{delta}-TCR are targeted to Golgi complexes and vacuoles of small intestinal enterocytes in active CD. The observed process may be involved in the pathogenesis of CD enterocytes. We propose a mechanism for the uptake of CD8, {alpha}/ß-TCR, and {gamma}/{delta}-TCR by the basolateral membrane of small intestinal enterocytes.—Zimmer, K.-P., Naim, H., Weber, P., Ellis, H. J., Ciclitira. P. J. Targeting of gliadin peptides, CD8, {alpha}/ß-TCR, and {gamma}/{delta}-TCR to Golgi complexes and vacuoles within celiac disease enterocytes. FASEB J: 12, 1349–1357 (1998)


Key Words: endoplasmic reticulum • T cell receptor • trans-Golgi network


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CELIAC DISEASE (CD)2 is a malabsorptive disorder characterized by an autodestructive response to small intestinal enterocytes secondary to the exposure of genetically susceptible individuals to dietary gluten. The ethanol-soluble wheat proteins are termed gliadins (1–3). Peptides encompassing amino acids 31–49 of A-gliadin have been found to exacerbate CD in vitro and in vivo (4–7). Gliadin is endocytosed (8) and translocated into HLA-DR antigen-containing endocytic vacuoles of small intestinal enterocytes in active CD; this colocalization is not detected for chicken egg and bovine albumin (9). It induces aberrant HLA-class II expression of crypt enterocytes within the small intestine in patients with CD (10–12). CD is strongly associated with the HLA-class II alleles DQ A1*0501 and B1*0201, which encode HLA-DQ2 (13). Increased numbers of CD8+ and {gamma}/{delta}-TCR+ intraepithelial lymphocytes have been observed in active untreated CD (14–16), although it is not known whether either cell population is involved in the disease pathogenesis. {gamma}/{delta}-TCR+ intraepithelial lymphocytes are able to lyse target cell lines in vitro (17); a direct cytolytic effector function toward enterocytes has not been demonstrated.

It is still obscure how cytolytic damage to the villous enterocytes occurs in CD, although apoptosis of villous enterocytes has been proposed as a possible mechanism (18). The cell biological processes between the uptake of gliadin and the T cell-mediated destruction of small intestinal enterocytes are poorly understood. It has been reported that exogenous antigens can be presented not only by HLA-class II antigens (19), but also by HLA-class I antigens (20, 21). The precise ultrastructural colocalization sites of gliadin peptides and T cell receptors (TCRs) in the small intestinal epithelium of the celiac lesion are important to aid our understanding of the pathogenesis of CD. In this immunofluorescence and immunoelectron microscopical study, we used a monoclonal antibody raised against a peptide corresponding to amino acids 3–56 of A-gliadin in frozen sections of small intestinal biopsies from patients with CD as well as control subjects. We sought to determine the subcellular compartments where this gliadin peptide colocalizes with {gamma}/{delta}-TCR, {alpha}/ß-TCR, CD3, CD4, CD8, LCA (leukocyte common antigen) and TIA. The aim of this study was to characterize the transport pathways of the proteins that play a crucial function in antigen presentation and immune reaction in CD within the intestinal epithelium.

Our results showed a high concentration of celiac toxic gliadin peptides in vacuoles and Golgi complexes in enterocytes of patients with active CD. In the same biopsies, CD8, {alpha}/ß-TCR, and {gamma}/{delta}-TCR were also detected in Golgi stacks and vacuoles of enterocytes in contrast to CD3, CD4, TIA, and LCA, whose localization sites were restricted to lymphocytes. In addition, we found {alpha}/ß-TCR, {gamma}/{delta}-TCR, and CD8 molecules adjacent to the basolateral membrane of CD enterocytes at contact sites with lymphocytes. This labeling pattern was not observed in enterocytes from treated CD patients or controls. Uptake and targeting of soluble {alpha}/ß-TCR, {gamma}/{delta}-TCR, and CD8 components from the basolateral membrane to the Golgi complex of small intestinal enterocytes in patients with active CD points to a mechanism that may impair the functional integrity of enterocytes in subjects with untreated CD.


   MATERIAL AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
We studied jejunal biopsies from children with CD diagnosed according to the criteria of the European Society of Paediatric Gastroenterology and Nutrition (ESPGAN) (22). Seven specimens were obtained from patients with untreated CD (between 1 and 12 years old). Three biopsies were taken from CD patients who had received a gluten-free diet for at least 6 months (2.5, 4.5, and 8 years old). Jejunal biopsies from patients subsequently diagnosed to be suffering from the irritable bowel syndrome (two patients, 3 and 4 years old) and postenteritic syndrome (two patients, 2 and 3 years old) served as controls. The patients (except for the treated CD patients) took a gliadin-containing meal 6 h before the biopsy was taken and fasted for the next 6 h. The patients did not receive any medication except for intravenous diazepam and metoclopramide before the biopsy procedure. The biopsies were fixed by 5% paraformaldehyde in 250 mM HEPES, pH 7.4.

Antibodies
The primary antibodies used in this study are summarized in Table 1. Binding sites were visualized at the electron microscopical level by gold conjugated goat anti-rabbit serum (diameter of 6 nm and 12 nm, DIANOVA/D-Hamburg, dilution of 1:50) and gold conjugated goat anti-mouse serum (diameter of 6 nm and 12 nm, DIANOVA/D-Hamburg, dilution of 1:10). Cross-reaction of the gold conjugated goat anti-mouse and anti-rabbit serum against human immunoglobulin G (IgG), IgA, and IgM was abolished by labeling the sections with goat anti-human IgG, IgM and IgA (1 mg/ml). For light microscopical level experiments, a Texas red conjugated goat anti-rabbit (CAPPEL/D-Eppelheim, dilution of 1:50) and a fluorescein conjugated goat anti-mouse antibody (CAPPEL/D-Eppelheim, dilution of 1:10) were used. Isotype control experiments were performed using mouse IgG1, mouse IgG2a, and mouse IgG2b (all from DIANOVA/D-Hamburg).


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  		Table 1. Antibodiesa

Biosynthetic labelings and immunoprecipitations
Caco-2 cells were incubated in methionine-free medium containing 20% dialyzed fetal calf serum and labeled continuously with [35S]methionine (ICN/D-Meckenheim) for 6 h at 150 µCi/100 mm culture dish. The cells were washed three times in phosphate-buffered saline (PBS) and solubilized in 100 mM Na2PO4, 1% Triton X-100 and 40 µg/ml phenylmethyl sulfonyl fluoride, pH 7.0 on ice for 1 h. The detergent extracts were immunoprecipitated with the WB8 antibody or the antibody against dipeptidylpeptidase IV (DPPIV) (27) according to the procedure described elsewhere (28). The control used non-immune mouse serum. One microliter of antibody or non-immune serum was used for each immunoprecipitation. In parallel experiments the WB8 antibody or non-immune serum were added to the detergent extracts and left on ice for 1 h. Subsequently cross-linking was performed by the addition of 3,3'-dithiobis(propionic acid N-hydroxysuccinimide ester) (PIERCE/Rockford, Ill.) to a final concentration of 1 mM. The reaction was stopped after 20 min by adding ethanolamine (45 mM final concentration) at room temperature. Cross-linked detergent extracts were then immunoprecipitated with the WB8, anti-DPPIV antibody or non-immune serum. Immunoprecipitates derived from the cross-linked samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions (28).

Immunofluorescence
Semithin frozen sections (0.5 µm) of the small bowel biopsies were prepared with a Sorvall cryoultramicrotome MT 6000 at -60°C to -70°C. They were mounted on glass coverslips that were coated with poly-L-lysine. The sections were permeabilized with 0.05% saponin to increase the signals of intracellular bindings sites, incubated with the primary antibodies for 1 h, and then with the secondary antibodies for 1 h at room temperature. The specimens were photographed in a Zeiss Axioskop fluorescence microscope.

Immunoelectron microscopy
Sectioning and labeling of ultrathin frozen sections (50 nm) using the technique of Tokuyasu were performed as described in detail elsewhere (29, 30). Small tissue specimens were cryoprotected by polyvinylpyrrolidone/sucrose, frozen in liquid nitrogen, and sectioned with a Sorvall cryoultramicrotome MT 6000 at -100°C to -110°C. Thawed sections were incubated at room temperature with antibodies for 45 min. After labeling with immunogold, the grids were contrasted, embedded in 2% methylcellulose, and examined in a Phillips 301 electron microscope (9).

In situ hybridization
Semithin frozen sections of small bowel biopsies were collected on poly-L-lysine-coated glass slides. The sections were denaturated at 90°C for 2 min and hybridization was performed at 37°C for 2 h. The DNA probes were used at a concentration of 1 ng/ml hybridization buffer (DIANOVA/D-Hamburg). The 45-mer oligonucleotide probe corresponding to nt 710-666 of the human CD8{alpha} gene was synthesized and conjugated with FITC (DIANOVA/D- Hamburg), and served to localize mRNA of the CD8 {alpha}-chain at the light microscopical level. It did not show any substantial homologies with known sequences contained within viruses, plants, or ribosomal RNAs. A 23-mer oligonucleotide (synthesized by DIANOVA/D- Hamburg) was used to detect mRNA of the TCR receptor {gamma} subunit in semithin frozen sections. Control experiments were carried out by using corresponding sense probes. All oligonucleotide probes were provided with the same degree of FITC labeling and used at a concentration of 1:50. The specimens were photographed in a Zeiss Axioskop fluorescence microscope.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Detection of gliadin peptides in vacuoles and Golgi complexes of CD enterocytes
On an ultrastructural level, the antibody WB8 labels vesicles and large vacuoles of enterocytes from patients with active untreated CD. The large vacuoles are either multivesicular bodies with a low concentration of gliadin peptides or oligomembranous vacuoles with a high accumulation of gliadin peptides ( Fig. 1a). In addition to the vacuolar staining, the WB8 antibody also reveals a strong labeling of Golgi complexes inside enterocytes in untreated CD ( Fig. 1b). In contrast to the polyclonal antibody against unfractionated gliadin (9), the binding sites of the WB8 antibody are decreased on the apical membrane of enterocytes and in amorphous material inside the gut lumen, but distinctly increased in the Golgi complexes. The monoclonal antibody WB8 exhibits a higher affinity and specificity against the peptide corresponding to amino acid residues 3–56 of A-gliadin than the polyclonal anti-unfractionated gliadin antibodies (23, 24). This antibody was raised against an A-gliadin peptide with known in vivo toxicity to patients with CD. Cisternae of the rough endoplasmic reticulum of the enterocytes do not contain any significant amount of gliadin peptides. A small but significant number of gliadin peptides are localized in the paracellular space close to the lateral membrane of enterocytes as well as between the basal membrane of enterocytes and protruding cell surface extensions of T lymphocytes. This result indicates that gliadin is endocytosed by CD enterocytes. Because the patients had fasted 6 h before the biopsy was obtained, it cannot be determined whether the gliadin accumulated in the large vacuoles originates by uptake from the apical or basolateral membrane.



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  		Figure 1. Localization sites of gliadin peptides and CD8 within CD enterocytes. a) Gliadin peptides in endocytic vesicles and vacuoles of CD enterocytes. Thin frozen section from an enterocyte of an untreated CD patient, labeled by the gliadin peptide antibody WB8 and goat anti- mouse IgG conjugated with 12 nm large gold particles. A high number of gliadin peptides accumulate in a large vacuole of an enterocyte. Few gliadin peptides are detected in vesicles close to the apical membrane. Lu, bowel lumen; EV, endocytic vacuole; D, desmosome; E, endocytic vesicle; C, centriole; LM, lateral membrane; Mi, microvilli. x30,000. b) Gliadin peptides present throughout the Golgi stacks of CD enterocytes. Thin frozen section from an enterocyte of an untreated CD patient, labeled by the BiP antibody and goat anti-rat 12 nm large immunogold particles as well as the gliadin peptide antibody WB8 and goat anti-mouse 6 nm large immunogold particles. The Golgi stacks contain many gliadin peptides. In contrast, there are no gliadin peptides inside the endoplasmic reticulum. ER, endoplasmic reticulum; CV, coated vesicles; G, Golgi stacks; M, mitochondria. x73,000.

Immunofluorescence microscopical evaluation confirms the intracellular staining of enterocytes that is observed with the antibody WB8 in jejunal biopsies from untreated CD patients. The intracellular labeling of enterocytes, the last being identified by colocalization of sucrase-isomaltase, is distinct after treatment of the sections with saponin and resembles the staining pattern of Golgi stacks and lysosomes on an immunofluorescent microscopical level.

Labeling of the Golgi region and vacuoles by the WB8 antibody in enterocytes is present in five jejunal biopsies obtained from patients with untreated CD that exhibit villous atrophy. The biopsies from two untreated patients do not reveal the strong labeling pattern of the WB8 antibody within enterocytes. We assume that these patients had commenced a gluten-free diet before the biopsy was taken. No labeling is observed with this antibody in enterocytes from patients with treated CD who had been receiving a gluten-free diet. In three biopsies from the control patients who were suffering from the irritable bowel syndrome or postenteritic syndrome, few but significant binding sites of the WB8 antibody are detectable in vesicular structures close to the apical surface of enterocytes. No background labeling was noted on nuclei, cytosol, and mitochondria of the different cell types present in the small intestinal biopsies. Sections labeled with gold-conjugated goat anti-rabbit serum and goat anti-mouse serum alone revealed no significant staining. Preincubation with the goat anti-human IgG, IgM and IgA blocked the binding of the gold-conjugated goat anti-sera against human IgG, IgM, and IgA present in situ. Isotype control experiments on semithin and ultrathin frozen sections of biopsies from patients with untreated CD were also negative.

Because a cross-reactivity between antibodies raised against gliadin to epitopes expressed by enterocytes has been reported (31), we wished to determine whether the WB8 antibody cross-reacted with Caco-2 cells that had not been exposed to gliadin. Our results indicate that the WB8 antibody does not cross-react with proteins in Caco-2 cells, as determined by immunoprecipitation and chemical cross-linking.

Active CD enterocytes take up CD8, {alpha}/ß-TCR, and {gamma}/{delta}-TCR molecules but no LCA, CD3, and CD4
CD8 and {gamma}/{delta}-TCR positive intraepithelial lymphocytes have been implicated in the cytolytic damage of enterocytes in untreated CD (14–16). We wished to explore the subcellular localization of lymphocyte receptors in the small intestinal biopsies described above. Therefore, we studied the subcellular localization sites of TCR subunits by using semithin and ultrathin frozen sections, which we labeled with monoclonal antibodies against CD8, CD4, CD3, {alpha}/ß-TCR, and {gamma}/{delta}-TCR. The labeling obtained with the monoclonal antibody against CD4 is restricted to the plasma membrane of lymphocytes in the lamina propria. The antibodies against CD3 and TIA only label intraepithelial and mucosal lymphocytes. In contrast, immunofluorescence and immunogold labeling results show that CD8 ( Fig. 2a, b, d), {alpha}/ß-TCR, and {gamma}/{delta}-TCR ( Fig. 3a) are present in large vacuoles and Golgi complexes of enterocytes from patients with active untreated CD in addition to the plasma membrane of the lymphocytes present within the small intestinal biopsies. This labeling pattern is obtained by two monoclonal antibodies against CD8 and {gamma}/{delta}-TCR and one monoclonal antibody against {alpha}/ß-TCR. The immunofluorescence labeling of enterocytes with the CD8, {alpha}/ß-TCR, and {gamma}/{delta}-TCR antibodies is enhanced after treatment of the semithin sections with saponin, which improves the penetration of antibodies. CD8, {alpha}/ß-TCR, and {gamma}/{delta}-TCR labeling inside enterocytes is found in the five untreated CD patients whose enterocytes reveal staining of Golgi complexes and vacuoles by the WB8 antibody. The WB8 negative enterocytes of two patients with untreated CD also fail to react with the CD8, {alpha}/ß-TCR, and {gamma}/{delta}-TCR antibodies. Because all enterocytes of the five untreated celiac patients react with the WB8, CD8 ( Fig. 2a, b), {alpha}/ß-TCR, and {gamma}/{delta}-TCR antibodies, we conclude that these proteins colocalize in these cells. The antibodies against CD8, {alpha}/ß-TCR, and {gamma}/{delta}-TCR do not label enterocytes from treated CD nor controls. Control experiments as described above confirm the specificity of labeling pattern.



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  		Figure 2. Detection of CD8 in Golgi complexes and vacuoles of enterocytes in bowel biopsies of untreated CD patients. a) Immunofluorescence pattern of CD8 in untreated CD enterocytes. Semithin frozen section showing a crypt with a lumen in the center. It is labeled first by the monoclonal mouse CD8 antibody (and fluorescein conjugated goat anti-mouse serum) and in a second step by a polyclonal rabbit antibody against sucrase-isomaltase (and Texas red conjugated goat anti- rabbit serum). The staining of the CD8 antibody is consistent with the pattern of lysosomes and Golgi complexes. Lu, bowel lumen. x320. b) Localization of CD8 in untreated CD enterocytes as well as IEL. Semithin frozen section through a crypt labeled by the CD8 antibody; border line indicates the lumen of the bowel. CD8 bindings sites are present in Golgi complexes and vacuoles of enterocytes as well as on the surface of IEL (asterisks). Lu, bowel lumen. x210. c) Absent expression of CD8 in enterocytes of active celiac disease. This semithin frozen section is labeled by a fluorescein conjugated oligonucleotide corresponding to the human CD8{alpha} gene. CD8a is expressed in three IELs; there is no expression of CD8{alpha} within enterocytes. Lu, bowel lumen; GC, Goblet cell; N, nucleus. x610. d) CD8 in the Golgi stacks and vacuoles of CD enterocytes. Thin frozen section of a small bowel biopsy from a patient with active celiac disease. The section is labeled by a monoclonal CD8 antibody and goat anti-mouse 12 nm large immunogold particles. G, Golgi stacks; V, vacuole. x54,000.



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  		Figure 3. Uptake of {gamma}/{delta}-TCR at the basolateral membrane of CD enterocytes. a) {gamma}/{delta}-TCR in the Golgi complex of CD enterocytes. Trans-Golgi network (TGN) of an enterocyte detected in a frozen section from a bowel biopsy of an untreated celiac disease patient. The section has been incubated with the monoclonal TCS 1 antibody against {gamma}/{delta}-TCR and 12 nm large immunogold particles. M, mitochondria; ER, endoplasmic reticulum; MVB, multivesicular bodies; G, Golgi complex; TGN, trans-Golgi network; x51,000. b) Detection of LCA on the surface of mucosal lymphocytes. Thin frozen section of a small bowel biopsy from an untreated celiac disease patient. It is labeled by a monoclonal mouse antibody against the leukocyte common antigen (LCA) and 12 nm large immunogold particles. Arrows indicate an invaginating process of a lymphocyte into an enterocyte. N, nucleus; L, lymphocyte; E, enterocyte. x46,000. c) {gamma}/{delta}-TCR on the surface of lymphocytes as well as enterocytes. An interdigitation (asterisk) of a lymphocyte into an enterocytes detected in a frozen section of a small bowel biopsy from a patient with untreated celiac disease. The section is labeled by the {gamma}/{delta}-TCR antibody and 12 nm large immunogold particles. The gold particles are located on the surface of the lymphocyte as well as the basolateral membrane and vacuoles of an enterocyte. The lymphocyte does not reveal surface labeling on its invaginating process (asterisk). Note the close contact of the lymphocyte surface with the basal membrane of the enterocyte at few membrane domains. BL, basolateral membrane; N, nucleus; V, vacuoles; M, mitochondria; E, enterocyte; L, lymphocyte. x29,000.

In situ hybridization experiments are performed to examine whether the {alpha}-chain of CD8 as well as the {gamma} subunit of the TCR are expressed in jejunal enterocytes in individuals with untreated CD. The results of these experiments reveal that mRNA of the {alpha}-chain of CD8 ( Fig. 2b) and the {gamma} subunit of the TCR are detected in lymphocytes of the lamina propria but not inside enterocytes of patients with CD, excluding the expression of these T cell receptor subunits by enterocytes of patients with active CD.

Examining the intercellular cleft that lies between enterocytes and lymphocytes in jejunal biopsies from untreated CD patients, we note interdigitating extensions of lymphocytes that deeply indent enterocytes ( Fig. 3b, c). Labeling ultrathin frozen sections by antibodies against CD4, CD3, and LCA, gold particles are detected only on the surface of the lymphocytes ( Fig. 3b). In contrast, CD8, {alpha}/ß-TCR and {gamma}/{delta}-TCR signals are located on the basal membrane of enterocytes, the intercellular space, and the plasma membrane of lymphocytes ( Fig. 3c). This labeling pattern is distinct in the vicinity of the interdigitating extensions of lymphocytes into enterocytes.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The monoclonal antibody raised against the A-gliadin peptide, corresponding to amino acids 3–56, labels gliadin peptides inside small vesicular and tubular structures close to the apical membrane of CD and control enterocytes. This indicates an endocytic uptake of gliadin by enterocytes. The question arises whether the gliadin peptides accumulated in large vacuoles and Golgi complexes of enterocytes originate from endocytosis at the apical or basolateral membrane. Due to the experimental design, with the last gliadin ingestion at least 6 h before the biopsy, there is doubt that gliadin peptides within vacuoles and Golgi complexes of enterocytes originate from the apical membrane. Uptake of gliadin peptides may also occur from the basolateral membrane possibly after presentation to lymphocytes. In vivo studies with time-dependent uptake of gliadin in the first 6 h are in progress. The endocytic transport of transferrin and the transferrin receptor to the trans-Golgi region has been reported (32). There is evidence that intracellular transport between the endocytic compartment and the trans-Golgi network occurs mainly through late endosomes (33). This transport process has been shown to proceed to the trans-Golgi cisterna (34). On the other hand, our results suggest that gliadin peptides in the paracellular space are transported, possibly complexed with CD8, {alpha}/ß-TCR, and {gamma}/{delta}-TCR, from the basolateral membrane to an endosomal compartment and the Golgi complex in CD enterocytes. Because we detected CD8, {gamma}/{delta}-TCR, and {alpha}/ß-TCR only in CD enterocytes that contain gliadin peptides, our results supply indirect evidence that gliadin takes on a driving role for CD8, {gamma}/{delta}-TCR, and {alpha}/ß-TCR uptake into vacuoles and Golgi complexes of CD enterocytes. It has not been shown that gliadin peptides can be presented by HLA-class I antigens to cytotoxic T lymphocytes. However, the presentation of exogenous antigens by HLA-class I antigens has been observed in experimental systems (20, 21).

A second intracellular transport process that appears to be restricted to CD enterocytes is deduced from the demonstration of CD8, {alpha}/ß-TCR, and {gamma}/{delta}-TCR in Golgi complexes and vacuoles of enterocytes from untreated CD patients. The presence of TCR subunits in epithelial cells has only been reported in endometrial glandular epithelium (35). Light microscopical studies using thick frozen sections failed to demonstrate the intracellular staining of {gamma}/{delta}-TCR, {alpha}/ß-TCR, and CD8 inside CD enterocytes (14). The reason for this discrepancy is that intracellular binding sites are not accessible for the applied antibodies due to their poor penetration in 5 µm thick sections in contrast to surface binding sites. Furthermore, the differentiation between enterocytes and intraepithelial lymphocyte is less clear at the light microscopical level. Because our experiments exclude the expression of CD8 and {gamma}/{delta}-TCR by CD enterocytes, we assume that the detected TCR components are targeted to vacuoles and Golgi complexes of CD enterocytes after internalization at the basolateral membrane. Our concept that CD8, {alpha}/ß-TCR, and {gamma}/{delta}-TCR that originate from cytotoxic T lymphocytes are transferred to enterocytes in active untreated CD is supported by the observation of interdigitating extensions of lymphocytes at the basal membrane of enterocytes ( Fig. 3b, c). Similar invaginating processes between lymphocytes and enterocytes have also been described in the small intestine of CD patients and the normal mucosa of guinea pigs (36, 37). Our observation hints at a model in which the delivery of perforin from granules of cytotoxic lymphocytes into target cells by membrane fusion or endocytosis was proposed (38). The internalization of CD8, {alpha}/ß-TCR, and {gamma}/{delta}-TCR at the basal membrane seems to be a highly selective process, because CD4, CD3, TIA, and LCA, which are also present on the cell surface of mucosal lymphocytes, are not detected in enterocytes. The precise mechanism of CD8, {alpha}/ß-TCR, and {gamma}/{delta}-TCR uptake by enterocytes in active untreated CD is unclear. However, CD8, {alpha}/ß-TCR, and {gamma}/{delta}-TCR molecules ( Fig. 3c) detected adjacent to the basal membrane of molecules, including CD8 and {alpha}/ß-TCR, are released into the extracellular space as soluble fragments (39, 40). Increased levels of soluble CD4 and CD8 have been demonstrated in the serum of CD patients (41, 42). Because CD3 mainly represents a membrane component of the TCR complex and is not involved in the binding of TCR to the HLA-class I antigen/peptide complex, it is unlikely that the extracellular domain of CD3 can be proteolytically cleaved as it has been shown for CD8, {alpha}/ß-TCR, and {gamma}/{delta}-TCR. Furthermore soluble forms of CD3 have so far not been reported.

In conclusion, there is evidence that (soluble) CD8, {alpha}/ß-TCR, and {gamma}/{delta}-TCR are targeted to Golgi complexes and vacuoles of small intestinal enterocytes in active celiac disease. The presence of CD8, {alpha}/ß-TCR, and {gamma}/{delta}-TCR inside these enterocytes is restricted to those containing gliadin peptides in Golgi complexes and vacuoles. We assume that the intracellular transport of CD8, {alpha}/ß-TCR, and {gamma}/{delta}-TCR to vacuoles and Golgi complexes starts from the basal membrane of CD enterocytes that were interdigitated by extensions of CD8, {alpha}/ß-TCR, and {gamma}/{delta}-TCR positive lymphocytes. This selective process has not been observed for LCA, TIA, CD3, and CD4. The demonstration of gliadin peptides together with CD8, {alpha}/ß-TCR, and {gamma}/{delta}-TCR in the Golgi complex of CD enterocytes points to the presence of these proteins in the biosynthetic pathway. It is possible that a great many of these proteins, especially after formation of complexes in the Golgi complex, affect the secretory functions of these cells, as has been suggested (43). It remains to be examined whether the biosynthetic pathway is impaired in CD enterocytes, representing an additional factor in the pathogenesis of the cytolytic damage of CD enterocytes.


   ACKNOWLEDGMENTS
 
We thank Dr. A. Zweibaum (Unité de Recherches sur la Différenciation Cellulaire Intestinale, Villejuif/France) for the antibody against sucrase-isomaltase and Dr. D. Bole (University of Michigan, Ann Arbor) for the antibody against BiP. We thank Cordula Westermann for excellent technical assistance. Grant Support: German Research Foundation (DFG-Az.: Zi 294/4–3, Zi 294/6–1); St. Thomas' Hospital Research (Endowments) Committee; National Institutes of Health (Ro1 DK47716); Semper AB, and Nutricia UK Ltd.


   FOOTNOTES
 
1 Correspondence: Universitätskinderklinik, Albert Schweitzer Str. 33, D-48149 Münster, Germany. E-mail: zimmerp{at}uni-muenster.de

2 Abbreviations: CD, celiac disease; DPPIV, dipeptidylpeptidase IV; Ig, immunoglobulin; PBS, phosphate- buffered saline; {gamma}/{delta}-TCR, {gamma}/{delta}-T cell receptor; {alpha}/ß-TCR, {alpha}/ß-T cell receptor; LCA, leukocyte common antigen; TIA, T cell-restricted intracellular antigen.

Received for publication May 18, 1997. Accepted for publication June 10, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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