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. 2003 Dec;134(3):516–524. doi: 10.1111/j.1365-2249.2003.02326.x

Gliadin and tissue transglutaminase complexes in normal and coeliac duodenal mucosa

R CICCOCIOPPO *, A DI SABATINO †, C ARA *, F BIAGI †, M PERILLI ‡, G AMICOSANTE ‡, M G CIFONE §, G R CORAZZA †
PMCID: PMC1808891  PMID: 14632760

Abstract

Tissue transglutaminase (tTG) seems to be the target self-antigen for endomysial antibodies in coeliac disease (CD) and to catalyse the critical deamidation of gliadin which strengthens its recognition by HLA-restricted gut-derived T cells. To date, it has not been demonstrated whether gliadin is cross-linked to tTG within the gut wall, a phenomenon known to occur in vitro. We therefore investigated the putative presence of tTG and gliadin complexes directly in duodenal mucosa. The immunoprecipitation and Western blotting experiments were performed on mucosal biopsies obtained from untreated, treated CD patients and biopsied controls, by using either anti-tTG or anti-gliadin antibodies, in both denaturating/reducing or nondenaturating/nonreducing conditions. A subset of experiments was performed by using anti-tTG antibodies purified by affinity chromatography from sera of untreated coeliac patients. The localization of tTG and gliadin was studied by immunofluorescence at confocal laser microscopy on seriate sections of diseased and normal duodenal mucosa by using the same antibodies of the coimmunoprecipitation section. The amounts of tTG and gliadin coimmunoprecipitated with anti-tTG monoclonal antibody in untreated CD mucosa were significantly increased compared to those of the other two groups. When performing the experiments in nondenaturating/nonreducing conditions, a high molecular weight band formed by both molecules, was evidenciated. Also the anti-tTG antibodies purified from patients' sera turned out to be able to coimmunoprecipitate the two molecules. The analysis by confocal microscopy showed that tTG colocalizes with gliadin at the epithelial and subepithelial levels in active CD, and only in the lamina propria of the villi in normal mucosa. Our findings firstly demonstrated that gliadin was directly bound to tTG in duodenal mucosa of coeliacs and controls, and the ability of circulating tTG-autoantibodies to recognize and immunoprecipitate the tTG-gliadin complexes.

Keywords: coeliac disease, duodenal mucosa, gliadin, tissue transglutaminase

INTRODUCTION

The intestinal lesions of coeliac disease (CD) are caused, in genetically susceptible individuals, by a T-cell response to epitopes of wheat dietary gliadin [1]. Although the pathogenesis of this condition is still obscure, a strong autoimmune component is recognized, as supported by the production of class A immunoglobulin (anti-endomysial antibodies) against reticular components of the extracellular matrix [2]. In 1997, Dieterich et al. [3] showed that immunoprecipitation of human fibrosarcoma cell lysates, using the immunoglobulin A fraction from coeliac sera, resulted in a single protein band of 85 kD, which, after sequence analysis, was assigned to the enzyme tissue transglutaminase (tTG). The demonstration that tTG was the predominant, if not the sole, endomysial autoantigen in CD was promptly confirmed [4].

tTG is a calcium dependent ubiquitous intracellular enzyme which catalyses the covalent and irreversible crosslinking of a glutamine residue in glutamine-donor proteins with a lysine residue in glutamine-acceptor proteins which results in the formation of an e-(γ-glutamyl)-lysine (isopeptidyl) bond. The reaction is a multistep process, in which the active site of the enzyme firstly reacts with the glutamine residue to form the acyl-enzyme intermediate under release of ammonia. In a second step, the complex reacts with a primary amine to form an isopeptide bond and liberate the reactivated enzyme. The final step consists of the formation of covalently crosslinked, often insoluble supramolecular structures [5]. Apart from crosslinking its substrates, tTG can also hydrolyse peptide-bound glutamine to glutamic acid either at lower pH, or when no acceptor proteins are available [6]. Accordingly, it has recently been shown that in slightly acidic environment, as occurs in chronic inflammation, tTG-induced gliadin peptide polymerization (transamidation) decreases, whereas deamidation increases [7]. Therefore, the tTG-mediated reactions with polyamines, such as gliadin, may result in protein modification possibly affecting their biological activity, antigenicity, and turnover. Interestingly enough, Bruce et al. [8] found an increased mucosal activity of tTG in untreated and treated CD patients compared to healthy controls, and that gliadin represented a preferential substrate for this enzyme. Taken together, these results point to the relevance of gliadin–tTG interaction in CD and a series of innovative studies reported that, by catalysing the critical and ordered deamidation of gliadin, tTG determines its strongly increased affinity for HLA-DQ2/DQ8 molecules on antigen presenting cells and, by consequence, enhances gliadin recognition by gut-derived T cells [9–13], even though a deamidation-independent response of gliadin-specific T-cells was found in paediatric cases of CD suggesting that the immunological reaction may be initiated toward native gliadin peptides [14].

On this basis, the generation of new antigens by an enzymatic post-translational modification may well represent a novel mechanism for tolerance breaking and autoimmune disease initiation [15]. Because the formation of tTG-gliadin complexes within the gut mucosal wall, although plays a central role in the pathogenesis of CD [9–13], has never been demonstrated, in the present study, we aimed to explore the putative presence of gliadin and tTG complexes in the duodenal mucosa of coeliac patients in comparison to healthy controls.

PATIENTS AND METHODS

Patients and biopsies

Multiple size-appropriate endoscopic biopsy specimens of the second part of the duodenum were obtained from 13 untreated coeliac patients (8 females; mean age 32·1 years, range: 21–44 years), 13 coeliac patients on a gluten-free diet for at least 12 months (7 females; mean age 34·4 years, range: 20–42 years) and 13 consenting subjects (7 females; mean age 38·3 years, range 18–46 years) undergoing upper gastrointestinal endoscopy for functional dyspepsia. Subjects using steroidal or nonsteroidal anti-inflammatory drugs or presenting an inflamed mucosa at histology were excluded from the study.

For each patient and control, two well-oriented biopsies were processed according to standard methods for conventional histology, four biopsies were frozen at −80°C and, when possible, two additional biopsies were collected in OCT compound Tissue-Tek (Sakura Finetek Inc., Torrance, CA, USA). All coeliac patients were diagnosed on the basis of commonly accepted histological and serological criteria, namely the presence of subtotal villous atrophy with crypt hyperplasia and mucosal lymphocyte infiltration at the duodenal biopsy and the positivity for anti-endomysial antibodies. In all patients a second biopsy, taken after a course of gluten-free diet, showed a significant improvement of these lesions.

The study was approved by the local Ethic Committee and each patient gave informed consent to the study.

Antibodies

Anti-Transglutaminase II mouse monoclonal antibody (Clone CUB 7402, NeoMarkers, Fremont, CA, USA), anti-gliadin rabbit polyclonal antibody (Sigma Chemical Co., St. Louis, CA, USA), anti-human fibronectin mouse monoclonal antibody (Sigma Chemical Co.) and anti-tTG antibodies, purified from untreated coeliac sera by affinity chromatography, were used. The separation of anti-tTG autoantibodies was performed as described by Lock et al. [16]. Briefly, cyanogen bromide-activated Sepharose 4B (Amersham Pharmacia Ltd, Milton Keynes, UK) was prepared by mixing 0·5 g of beads in 20 ml 1 mm HCl for 1 h at room temperature. The beads were washed once in 40 ml 1 mm HCl and twice in 10 ml coupling buffer (0·1 m sodium hydrogen carbonate, 0·5 m sodium chloride pH 8·3). Guinea pig tTG (Sigma Chemical Co.; 1·4 mg) was dissolved in 3 ml coupling buffer and incubated with the Sepharose 4B overnight at 4°C. The beads were blocked with 0·2 m glycine pH 8 for 2 h at room temperature and then washed twice with 20 ml 0·1 m Tris + 0·5 m sodium chloride pH 8, and twice with 20 ml acetate buffer (0·1 m sodium acetate + 0·5 m sodium chloride pH 4). The material was packed into a glass column, stored at 4°C in PBS with 0·1% azide, and equilibrated with PBS before use. Samples were applied at 0·5 ml/min and the flow-through collected. Bound material was eluted with 0·1 m glycine pH 2·6 and collected in 1 ml fractions. tTG ELISA (IPR, Catania, Italy) was used to determine the fraction with peak reactivity for each serum. The active fraction and the flow through containing unbound protein were analysed in parallel with the original serum.

FITC-conjugate goat anti-mouse IgG and Cy3-conjugate goat anti-rabbit IgG were purchased from Zymed Laboratories Inc. (San Francisco, CA) and used for immunofluorescence experiments.

Immunoprecipitation and immunoblotting

After homogenization, lysis and sonication of the biopsy samples, the protein content was assayed by the bicinchoninic acid procedure (Pierce, Rockford, IL, USA). Then, 0·5 mg of lysate from each sample and from a culture of murine NIH 3T3 fibroblasts, used as positive control of the presence of tTG and as negative control for the presence of the gliadin, were incubated for 6 h at 4°C with the anti-tTG II monoclonal antibody (NeoMarkers; 2 µg/mg protein lysate). The immunoprecipitates were treated with 20 µl A/G plus agarose overnight at 4°C, and washed four times with RIPA buffer (PBS, 1% NP40, 0·5% sodium deoxycholate, 0·1% sodium dodecyl sulphate, protease inhibitors). Thereafter, those from seven untreated coeliacs, seven treated coeliacs, seven biopsied controls, and from the fibroblast cell culture were dissolved in 50 µl sodium dodecyl sulphate sample buffer under reducing/denaturating conditions, boiled and loaded into the slots of 12·5% sodium dodecyl sulphate polyacrylamide gels. Separately, the precipitated proteins from six untreated coeliacs, six treated coeliacs and six biopsied controls were divided into two parts and run on two fixed 8% (wt/vol) polyacrilamide gel electrophoresis in nondenaturing/nonreducing conditions with a Mini-Protean II apparatus (Bio-Rad Laboratories, Richmond, CA, USA). The proteins from one of these gels were stained with AgNO3, as described by Damerval et al. [17], while the proteins from the other gel were processed as described above. In both reducing/denaturating and nonreducing/nondenaturating gels, guinea pig transglutaminase, gliadin, pepsin-trypsin digested gliadin, and human fibronectin (all from Sigma Chemical Co.) were loaded as positive controls. The proteins from all gels, except those from gel stained with AgNO3, were blotted onto nitrocellulose membranes and immediately stained with Ponceau red (Sigma Chemical Co.). Destaining was carried out by immersing the membranes in distilled water until bands became visible. Then, the membranes were processed first 1 h with TBS-T + 10% powdered milk, and parallely incubated overnight with the anti-tTG II monoclonal antibody (NeoMarkers; 1 : 500 dilution) or with the anti-gliadin polyclonal antibody (Sigma Chemical Co.; 1 : 1000 dilution). As positive control of the occurred tTG coimmunoprecipitate the anti-human fibronectin antibody (Sigma Chemical Co.; 1 : 500 dilution) was used, being fibronectin another preferred substrate of tTG [18]. After washing and treatment for 1 h with TBS-T + 10% powdered milk, the blotted bands were incubated with the relevant horseradish peroxidase-linked immunoglobulin G for 1 h in the same buffer. After extensive washing, immunostaining was recorded photographically, using an enhanced chemiluminescence kit (Amersham Pharmacia Ltd). The subsequent steps were the same as described above.

In a subset of experiments, immunoprecipitation was performed by using anti-tTG antibodies purified from four adult untreated coeliac patients, in which high serum titres of these class A antibodies had previously been established by ELISA (IPR).

Bands were quantified by scanning densitometry using an LKB Ultrascan XL Laser Densitometer (Kodak Ltd, Hemel Hempstead, UK) and measured in terms of intensity and surface area of each band corresponding to tTG, or in terms of the sum of intensity and area of the bands relative to gliadin and its fragments.

Confocal immunofluorescence microscopy

Cryostat seriate sections (4 µm) of OCT-embedded endoscopic specimens from eight untreated, seven treated coeliacs and seven biopsied controls were fixed in methanol for 10 min at −20°C, and incubated either with the anti-tTG II monoclonal antibody (NeoMarkers; 1 : 40 dilution) or with the anti-gliadin polyclonal antibody (Sigma Chemical Co.; 1 : 50 dilution) plus 1% bovine serum albumin for 1 h at room temperature in a moist chamber. After washing with PBS, the sections were incubated with a FITC-conjugate goat anti-mouse IgG, or Cy3-conjugate goat anti-rabbit IgG (both from Zymed Laboratories Inc.), respectively, and 10% normal goat serum for 1 h. Confocal immunofluorescence microscopy was performed by using a SARASTRO 2000 confocal scanning laser microscope (Molecular Dynamics). The light source was an argon ion laser (25 mW) giving excitation wavelength in the region 458–584 nm. FITC was excited at 488 nm. Cy3 was excited at 550 nm. Digital images of single optical sections were acquired using a 63× Nikon Plan-Apo objective; image sizes were 512 × 512 (pixel size: 0·17 µm). Negative control cover-slides were set by exposing the seriate sections under similar conditions but without the primary antibody.

Statistics

Statistical analysis by Student's t-test was performed by the STATPAC Computerized Program and a P-value ≤0·05 was used as the significance criterion.

RESULTS

Co-immunoprecipitation experiments

By immunoprecipitation and blotting of duodenal biopsies of untreated, treated CD patients and control subjects with the anti-tTG monoclonal antibody in reducing and denaturating conditions, we found, in all cases, a band of 85 kD (Fig. 1a), which was identified as tTG by using a tTG extract from guinea-pig liver and a lysate of the murine fibroblast cell line 3T3 (data not shown) as positive controls. It is noteworthy that this band had the same molecular weight of the original protein immunoprecipitated by Dieterich et al.[3] from human fibrosarcoma cell lysate by using the immunoglobulin A fraction of CD sera, which, after sequence analysis, was assigned to tTG. The quantification of the bands showed a significantly increased level of tTG in untreated CD patients compared to treated CD and healthy controls, such as in treated CD compared to healthy controls (Fig. 1a). The blotting of the tTG-immunoprecipitates with the anti-gliadin antibody revealed the coimmunoprecipitation of both whole gliadin and its fragments directly in the gut mucosa of active CD mucosa. In particular, other than some aspecific high molecular bands (>95 kD, data not shown), bands of 52 and 44 kD corresponding to undigested gliadin, of 33 and 28 kD, together with an area ranging from 13 to 9 kD molecular weight corresponding to digested gliadin fragments (the lane of pepsyn-trypsin treated gliadin was entirely occupied by a smear: data not shown) were evident. In the control mucosa the levels of these bands were lower than untreated CD, whereas in treated CD mucosa, only a weak positivity in correspondence of the 33 and 28 kD bands was present (Fig. 1b). As expected, no gliadin band was revealed in the fibroblast lysate (data not shown). Quantitative analysis assessed that in mucosal samples of active coeliacs, the sum of both areas and intensities of gliadin bands was significantly higher in comparison to control biopsies, while in treated coeliacs the amount was very scanty compared to the other two groups (Fig. 1b). Concerning the results obtained by immunoblotting with the anti-fibronectin antibody, a band corresponding to the fibronectin in both healthy and diseased mucosa was found (data not shown).

Fig. 1.

Fig. 1

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a. Western blot and densitometric analysis of immunoprecipitates obtained by the anti-tissue transglutaminase (tTG) monoclonal antibody from duodenal biopsies of three representative cases of untreated (UCD), treated coeliac patients (TCD) and biopsied controls (BC). The samples were dissolved in sodium dodecyl sulphate sample buffer under reducing and denaturating conditions and loaded into the slots of 12·5% sodium dodecyl sulphate polyacrylamide gel electrophoresis. The band of 85 kD (arrow) is evident in all cases, and the quantification demonstrated a significantly increased level in UCD compared to TCD and BC, such as in TCD compared to BC. b. The blotting with the anti-gliadin polyclonal antibody of the same samples revealed the presence of bands of 52, 44, 33, 28 kD, together with an area ranging from 13 to 9 kD molecular weight in UCD mucosa, whereas these bands were weaker in BC and only a light positivity in correspondence of the 33 and 28 kD bands was present in TCD. Densitometric analysis of both band intensity and area is also shown. Mean values ± SEM are reported (*P < 0·05; **P < 0·001).

The experiments carried out in nondenaturing and nonreducing conditions revealed the presence of a band of 176 kD molecular weight in the gel stained with AgNO3 in the duodenal mucosa of untreated CD and biopsied controls, whereas no band was evident corresponding to the positive control tTG (Fig. 2a), suggesting that all tTG is presumably engaged in forming supramolecular complexes. When the same samples, loaded in the other nondenaturating and nonreducing gels, were blotted with the anti-tTG monoclonal antibody (Fig. 2b) and with the anti-gliadin antibody (Fig. 2c), the high molecular weight band was evident, demonstrating the bond between tTG and gliadin peptides. In these experimental conditions, no significant difference within the groups was revealed by densitometric analysis (data not shown). When considering treated CD mucosal samples, even though the 176 kD band was also evident either in the gel stained with AgNO3 or in the gel blotted with the anti-tTG monoclonal antibody, no band appeared after anti-gliadin antibody incubation, suggesting that in this condition all tTG is involved in binding other molecules (data not shown).

Fig. 2.

Fig. 2

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(a) Western blot analysis of immunoprecipitates obtained by the anti-tissue transglutaminase (tTG) monoclonal antibody from duodenal biopsies of four representative cases of untreated coeliac patients (UCD), and two biopsied controls (BC). The samples were loaded into the slots of 8% sodium dodecyl sulphate polyacrylamide gel electrophoresis in nondenaturing and nonreducing conditions as described in the Methods section. The presence of a band of 176 kD at the gel stained with AgNO3, directly in the duodenal mucosa of the two groups studied was found, whereas no bands were evident corresponding to the positive control tTG. The same samples loaded in nondenaturing and nonreducing conditions, were blotted either (b) with the anti-tTG monoclonal antibody or (c) with the anti-gliadin polyclonal antibody. These results revealed that tTG is engaged with gliadin to form a supramolecular complex of 176 kD.

The obtained results represent the first direct evidence that the action of tTG in duodenal mucosa leads to the formation of covalently crosslinked supramolecular structures which, in the subjects following a normal diet, comprise gliadin molecules. The covalent bonds that give rise to these complexes could be dissolved by using strong denaturating and reducing conditions, thus enabling us to observe tTG and gliadin in a separate migration pattern, as shown in Fig. 1.

In the second set of experiments, the purified anti-tTG fraction from sera samples of untreated CD patients were able to recognize and coimmunoprecipitate detectable levels of tTG (Fig. 3a) and gliadin (Fig. 3b) in duodenal mucosa of both active CD and control conditions. Of note, just two gliadin bands, with a molecular weight of 52 and 44 kD, were detectable by using anti-tTG sera samples (Fig. 3b) instead of at least six bands shown by using the anti-tTG monoclonal antibody (Fig. 1b). No significant difference between healthy controls and CD patients was revealed by densitometric analysis of both tTG and gliadin levels (data not shown).

Fig. 3.

Fig. 3

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a. Western blot analysis of immunoprecipitates obtained by the purified anti-tissue transglutaminase (tTG) autoantibodies from an adult patient with active coeliac disease, of duodenal biopsies of four representative cases of untreated coeliac patients (UCD), and two biopsied controls (BC). The band of 85 kD (arrow) is evident in all cases. b. The blotting with the anti-gliadin polyclonal antibody revealed the coimmunoprecipitation of two different bands of 52 and 44 kD in the mucosa of the two groups.

Co-localization experiments

Confocal immunofluorescence microscopy analysis was performed to study the localization of tTG and gliadin in normal and diseased duodenal mucosa, by processing seriate sections with the same antibodies used in the coimmunoprecipitation experiments. In the normal mucosa, tTG positivity was found in close connection to a population of cells situated just beneath the epithelium either in the crypts (Fig. 4a) or in the villi (Fig. 4b). In untreated coeliac patients the labelling intensity was increased and appears to be like clods at the level of the extracellular matrix (Fig. 4e), and the epithelium becomes slightly positive at the surface level (Fig. 4f). The intestine of treated coeliac patients showed at these localizations, a labelling pattern between that of the normal and the active diseased samples (Fig. 4c,d). As demonstrated by our previous immunohistochemical study, the subepithelial tTG containing cells closely resemble fibroblasts [4].

Fig. 4.

Fig. 4

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Confocal images of biopsy specimens from one representative case of biopsed control (a, b), one representative case of treated coeliac patient (c, d) and one representative case of untreated coeliac patient (e, f) incubated with the anti-tTG monoclonal antibody. In (a, c, e) the crypt regions are shown, whereas in (b, d, f) the epithelial surface is shown. The marker corresponds to a length of 10 µm.

As concerns the sections incubated with the anti-gliadin polyclonal antibody, in the biopsied controls we observed the positivity within enterocytes either in the crypts (Fig. 5a) or in the villi, other than in the lamina propria of the villi (Fig. 5b); in untreated CD the enterocyte staining was highly evident and lamina propria resulted positive either at the crypt or surface level (Fig. 5e,f). In treated CD we found only a weak positivity of the brush border of the enterocyte present at the apex of the villi and few spots in the lamina propria of the crypts (Fig. 5c,d). These observations allow us to conclude that tTG colocalizes with gliadin at the epithelial and subepithelial levels in active CD, whereas the colocalization is observed only in the lamina propria of the villi in normal mucosa.

Fig. 5.

Fig. 5

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Confocal images of biopsy specimens from one representative case of biopsed control (a, b), one representative case of treated coeliac patient (c, d) and one representative case of untreated coeliac patient (e, f) incubated with the anti-gliadin polyclonal antibody. In (a, c, e) the crypt regions are shown, whereas in (b, d, f) the epithelial surface is shown. The marker corresponds to a length of 10 µm.

DISCUSSION

Growing evidences indicate that tTG is involved in the pathogenesis of CD [3,9–13], and that gliadin would represent a highly preferred substrate because of its high content of glutamyl residues [8,19]. tTG is best known for its ability to catalyse transamidation reactions in a multistep process [5], however, at low pH or when no acceptor proteins are available, deamidation of glutamines is favoured over their incorporation into isopeptide bonds [20]. The specific and ordered conversion of a glutamine residue into a negatively charged glutamic acid mediated by tTG [9,10], allows its selective binding to HLA-DQ2/DQ8 molecules and enhances gliadin recognition by gut-derived T cells from coeliac patients [9–11]. Furthermore, the development of autoantibodies to tTG has been suggested to result from the covalent incorporation of tTG into high molecular weight complexes with gliadin, a reaction consequent to the autocatalytic activity of tTG [20]. Only recently, it has been demonstrated that T cells within coeliac lesions, other than react toward native gliadin epitopes [14], recognize deamidated gliadin epitopes too that are formed in situ by endogenous tTG [21]. However, to date, no direct evidence is available that gliadin is cross-linked to tTG directly in the gut wall, a phenomenon known to occur in vitro[3,9].

Our findings revealed that in CD mucosa of both untreated and treated patients, the levels of tTG are increased in comparison to healthy mucosa, supporting a previous study in which the activity of this enzyme has been found raised in the same conditions [8], whereas those of coimmunoprecipitated gliadin result increased in active CD compared with normal mucosa and are almost absent in treated CD patients that follow a gluten free diet. Furthermore, our study firstly indicated that tTG and gliadin are bound to form supramolecular complexes directly in the duodenal mucosa of both untreated CD patients and biopsied controls. Specifically, the immunoprecipitates obtained from the biopsy specimens by using either the commercial monoclonal anti-tTG antibody or the purified anti-tTG antibodies, revealed the presence of the enzyme, as far as that gliadin was bound to tTG at Western blot analysis. However, by using the anti-tTG monoclonal antibody, in adult active CD, many gliadin fragments, with different molecular weights, were coimmunoprecipitated, whereas it does not appear with the purified anti-tTG antibodies, suggesting a different immunoreactivity of the antibody preparations [22]. When performing the experiments in nondenaturating and nonreducing conditions the covalent bond which links the molecules remains intact, allowing the demonstration that tTG is prevalently engaged with gliadin in the formation of supramolecular complexes in the subjects following a gluten-containing diet, as well as with other molecules in the subjects following a gluten-free diet.

The confocal microscopy experiments designed to assess the localization of tTG and gliadin in mucosal samples showed that in the normal mucosa tTG is expressed only in the subepithelial region from cells that resemble the features of myofibroblasts, as we have previously demonstrated [4]. It has been shown that at this level, tTG is closely associated with the β-integrin functioning as a cell surface adhesion molecule independently from its catalytic activity [23]. However, upon mechanical stress, inflammation, infection, or during apoptosis, the enzyme is secreted into the extracellular matrix and expresses its catalytic activities crosslinking several proteins, then giving these molecules greater resistance to degradation [24]. Accordingly, in active CD we observed either an increased labelling intensity of tTG at the level of subepithelial region with a pattern similar to clods that probably reflects an extracellular localization, or the presence of positivity in the epithelium that was absent in treated CD and normal mucosa. The increased positivity observed in the submucosa of both untreated and treated CD, together with the immunoprecipitation results, could reflect either a clonal proliferation of myofibroblasts or the induction of tTG synthesis and secretion by the action of cytokines. The findings that tTG is regulated by TNF-α[25] and IL-6 [26], and the demonstration by Nilsen et al. [27] that these two cytokines are up-regulated in the CD mucosa, argues in favour of the latter hypothesis. Furthermore, the epithelial labelling present in active CD, might be explained by the presence of abnormal apoptosis levels in the diseased intestine [28], being this enzyme involved in the programmed cell death cascade [29].

As concern gliadin localization, we observed that in untreated coeliac mucosa there is a strong fluorescence both in the epithelium and in the submucosa, whereas in the normal mucosa the labelling is weaker and totally absent in the extracellular matrix at the crypt level. The red spots present in the lamina propria of treated CD mucosa may be explained by the prolonged trapping of antigens by dendritic cells [30], whereas the positivity of the brush border could be attributed to a continuous exposure to trace amounts of gliadin in the diet or to an unspecific staining, accordingly with the results reported in the coimmunoprecipitation section. Therefore, a colocalization of gliadin with tTG is evident in untreated CD patients and healthy controls, even though possible differences in tTG catalytic activity among these groups cannot be excluded.

Taken together, our results suggest that tTG and gliadin form supramolecular complexes directly in normal and diseased duodenal mucosa and that in active CD the levels of both molecules are increased. On the other hand, we cannot exclude the possibility that the tTG coimmunoprecipitates found in our study might include other substrates such as peptides of the extracellular matrix, or some viral, bacterial or other nutritional proteins [31], whose identity needs further investigations. Finally, our findings prompt us to hypothesize that, since gliadin is bound to tTG in biopsied controls too, this event is necessary, but not sufficient for the development of enteropathy.

Acknowledgments

The Authors wish to acknowledge the Associazione Italiana Celiachia-Sezione Lombardia for financial support, and Mr Prospero Colimberti for his skilled technical assistance.

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