IgA (green) and transglutaminase 2 (TG2, red) in the normal jejunum (A, D, G), in the early developing stage of coeliac disease (B, E, H) and in the coeliac flat lesion (C, F, I). (B) Subepithelial IgA deposits in the villi and around crypts in the mucosa with a normal architecture and (C) in the flat mucosa developed four months later in the same patient. IgA deposits colocalised with TG2 (E, F, H. I), as indicated by the yellow merging of the labels. Both deposited IgA and TG2 were closely related to vessels shown in blue (I, J, arrows). (K) Similar jejunal IgA deposits in a patient with dermatitis herpetiformis and negative serum endomysial antibodies. (L) Crypt basement membrane laminin (red band) with coeliac IgA (green) deposits. There was only partial overlap (yellow) on the extracellular surface of the laminin. Bar = 50 m m. 

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... frozen tissue samples from six additional patients who underwent biopsies from organs other than the small bowel while they had active coeliac disease. All of these biopsies had been performed based on clinical indications independent of the evaluation of coeliac disease (table 2). Samples were traced retrospectively and included two lymph nodes, one skeletal muscle, two liver biopsy samples, and a non-inflamed appendix. As controls, one lymph node, three liver, and six appendix samples from non-coeliac patients were included. These had been removed in unrelated surgical interventions and were histologically normal. All biopsies were performed for diagnostic purposes and informed consent was obtained. Unfixed 5 m m thick sections of patient tissues were investigated for deposited immunoglobulins by direct immunofluorescence using fluorescein isothiocyanate labelled rabbit antibodies against human IgA, IgG, and IgM (Dako AS, Glostrup, Denmark) at a dilution of 1:40 in phosphate buffered saline (PBS), pH 7.4. These stainings were also performed as multicolour procedures where patient immunoglobulins were shown in green and extracellular matrix proteins in red using rhodamine conjugated antimouse or antirabbit secondary antibodies, as described elsewhere. 18 19 The primary antibodies used were monoclonal mouse antibodies against TG2 (CUB7402; NeoMarkers, Fremont, California, USA), and rabbit antibodies against fibronectin (Dako) and laminin (Dako), respectively, diluted 1:200 in PBS. For investigation of the relation of patient immunoglobulins to blood vessels, green was used for detection of patient IgA, red for TG2, and blue for an endothelial marker (rabbit antibodies against von Willebrand factor (Dako) followed by AMCA conjugated secondary antirabbit antibodies (Vector Laboratories, Burlingame, California, USA)). Unfixed cryosections were washed twice in PBS to remove blood and related antibodies. A solution of 0.25% chloroacetic acid (Fluka Chemie AG, Buchs, Switzerland) with 0.2 M/l NaCl, pH 2.7, was then used as the eluent 28 and applied to the sections for 20 minutes. The eluates with chloroacetic acid were neutralised with 1 M/l imidazole, washed in PBS, concentrated by centrifugation on 10K membranes (Microsep, Pall Corporation, Ann Harbor, Michigan, USA), and lyophilised. Treated sections were washed twice in PBS and examined by immunofluorescence for the remaining IgA and TG2 in tissues, as described above. Sections from extraintestinal samples, from six coeliac and three dermatitis herpetiformis jejunum samples, were investigated by immunofluorescence after choroacetic acid treat- ment. Lyophilised tissue eluates were prepared from a coeliac liver, lymph node, and a non-coeliac liver. Endomysial antibodies were investigated in serum samples and tissue eluates by an indirect immunofluorescent method on monkey oesophagus, normal human umbilical cord, jejunum, and appendix sections, as described elsewhere. 18 The initial dilution was 1:2.5 in PBS, and samples non-reactive at this dilution were considered negative. Eluates were also tested on TG2 knockout mouse (TG2 2 / 2 ) jejunum as a substrate, and on TG2 2 / 2 jejunum coated with human recombinant TG2 expressed in Escherichia coli , 29 as previously described. 19 Tissue eluates were tested for the presence TG2 and IgA by western blot 30 and for TG2 specific IgA by ELISA using microtitre plates coated with fibronectin (Sigma, St Louis, Missouri, USA) and recombinant TG2, as described by Achyuthan and colleagues. 31 Also, plates coated with rabbit antibodies against mouse IgG1 (ICN, Aurora; dissolved in 0.03 M bicarbonate buffer, pH 9.6) and CUB7402 monoclonal TG2 specific capture antibodies were applied to further verify the presence of both TG2 specific IgA and TG2 in the eluates. Bound IgA was detected with peroxidase conjugated rabbit antibodies against human IgA, as described previously. 30 In non-coeliac jejunal biopsy samples, IgA was detected only inside plasma cells and epithelial cells (fig1A). In contrast, clear subepithelial IgA positivity was found along the villous and crypt basement membranes in jejunal samples from all 10 coeliac patients, both in the developing stage of the disease when the villous architecture was still normal and also later when the mucosa had deteriorated to the typical flat lesion (fig 1B, C, same patient). This IgA positivity pattern was identical to the normal tissue localisation of TG2 (fig 1D, G; red) which appeared merged into yellow in the coeliac mucosa due to colocalisation with IgA deposits labelled in green (fig 1E, F, H, I). This colocalisation was also seen on reticulin fibres of lymphoid follicles (fig 1H, asterisk) and on trabecular structures which appeared fragmented in the flat lesion (fig 1F, arrow). IgA in plasma cells or inside crypt epithelial cells did not merge into yellow in either normal or coeliac mucosa. Both coeliac IgA and TG2 were arranged around mucosal vessels, shown in blue (fig 1 I, J, arrows). Coeliac IgA was located on the extracellular surface of laminin (fig 1L), and also showed a close relation to fibronectin (not shown). Similar IgA deposits were present in seven of 11 patients with dermatitis herpetiformis who had a normal jejunal villous structure at diagnosis. Two dermatitis herpetiformis patients who had clear jejunal IgA deposits were negative for circulating IgA class serum endomysial antibodies (fig 1K). Jejunal IgM and IgG positivity did not differ between coeliac and control samples. To establish whether coeliac IgA can reach TG2 in extraintestinal tissues, we examined by double immunofluorescence two coeliac lymph nodes, two liver samples, one appendix, and one skeletal muscle specimen from coeliac patients (table 2, figs 2, 3). Specimens were normal (cases 1, 2, 3, 5, and 6 in table 2) or showed only non-specific changes (case 4, liver steatosis; table 2) by conventional microscopy. All of these tissues contained abundant TG2 in endomysial localisations or around reticulin fibres both in non-coeliac controls (fig 2A, 3B) and in coeliacs (fig 2G, J; fig 3D, F, H, red). Tissue IgA was not related to endomysial or reticulin structures in non-coeliac lymph node (n = 1), appendix (n = 6), or liver (n = 3) samples and appeared only inside epithelial cells or in the centre of lymphoid follicles (fig 2A, D; fig 3A, B, green). However, in coeliac samples there were heavy IgA deposits on reticulin fibres also (lymph nodes, appendix, liver (fig 2B, E, H; fig 3C)) and on endomysial structures (muscular layer of the appendix, skeletal muscle (fig 2F, K)) which were colocalised with tissue TG2 (fig 2C, I, L; fig 3D). In the case of IgA deficiency (cases 5 and 6 in table 2), IgA deposits were missing (fig 3E, F) but TG2 related IgG deposits were seen in the liver (fig 3G, H) and also on archive electron microscopic kidney pictures of case 6 (not shown). Colocalisation of in vivo deposited antibodies with TG2, both in the jejunum and also in extraintestinal tissues, strongly suggested that the specific target for coeliac antibody binding is TG2. To obtain direct evidence, we eluted deposited IgA from extraintestinal tissues for further studies. We used chloroacetic acid to disrupt TG2 binding to fibronectin 28 and to release the in vivo deposited IgA together with released TG2. Treatment of the sections resulted in disappearance of extracellular IgA deposits in sections from both the extrajejunal and jejunal coeliac samples, while IgA in plasma cells and epithelial cells remained visible on immunofluorescence (fig 4A, B). Likewise, in tissues, TG2 became undetectable by monoclonal TG2 specific autoantibodies after elutions with choroacetic acid. To ascertain that both TG2 and IgA are found in eluates, western blot and ELISA studies were conducted with concentrated eluates prepared from a coeliac and a non- coeliac control liver, and from a coeliac lymph node. A clear band at 80 kDa, corresponding to the molecular weight of TG2 was, as expected, equally detectable with monoclonal TG2 antibodies both in coeliac and non-coeliac eluates (fig 4C). Human IgA was also detected both in coeliac and non-coeliac eluates (data not shown). However, when the eluates were tested by ELISA for TG2 specific IgA class antibodies, high optical density values were obtained only with IgA eluted from the coeliac liver and lymph node (0.578 and 2.551, respectively) but not with IgA eluted from the control liver (0.052, representative values from two independent elution experiments). This finding indicates that IgA originating from non-coeliac tissues was not directed against TG2. The result was confirmed in an additional experiment where the eluates were added to ELISA plates coated with TG2 specific monoclonal antibodies and IgA was measured from the bound TG2 human IgA complexes; coeliac liver eluate gave an optical density of 0.493 versus 0.107 obtained with the control liver eluate (fig 4D). Similarly, IgA eluted from coeliac tissues bound to human recombinant TG2 applied on TG2 2 / 2 mouse tissues (fig 4E, F), and also to the endomysial and reticulin structures of human umbilical cord (fig 4I), monkey oesophagus (fig 4J), and normal appendix (fig 4K) sections. In contrast, IgA eluted from the non-coeliac liver did not bind to any of these structures (fig 4G, H). Furthermore, there was no IgA binding to the TG2 2 / 2 mouse tissues with either the coeliac or non-coeliac eluates. The experiments above collectively demonstrate that deposited IgA seen in coeliac tissues is targeted against TG2. In the present study, we showed for the first time that in vivo targeting of TG2 in coeliac disease occurs in the form of in situ endomysial, reticulin, and jejunal subepithelial autoantibody binding. Patient IgA found deposited on extracellular TG2 in the liver, lymph nodes, and muscles indicates that the coeliac disease autoantigen is widely accessible to the intestinally produced 21 circulating ...