Coeliac disease is an autoimmune disorder characterised by production of a large array of antibodies against tissues structures, matrix, and cellular antigens.1,3,8–11,19
According to the present findings, TG2 specific autoantibodies would appear to be of primary importance. Our results with TG2 knockout tissues for the first time directly prove that the diagnostically relevant EMA, ARA, and JEA binding patterns in serum samples from both coeliac and dermatitis herpetiformis patients are clearly and exclusively TG2 autoantibody dependent and target the fibronectin bound extracellular TG2 in normal human and rodent tissues. In the large series of serum samples we tested on TG2 knockout mouse tissues, TG2 unrelated non-specific EMA positivities were not observed. Complementing these results, we also experienced the same with EMA positive subjects in whom coeliac disease remained unconfirmed (data not shown). Thus the typical coeliac-type immunofluorescent positivity has an outstanding specificity for TG2 antibodies. Non-TG2 antibodies that produce EMA binding, albeit theoretically possible, seem to be very rare.
Serum samples from some seriously ill coeliac patients often show complex antibody binding. Due to high background binding to epithelial or smooth muscle cells, such “difficult” samples may appear negative for EMA in low serum dilutions20
but react with TG2 in ELISA. Smooth muscle cells contain high amounts of TG2. However, our results do not support the notion that reactivity with smooth muscle cells represents antibodies to intracellular TG2, as it was similarly observed in TG2 deficient tissues. We identified a subtype of smooth muscle antibodies as antibodies to actin. When present together with EMA, actin antibodies bound to the contractile bundles of the villi may modify the appearance of the coeliac-type positivity on the jejunum and may be responsible for the additional non-TG2 related components observed by other investigators.12
However, actin antibodies alone were not able to elicit EMA-type positivity in the absence of TG2.
We frequently found non-TG2 related antibodies in serum samples from coeliac and dermatitis herpetiformis patients but in low titres compared with endomysial autoantibodies. Apart from antibodies against actin, their binding lacked any specific morphological appearance and they occurred in less than half of all patients. Gliadin antibodies were associated both in coeliacs and controls with a higher rate of antibody binding to jejunal epithelial cells. These features support the view that these antibodies may be secondary and variably induced by tissue damage facilitating increased presentation of different self antigens to the immune system.19
As we now observed, coeliac disease specific autoantibodies react primarily with human TG2. Cross reaction with mouse TG2 was prevalent (93.4%) but not universal in our coeliac patients. The slightly different rate of coeliac autoantibody positivities in primate and rodent tissues was for a long time a subject of diagnostic debate and led to the misconception that ARA (rodent specific) and EMA (primate specific) tissue autoantibodies represent different antibody populations.21
However, with demonstration that a recombinant human TG2 protein inserted into mouse tissues comprises as good a coeliac autoantigen as natural primate tissues, we provide additional evidence that the “reticulin” and “endomysial” binding patterns are tissue-type and not species dependent detection methods of the same TG2 autoantibodies.
Our current understanding of the differences in clinical outcome between classical coeliac disease and dermatitis herpetiformis, where apart from gluten enteropathy dermal IgA deposits also occur, remains incomplete. It has been hypothesised that dermal IgA may represent immune complexes or additional antibodies against dermal proteins.2
Recent interest has focused on potential cross reaction with other structurally related transglutaminases, of which several are expressed in the epidermis (TG1, TG3, TG5, TG7).4,22,23
While serum antibodies from coeliac and dermatitis herpetiformis patients do not recognise TG1, it has been shown recently that a subset in both diseases cross reacts with TG3 (epidermal transglutaminase).23
In dermatitis herpetiformis, patients also develop high avidity antibodies specific for TG3, and TG3 is a component of dermal deposits and seems to be the major autoantigen involved in skin manifestations.23
However, dermatitis herpetiformis patients also produce autoantibodies that primarily react with TG2 and relate to the enteropathy. As we have shown here, TG3 specific antibodies do not contribute to the EMA positivity of these sera which requires targeting of TG2.
In similar ways, other particular presentation types of coeliac disease may potentially be associated with targeting of both TG2 and other transglutaminases. Studies with mRNA suggest that TG5,7 as well as TG3 are widely distributed in various tissues4,22,23
but the protein amounts and their histochemical localisation in non-dermal tissues are at present still unknown. Tissues of TG2 knockout mice are particularly suitable in the search for such autoantibody targets because the apparently normal condition of these mice is most probably maintained by other upregulated transglutaminases. 13
As we also observed, these mice do not develop jejunal villous atrophy even in the absence of TG2 function. However, with the conventional immunofluorescent technique, we did not find any staining differences between samples from coeliac and dermatitis herpetiformis patients. This is not in contrast with previous results because the autoantibodies in both conditions can cross react with TG3 in ELISA and they differ only in their relative avidity and specificity23
which, however, may not lead to visible differences in immunofluorescence. We were also unable to demonstrate staining differences between patients and controls in TG2 deficient tissues. Several explanations are possible, such as the small fraction of TG3 reactive autoantibodies23
or the low amounts of TG3 protein. In fact, TG3 specific rabbit antibodies, working in western blot or on skin sections, also failed to show jejunal immunofluorescent positivity in other studies.22,23
Therefore, further search for additional transglutaminase targets will necessitate more sensitive detection tools or unmasking of antigens.
In conclusion, our results confirm the identity of EMA, ARA, and JEA with TG2 specific autoantibodies and directly prove the exclusive role of TG2 in the specific and diagnostic coeliac-type immunofluorescent reactions.