Summary

Dieterich et al show that immunoprecipitation of human fibrosarcoma cell lysates (cell line HT1080) using the IgA fraction from serum samples of patients with coeliac disease results in a single protein band with a molecular weight of 85 kDa. Immunoprecipitation occurred exclusively with 25 coeliac disease serum samples, but with none of the 25 control samples. The 85 kDa antigen was cleaved with endoproteinase Asp-N, and after amino-terminal sequence analysis, the three cleavage products tested all yielded sequences that could be assigned to tissue transglutaminase (EC2.3.2.13; tTG). In order to prove that tTG obtained from the fibrosarcoma cells binds to the endomysial antibody fraction of coeliac serum IgA, the authors performed indirect immunofluorescence with high titre coeliac disease serum samples on monkey oesophagus with or without prior incubation of those samples with a commerically available tTG extract (Sigma, Deisenhofer, Germany). Pretreatment with tTG almost completely abolished endomysial antibody labelling. Also, when the Sigma tTG was used as antigen in an enzyme linked immunosorbent assay (ELISA), 12 coeliac disease patient samples but none of the seven control serum samples displayed increased IgA immunoreactivity. Dieterich et al conclude they have identified tTG as the unknown endomysial autoantigen.

Comment

Coeliac disease is brought about by ingestion of cereal gluten.1 At the same time, gliadin triggers the production of certain IgA class serum tissue autoantibodies.2 The antigen recognised by these antibodies is highly preserved in rodents and primates. In humans the antigen is present in the extracellular matrix of most tissues,3 and in coeliac disease the antibodies are thought to be target organ related.4 The antigen is also intensively expressed in monkey oesophagus5 and human umbilical cord vessel6smooth muscle fibre encased connective tissue, known as endomysium. Identification of the autoantigen(s) has been the aim of researchers for many years. In 1973, Pras and Glynn isolated a non-collagenous reticulin component from kidney tissue,7 but later studies suggested that reticulin was not a single entity.8Our group identified and purified extracellular matrix non-collagenous protein molecules from human fetal lung tissue that bound specifically to serum reticulin and endomysial IgA from patients with coeliac disease.9 We hypothesised that autoimmune mechanisms are important in the pathogenesis of coeliac disease. In a later study we showed that the autoantigen is expressed by human fibroblasts.10 Whelan et al showed that an antigen found in human umbilical vein endothelial cells is antigenically similar to that found in reticulin and endomysium.11 Karska et al again suggested that calreticulin is a potential autoantigen12 and Borneret al isolated several structural protein components from various animal tissues.13

Now Dieterich and colleagues have shown that tTG is the predominant, if not the sole, endomysial autoantigen characteristic for coeliac disease. Without trying to underrate the importance of this finding, some methodological aspects seem worthy of comment. The authors did not show that the immunoprecipitated 85 kDa protein possesses tTG activity, nor did they present a positive immunoblot with monoclonal tTG antibody. Nevertheless, after cleavage, the molecule was identified as tTG on sequencing. What evidence is there that tTG binds solely to reticulin/endomysial IgA (perhaps 10% of all the coeliac IgA) and not to gliadin, other food proteins, or other autoantigen IgA in the same serum sample? Here there may be a potential pitfall. Inhibition of endomysial antibody positivity was not performed using a pure synthetic or human recombinant tTG but with Sigma tTG, a crude extract from guinea pig liver, containing tTG and possibly other matrix proteins. In fact, Sigma tTG is comprised of at least 14 different components, as seen on silver staining. Also, the liver, both in rodents and primates, expresses the reticulin/endomysial autoantigen and absorbs from coeliac serum samples the IgA responsible for reticulin/endomysial antibody positivity.2 ,14 Is the inhibition of endomysial antibody positivity due to other unidentified reticulin/endomysial molecules in the liver extract? In parallel, the ELISA results may also be due to liver reticulin and not only to tTG in the commercially available tTG extract.

Nevertheless, tTG fits the picture. It is widely distributed in human organs. The precise physiological function of tTG is still unclear, but evidence is accumulating that there is a close correlation between tTG activity and cell proliferation, differentiation and death. tTG may have an important role in the formation of the extracellular matrix. In this context the biological events occurring in the small bowel crypt–villus axis in normal intestine and coeliac disease is of interest. In wound healing the provisional extracellular matrix is stabilised by tTG. Interestingly, coeliac disease research has concentrated on transglutaminases for many years.15-18Furthermore, gliadin is a preferred substrate for tTG, as shown by Dieterich et al. This provides new insights into the findings of Unsworth et al 19 who showed specific gliadin binding in a reticulin-like manner to connective tissue fibres of mammalian tissues.

It is now tempting to speculate that ingested and digested gliadin, known to have high affinity for jejunal mucosal lamina propria reticulin and tTG, unmasks cryptic tTG epitopes leading to the production of reticulin/endomysial autoantibodies.20 At the same time specific HLA class II molecules on antigen presenting cells could present the tTG self-peptides, thus activating the autoreactive T cell population. Continuing gliadin ingestion, analogous to recurrent infection, may be responsible for the maintenance of the disease by continuously revealing disease triggering self-epitopes. Coeliac disease is indeed self-perpetuating and irreversible if the environmental trigger, gliadin, is not removed. It is not known what the outcome in other autoimmune diseases would be if all the environmental triggers could be removed early enough.