Biological functions of coeliac disease autoantibodies

Abstract

Keliakia on sairaus, jossa vehnän, rukiin ja ohran valkuainen, gluteeni, laukaisee immunologisen reaktion ohutsuolessa tietyn perimän omaavilla ihmisillä. Jatkuva viljan käyttö ravintona ylläpitää reaktiota ja sen seurauksena ohutsuolen rakenne vaurioituu, jolloin myös elimistön tarvitsemien ravintoaineiden imeytyminen häiriintyy. Viljojen aiheuttaman keliakian tarkkaa taudinaiheuttamismekanismia ei vielä tunneta. Tampereen yliopiston Lääketieteen laitoksen Lastentautien tutkimuskeskuksessa toimivan, prof. Markku Mäen johtaman keliakiatutkimusryhmän hypoteesin mukaan kyseessä on autoimmuunitauti. Keliakiapotilaan veressä kiertävät vasta-aineet tunnistavat ihmisen kudoksissa olevan entsyymin, kudostranglutaminaasin.

Tutkimuksessani olen hyödyntänyt kehittämääni suolen limakalvon biologiaa jäljittelevää soluviljelymallia. Sen avulla olen voinut osoittaa, että keliakiapotilaan verestä eristetyt IgA-luokan kudosvasta-aineet todella puuttuvat solujen väliseen vuorovaikutukseen ja osallistuvat siten keliakialle tyypillisen limakalvovaurion muodostumiseen. Lääketieteessä ei aikaisemmin ole voitu osoittaa, että autoimmuunitaudeille tyypillisillä veren vasta-aineilla olisi biologista vaikutusta, siten tutkimustulokseni ovat biologiassa ja lääketieteessä uraauurtavia. Tutkimukseni, jossa on ensimmäistä kertaa osoitettu keliakiataudissa esiintyvien autovasta-aineen rooli keliakian taudinaiheuttamismekanismissa, ovat tuoneet täysin uuden näkökulman vilkkaaseen kansainväliseen keskusteluun. Vehnän ja muiden viljojen aiheuttaman keliakian taudiniheuttajamekanismien mahdollisimman tarkka tuntemus helpottaa myös hoitamattomaan keliakiaan liittyvien ja viljojen syömisen aiheuttamien muiden kansanterveyttä rasittavien sairauksien, kuten esim. luukadon, diabeteksen ja syöpätautien tuntemusta.

In the present study a three-dimensional mesenchymal-epithelial cell co-culture model mimicking the epithelial cell biology on the crypt-villus axis was developed. With this model it was shown that coeliac disease-specific autoantibodies, targeted against tissue transglutaminase, do indeed have biological effects on epithelial and mesenchymal cells. The present data show the calcium-activated tissue transglutaminase autoantibody ELISA to be highly sensitive and specific in detecting coeliac disease. In double immunofluorescence staining the colocalization of epitopes of monoclonal tissue transglutaminase antibodies with coeliac disease patients serum IgA was clearly demonstrated. Together these results confirm that tissue transglutaminase is the major autoantigen in coeliac disease. The need for calcium for a specific antibody-antigen reaction to occur suggests that coeliac disease autoantibodies detect conformational epitope(s) of tissue transglutaminase.

The transformed human intestinal chloride-secreting crypt-like T84 epithelial cells organized into two kinds of luminal structures when co-cultured with fibroblasts. Around all organized T84 cell colonies the basement membrane (silver staining) including laminin was well deposited. In half of all T84 cell colonies the epithelial cells were morphologically differentiated to columnar enterocyte-like cells with upregulated alkaline phosphatase brush border activity. This IMR-90 fibroblast induced differentiation was mediated via TGF-b. The co-culture model developed here was applied to study whether coeliac disease-specific serum IgA interferes in reciprocal interactions between epithelium and fibroblasts and whether tissue transglutaminase is involved in this process. When antibodies against tissue transglutaminase, both commercial (CUB7402) and purified total IgA fraction from coeliac disease patients sera were added to the co-cultures, a significant inhibition in differentiation of the secretory crypt-like T84 cells to columnar, enterocyte-like T84 cells was observed. The percentage of differentiated epithelial cell colonies decreased significantly from the control level of 49% to 26% in CUB7402-treated co-cultures and to 25% in cultures treated with coeliac disease patients IgA. A simultaneous, slight but significant increase in epithelial cell proliferation was also observed.

In the co-cultures tissue transglutaminase antibodies induced an astral growth pattern in IMR-90 fibroblasts, suggesting disturbed interaction between the fibroblasts and the underlining extracellular matrix. In fact, antibodies against tissue transglutaminase significantly inhibited IMR-90 fibroblast motility and collagen gel contraction, while increasing type I collagen degradation. In conclusion, the present study shows now, for the first time, that coeliac disease IgA-class autoantibodies targeted against tissue transglutaminase interfere in the mesenchymal cell-epithelial cell cross-talk in an in vitro model. The autoantibodies inhibited TGF-b-mediated epithelial cell differentiation and increased epithelial cell proliferation. Interferences in the regulation of epithelial cell differentiation and proliferation are also typical in vivo of the gluten-triggered coeliac small-bowel mucosal lesion (villous atrophy), where only the proliferative component seems to remain at the crypt-villus axis (crypt hyperplasia). Furthermore, these autoantibodies significantly affected mesenchymal cell biological performance by inhibiting motility and increasing matrix degradation. Provided that coeliac disease-specific autoantibodies, heavily deposited in the basement membrane area, exert the same effects in vivo as in vitro, it is highly likely that increased degradation of basement membrane components dramatically affects not only the biological functioning of intestinal myofibroblasts but equally of epithelial cells. Taken together, the presented data suggest that coeliac disease autoantibodies have an important role in the formation of the mucosal lesion typical of coeliac disease.