In vivo targeting of intestinal and extraintestinal transglutaminase 2 by coeliac autoantibodies

Abstract

Background: IgA class serum autoantibodies against type 2 (tissue) transglutaminase (TG2) bind to both intestinal and extraintestinal normal tissue sections in vitro, eliciting endomysial, reticulin, and jejunal antibody reactions. It is not known whether similar binding also occurs in coeliac patients in vivo, and may thereby contribute to disease manifestations.

Aims: To investigate intestinal and extraintestinal coeliac tissues for the presence of in vivo bound TG2 specific IgA and its relation to small intestinal mucosal atrophy.

Patients: We investigated jejunal samples with normal villous morphology from 10 patients with developing coeliac disease who subsequently progressed to a flat lesion, from 11 patients with dermatitis herpetiformis, and from 12 non-coeliac controls. Six extrajejunal biopsy samples (liver, lymph node, muscle, appendix), obtained based on independent clinical indications from patients with active coeliac disease, were also studied.

Methods: Double colour immunofluorescent studies for in situ IgA, TG2, and laminin were performed. IgA was eluted from tissue sections and tested for TG2 specificity by enzyme linked immunosorbent assay and indirect immunofluorescence.

Results: IgA (in one IgA deficient case IgG) deposition on extracellularly located TG2 was detected in jejunal and extrajejunal specimens of all coeliac patients, and also in seven of 11 dermatitis herpetiformis patients, of whom two had no circulating endomysial antibodies. IgA eluted from extraintestinal coeliac tissues was targeted against TG2.

Conclusions: Coeliac IgA targets jejunal TG2 early in disease development even when endomysial antibodies are not present in the circulation. Extraintestinal target sites of coeliac IgA further indicate that humoral immunity may have a pathogenetic role.

Figure 1

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 μm.

Figure 2

(A) Non-coeliac lymph node double stained for IgA (green) and transglutaminase 2 (TG2, red). (B) In vivo deposited IgA (green) on reticulin fibres in a lymph node from a coeliac patient (case 1 in table 2 ▶). (D) Normal appendix with IgA in epithelial cells. (E–I) Appendix from a coeliac patient (case 2) with in situ IgA deposits (green) on reticulin fibres (E), endomysium (F, arrow), and pericryptal subepithelial layer (H), corresponding to TG2 localisation in red (G). (J–L) Skeletal muscle from a coeliac patient (case 3) with an endomysial TG2 pattern (J) and endomysial IgA deposits (K, green). Colocalisation of IgA deposits with TG2 in all three coeliac specimens is indicated by yellow (C, I, L). There was no merging for IgA in the germinal centres or epithelial IgA (green in C and I). Bar = 50 μm.

Figure 3

IgA or IgG (green) and transglutaminase 2 (TG2, red) in the liver of a non-coeliac patient (A–B), and from an IgA competent (C–D, case 4 in table 2 ▶) and IgA deficient (E–H, case 5) coeliac patient. In coeliac patients, there was IgA (in IgA deficiency IgG) deposition on reticulin fibres (C, G). Yellow in (D) and (H) indicates colocalisation of deposits with TG2. Bar = 50 μm.

Figure 4

Coeliac liver (A) and coeliac lymph node (B) double stained for IgA (green) and transglutaminase 2 (TG2, red) after elution with choroacetic acid which releases tissue TG2. (C) Demonstration of TG2 in the eluates by western blotting. (D) TG2 specific IgA detected in the eluates by ELISA using human recombinant TG2 antigen (bars 1–3) or anti-TG2 capture antibody (bars 4, 5): 1, 4: IgA eluted from a normal liver; 2: IgA eluted from the coeliac lymph node; 3, 5: IgA eluted from the coeliac liver. (E–K) Indirect immunofluorescent studies with the eluates. (E) IgA (green) eluted from coeliac liver bound to recombinant human TG2 coated on TG2 knockout mouse jejunum and merged into yellow (F) when TG2 was labelled in red, while IgA eluted from a normal liver did not bind (G–H). (I) Endomysial binding of IgA eluted from the coeliac lymph node to human umbilical cord. Binding of IgA eluted from the coeliac liver to monkey oesophagus endomysium (J) and human appendix subepithelial band (K). Bar = 50 μm.