Of the 177 patients with coeliac disease, 26 (15%) had negative serum EmA, and 4 of these were IgA‐deficient. Thus, 22 EmA‐negative patients with coeliac disease constituted the study group. HLA DQ2 or DQ8 was detected in each of 12 patients with available sample (HLA DQ2 in 11 and DQ8 in 1). Among EmA‐negative patients with coeliac disease, 13 (59%) were men and the median age was higher than in EmA‐positive patients (table 1).
Table 1Demographic data and signs and symptoms leading to suspicion of coeliac disease in 173 immunoglobulin A‐competent patients with coeliac disease
Abdominal symptoms were significantly more common in the EmA‐negative group. Three EmA‐negative patients with coeliac disease were found to have enteropathy‐associated T cell lymphoma (EATL), which was detected at the same time as the diagnosis of coeliac disease was established. Two of these patients had HLA DQ2 and in one there were no data available. All three patients had proximal small‐bowel villous atrophy and crypt hyperplasia compatible with coeliac disease while on a gluten‐containing diet. Furthermore, two of these patients had small‐bowel biopsy taken earlier, 2 and 6 years before the diagnosis of coeliac disease and EATL. Even at that time, both showed partial villous atrophy and crypt hyperplasia, but the diagnosis of coeliac disease was overlooked. In all, 6 (27%) of the 22 EmA‐negative patients and 6 (4%) of the 151 EmA‐positive patients with coeliac disease died after the diagnosis of coeliac disease.
No differences were observed between EmA‐negative and EmA‐positive patients with coeliac disease in Marsh classification (table 2) or villous height:crypt depth ratios (fig 1).
Table 2Severity of small‐bowel mucosal villous atrophy according to Marsh classification in 173 immunoglobulin A‐competent patients with coeliac disease
Figure 1Villous height:crypt depth ratios in immunoglobulin A (IgA)‐competent endomysial antibody (EmA)‐negative and EmA‐positive patients with coeliac disease. Median values are shown by solid lines. In the EmA‐negative (more ...)
The median density of CD3+ IELs (fig 2A) was similar (p
0.292), whereas the density of γδ+ IELs was statistically significantly higher (p
0.007) in EmA‐positive than in EmA‐negative patients (fig 2B). Of the three EmA‐negative patients with EATL, two had normal densities of γδ+ IELs.
Figure 2The density of CD3+ (A) and γδ+ (B) intraepithelial lymphocytes (IELs) in immunoglobulin A (IgA)‐competent endomysial antibody (EmA)‐negative and EmA‐positive patients with coeliac (more ...)
Small‐bowel mucosal IgA deposits in colocalisation with extracellular TG2 were detected in all EmA‐negative (n
18) and EmA‐positive (n
17) examined patients with coeliac disease (figs 3 and 4). The intensity of intestinal IgA deposits did not correlate with the severity of the mucosal lesion—that is, villous height:crypt depth ratios. For example, three EmA‐negative patients with coeliac disease with villous height:crypt depth ratios close to 1.5 had IgA deposits with 2.5+ to 3+ intensity. Figure 4 shows that the intensity of mucosal TG2‐targeted IgA deposits decreased after adopting a GFD. In contrast, TG2‐targeted IgA deposits were not detected in any of the controls with intestinal diseases, not even in patients having autoimmune enteropathy with severe villous atrophy (fig 4).
Figure 3Subepithelial coeliac‐type small‐bowel mucosal immunoglobulin A (IgA) deposits (A, D, green, arrow) in IgA‐competent patients with coeliac disease, with human leucocyte antigen DQ2 and negative serum endomysial (more ...)
Figure 4Transglutaminase 2 (TG2)‐specific immunoglobulin A (IgA) deposits in the small‐bowel mucosa of IgA‐competent, endomysial antibody (EmA)‐negative patients with coeliac disease and age‐matched and (more ...)
To obtain direct evidence of the TG2 specificity of IgA deposits, further experiments were carried out. The small‐bowel mucosal subepithelial and pericryptal IgA deposits along TG2 in both EmA‐negative and EmA‐positive patients with coeliac disease remained unchanged after treatment with citrate buffer and 0.5–1 M KSCN (fig 5A). In contrast, the amount of IgA deposits substantially decreased in eight samples and almost completely disappeared in five samples (fig 5B) when the sections were treated additionally with chloroacetic acid, which removes TG2 from its fibronectin binding sites. The amount of detectable TG2 also decreased in parallel (fig 5C), whereas IgA in the brush border of epithelial cells remained essentially unchanged (fig 5A, B; asterisks). Chloroacetic acid had similar effects in EmA‐negative and EmA‐positive samples.
Figure 5Investigation of specificity of deposited immunoglobulin A (IgA) for transglutaminase 2 (TG2). (A) Pretreatment of coeliac duodenum section with 0.5 M potassium thiocyanate (KSCN) does not affect extracellularly deposited IgA (more ...)
When the small‐bowel sections were incubated in vitro with human recombinant GST‐TG2, binding of GST‐TG2 was observed both in coeliac and in non‐coeliac tissue sections along fibronectin (data not shown). This non‐specific binding to fibronectin could be blocked by pre‐incubating GST‐TG2 with a soluble 45‐kDa fragment of fibronectin as well as the G92 monoclonal anti‐TG2 mouse antibodies. Under these conditions, GST‐TG2 bound only to the coeliac tissue, colocalising with the IgA deposits (fig 5D,E), but did not bind to the duodenum sections from controls without extracellular IgA deposition (fig 5F). Small‐bowel sections from the seven serum EmA‐negative patients with coeliac disease gave similar results as the six EmA‐positive coeliac samples. These experiments collectively show that coeliac IgA antibodies were specifically bound in situ to TG2 target antigen in the duodenum samples of both serum EmA‐negative and EmA‐positive patients with coeliac disease.
Serum TG2 antibody test results were available in 14 of 22 EmA‐negative patients with coeliac disease; five were tested using guinea pig liver and nine using human recombinant as antigen. Four were positive and 10 negative for TG2 antibodies. Three of four EmA‐negative TG2 antibody‐positive patients had only low TG2 antibody levels using human recombinant as antigen (5.4, 6.8 and 12.8 U; normal values <5 U, median titre in untreated patients with coeliac disease 70.3, range 8.8–680).16
Only one had a high TG2 antibody level using guinea pig liver as antigen (159 U, normal value <20 U). In the EmA‐negative group, patients having positive TG2 antibodies in the serum did not show more intense intestinal IgA deposits than in TG2 antibody‐negative patients.
After a median of 13 months on a GFD, there were no differences in small‐bowel histological recovery between EmA‐negative and EmA‐positive patients with coeliac disease. Histological improvement was observed in all patients who underwent small‐bowel biopsy while on a GFD, except in the three affected by EATL. Three EmA‐negative and four EmA‐positive patients did not undergo small‐bowel biopsy, but clinical recovery on a GFD was evident in all; one EmA‐negative patient and one EmA‐positive patient were lost to follow‐up.