All subjects underwent upper-duodenal endoscopy. Those with GS revealed normal to mildly inflamed mucosa (Marsh 0–1), while all CD patients showed partial or subtotal villous atrophy with crypt hyperplasia according to the ESPGHAN criteria [13
]. Figure shows representative photomicrographs of CD3 staining on mucosal biopsy specimens from dyspeptic control patients, active-CD patients, and GS patients. As expected, CD patients (fig. ) had increased numbers of CD3+ IELs (>50/100 enterocytes) compared to controls (fig. ), while GS patients had a number of CD3+ IELs intermediate between CD patients and controls in the context of relatively conserved villus architecture (fig. ). The numbers of TCR-γδ IELs were only elevated in CD subjects (>3.4/100 enterocytes), while in GS patients the numbers of γδ IELs were similar to those in controls. More details on these findings are provided elsewhere [Sapone et al., manuscript in preparation].
Representative microphotographs of mucosal CD3 expression in a dyspeptic control patient (a), a gluten-sensitive (GS) patient (b) and a celiac disease (CD) patient with active disease (c) (40× magnification).
As shown in figure , IL-17A gene expression, measured by real-time quantitative PCR in biopsy specimens, was significantly elevated in active CD patients (mean ± SE: 0.0088 ± 0.002% of 18S) compared to GS patients (0.0025 ± 0.0008%; p = 0.03) and controls (0.0022 ± 0.0013%; p = 0.024). The level of variance across the groups was also statistically significant (p < 0.025). Interestingly, as figure shows, this difference appeared to be sustained by a subgroup (7 out of 13) of CD patients. The significance of this finding is at present unclear, as this subgroup did not seem to differ from the other CD patients with respect to clinical, histological, and serological parameters or the MHC haplotypes, with the exception of a trend toward increased positivity for anti-gliadin antibodies. Immunofluorescence staining of intestinal mucosal sections from this IL-17high subgroup documented an increased percentage (35.6 ± 3.7%) of T cells expressing the constitutive Th17 surface marker CCR6 relative to IL-17low CD (24.8 ± 8.1%) or control groups (24.0 ± 3.6%; a typical staining is shown in fig. ), but these differences did not reach statistical significance, presumably due to the small sample size and a high degree of interindividual variability. It can be speculated that this variability might reflect different stages of the condition, or imply an as yet unappreciated level of heterogeneity in the immune mechanisms involved in CD pathogenesis. Longitudinal studies in larger populations might help elucidate these issues.
Mucosal IL-17A mRNA expression in the 3 groups. IL-17A expression is significantly increased in CD patients (n = 13) compared to controls (n = 7; p = 0.024) and GS patients (n = 11; p = 0.03).
Fig. 3 Representative immunofluorescence staining of mucosal CD3 (FITC; green) and CCR6 (Alexa Fluor® 568; red) in a control subject (a) and a CD patient expressing high levels of mucosal IL-17A (b). Shown are merged confocal microscopy images (60× (more ...)
Together, these data show that the expression of IL-17, a cytokine that is thought to be involved in inflammatory and autoimmune processes [8
], is elevated in CD but not in GS. Our work confirms and extends the study by Castellanos-Rubio et al. [11
] showing gluten-dependent expression of IL-17A in active CD. In addition, combined with the other observed clinical and histological differences, it further supports the idea that CD and GS are distinct entities and that the immune system deals with gluten in different ways, possibly depending on the genetic makeup [2
]. Where in GS as well as in wheat allergy the gluten-induced response leads to immunity toward a non-self diet component, i.e. gliadin, in CD a deviated, self-directed adaptive response leads, in addition, to the onset of a full-fledged autoimmune process. In clear contrast to GS, CD results from a complex, as yet undetermined, interplay of increased intestinal permeability, mucosal damage, environmental factors additional to gluten, and genetic predisposition, which involves both MHC and non-MHC genes [Sapone et al., manuscript in preparation] [6
]. Based on our recent work, we can speculate that gliadin may directly affect the expansion of Th17 clones by inducing the expression of IL-23 among other cytokines [12
]. Studies are needed to elucidate whether the unique involvement of these cells in CD versus GS reflects differential expansion in response to gliadin-induced factors versus other mechanisms, including mucosal recruitment.
In conclusion, here we present for the first time evidence of differential intestinal mucosal immune responses to gluten between CD and GS, further supporting the notion that CD is the only clinical form of gluten reactivity involving autoimmune mechanisms.