The foods we eat contain numerous nonself molecules that do not normally stimulate a proinflammatory immune response in healthy individuals (35
). In some diabetes-prone rodents (6
) and humans (19
), there is evidence that the gut mucosa is mildly inflamed and the epithelial barrier is leaky, providing a potential entry point for nonself antigens in the context of a proinflammatory cytokine imbalance that could promote autoimmunity (4
). The present results support this view. Although the origin of the antigens and immune cells that initiate or drive the β-cell–specific autoimmune response is not known, diet is an important factor influencing diabetes outcome, possibly by supplying a constant source of stimulatory antigens to the gut immune system (4
There was moderately increased T-cell proliferative response to other dietary antigens such as ovalbumin, gliadin, and the celiac toxic gliadin 33-mer peptide (), but this was less pronounced than the response to WP. These data suggest a general impairment of oral tolerance in some type 1 diabetic patients (36
), with the strongest and most frequent abnormal proliferative response induced by the mixture of WPs.
We are aware of only one other study of WP response in patient PBMNCs (29
). Klemetti et al. (29
) showed an increased cell-mediated immune response to high concentrations of wheat gluten (400 μg/ml) in 24% of newly diagnosed type 1 diabetic patients. The proliferative responses in this study were low compared with those in the present study, possibly as a result of the high polypeptide concentration, different gluten fractions, culture conditions, proliferation assay, and/or different genetic background of the subjects as well as different duration of diabetes. In keeping with reports that high concentrations of gliadin are cytotoxic (37
), we found that 25 μg/ml of wheat protein extract inhibited response of PBMNC, whereas 6.2 μg/ml was optimum for our assay (29
). The CFSE assay used here not only permits identification of individual cell populations by flow cytometry but is also more sensitive than the thymidine assay (33
). Therefore, the present analysis permitted us to detect wheat-specific CD3+
T-cell proliferation using a low, noninhibitory dose of WP.
Additional evidence linking the development of diabetes in humans to wheat comes from epidemiological studies (27
) and reports of immune responses of patient tissues to WP (29
). Anti-gliadin antibodies have been reported in newly diagnosed children with type 1 diabetes (38
), and prospective studies of infants at high risk indicate that early exposure to cereals, particularly wheat, was linked to appearance of islet autoantibodies (27
). Furthermore, a significant subset of type 1 diabetic patients displays celiac disease autoantibodies (39
lamina propria and intestinal epithelial lymphocytes were increased in 20% of type 1 diabetic patients receiving rectally instilled gliadin (40
). Immunohistochemical evaluation and culture of jejunal biopsies from tissue transglutaminase antibody–negative type 1 diabetic children with WP increased frequency of activated CD25+
cells in the lamina propria as well as expression of HLA-DR in the crypts in association with enhanced infiltration of the epithelium by CD3+
). Therefore, the present results are consistent with those of Auricchio et al. (30
) showing that a subset of type 1 diabetic patients displayed signs of inflamed gut mucosa and increased immune reactivity to WPs.
Type 1 diabetes has been traditionally thought of as a T-cell–mediated disease associated with high levels of the Th1 cytokine IFN-γ. It now seems likely that type 1 diabetes is the result of dysregulation within a broader network of immune cell types (41
). For example, the role of the recently discovered Th17 cells and the extent to which there is a dysregulation of communication between Th1, Th2, and Th17 cells in human diabetes remain unclear. We observed WP-induced T-cell proliferation in nearly half of our type 1 diabetic patients but not in healthy control subjects. This response was mixed in nature, accompanied by increased production of proinflammatory cytokines IFN-γ, TNF, IL-6, and IL-17A as well as the counterinflammatory Th2 cytokine IL-4. The high concentrations of IFN-γ and IL-17A suggest that Th1 and Th17 cytokine–producing, WP-responsive CD3+
cells were activated in association with a proinflammatory condition in the gut (6
). IL-6 was present in WP culture supernatants at very high levels, four to six times those of IFN-γ. TNF was also increased but to a much lesser extent. IL-6 and TNF are proinflammatory cytokines that promote the secretion of IL-17 by Th17 cells and block the production of Foxp3+
regulatory T-cells (42
). We did not observe a significant increase in IL-10 in response to WP, making it unlikely that IL-17 originated from Th17 regulatory cells that produce both IL-10 and IL-17. It seems unlikely that such high amounts of IL-17 could originate from CD8+
T-cells because expansion of WP-responsive T-cells was predominantly from CD4+
cells and IL-17A production was blocked by anti-DR antibody.
High levels of IFN-γ were produced in response to WP, whereas the concentration of the Th2 cytokine IL-4, although significantly increased, was only one-third that of IFN-γ, suggesting a predominance of Th1 over Th2 cells. Although both of these cytokines can inhibit development of Th17 cells from naive precursors (42
), it has been suggested that committed Th17 cells are not affected (41
). Therefore, we favor the interpretation that WP stimulates the production of IL-17A from previously committed Th17 cells. While IL-17A can be produced under certain circumstances by CD8+
T-cells and members of the innate immune system (γδT-cells and natural killer T-cells), Th17 cells are the major producers of IL-17A. Thus, the overall cytokine pattern observed in WP-stimulated PBMNC from type 1 diabetic patients suggests a predominant Th1 and Th17 proinflammatory state consistent with the speculation that autoimmunity can occur when there is inappropriate cross-regulation between Th1 and Th17 cytokine networks (42
The role of IL-6 in type 1 diabetes remains controversial (43
). In the present study, patient PBMNC cultured with WP secreted large amounts of IL-6, the origin of which is presently unclear. Nonetheless, some points are worth noting: WP response was mainly from CD4+
and not CD8+
cells, most responders were HLA-DR4+
, and treatment with anti-DR did not block production of IL-6. These results suggest that IL-6 originated from non–T-cells. Others report that IL-6 gene expression was upregulated threefold in monocytes from adult-onset type 1 diabetic subjects (44
). Furthermore, overexpression of IL-6 in pancreas was correlated with islet inflammation, which could contribute to the development of autoimmune disease (43
). The high concentration of WP-induced IL-6 further supports our previous proposal of a potential mechanism by which wheat can promote the development of diabetes involving induction of proinflammatory cytokines such as IL-6, IFN-γ, and TNF-α by wheat antigens (4
The HLA genes are the major genetic determinant of type 1 diabetes. More than 90% of Caucasian type 1 diabetic patients carry either HLA-DR3 or HLA-DR4 haplotype, and a synergistic effect on type 1 diabetes risk is observed in HLA-DR3/4 heterozygous individuals (46
). Patients with type 1 diabetes and celiac disease share some HLA-associated risk genes such as HLA-DQ2 (HLA-DQB1*02) and HLA-DQ8 (HLA-DQB1*0302). The frequency of HLA risk genes is different between the two diseases: 90–95% of celiac disease patients are HLA-DQ2 and 5–10% are HLA-DQ8 (47
), whereas up to 50% of diabetic patients are HLA-DQ8/DQ2 heterozygous (48
). In our type 1 diabetic population, increased immune response to WPs was not explained by shared genetic risk for celiac disease. The frequency of HLA-DQB1*02 was not significantly different between type 1 diabetic patients and control subjects (P
= 0.6), and we did not detect any significant differences for HLA-DQB1*02 between WP responders and nonresponders (P
= 0.75) (). We also evaluated the HLA-DR and -DQ haplotype by high-resolution analysis in type 1 diabetic patients. A positive response to WP in patients was more frequently associated with the HLA-DR4 (DRB1*0401/4) DQB1*0301/2 haplotype, and HLA-DR3/DQB1*0201 appeared to magnify the response (, ).
A genetic basis for WP response in type 1 diabetic patients is suggested by the finding that T-cell proliferation and inflammatory cytokine production were blocked by anti-DR antibodies and responders were nearly all HLA-DR4+
. Others have observed enhanced expression of HLA-DR in the intestinal mucosa of children with type 1 diabetes (20
), and gluten-induced T-cell proliferation was blocked by anti-DR antibodies in two patients in a previous study (29
). Importantly, WP-induced T-cell proliferation was not explained by an overrepresentation of the major celiac disease risk gene HLA-DQ2. HLA-DQ2 prevalence was not different between patients and control subjects or between WP responder and nonresponder patient groups, and the T-cell response was not attributable to the presence of subclinical celiac disease given that all our patients with type 1 diabetes were negative for antibodies against the pathognomic celiac disease autoantigen, tissue transglutaminase. These findings are consistent with other reports showing that the gut inflammation observed in type 1 diabetic patients is different from that seen in patients with celiac disease (20
) and not necessarily related to HLA-DQ2 or HLA-DQ8 (30
). Therefore, the tolerogenic function of the gut immune system with respect to WPs was compromised in a large subset of patients with type 1 diabetes in an HLA-DR–restricted, diabetes-specific manner.
In summary, almost half of our type 1 diabetic patients displayed increased proliferation when PBMNCs were cultured in vitro with nontoxic concentrations of WP. The cytokine pattern was mixed having characteristics of a predominant Th1 and Th17 response with a lesser contribution of the Th2 cytokine IL-4. The WP proliferative response occurred mainly in HLA-DR4 individuals and was blocked with anti-DR antibodies but was not due to the major celiac disease gene HLA-DQ2. This demonstrated the presence of a mixed proinflammatory Th1/Th17 response to dietary WPs that appeared to be diabetes specific.