Immunologic Activity in the Small Intestinal Mucosa of Pediatric Patients With Type 1 Diabetes Mia Westerholm-Ormio, 1 Outi Vaarala, 2,3 Pa ¨ ivi Pihkala, 2 Jorma Ilonen, 4 and Erkki Savilahti 1 Involvement of gut immune system has been implicated in the pathogenesis of type 1 diabetes. However, few studies have been performed on the gut mucosa from patients with type 1 diabetes. Thus, we characterized the stage of immune activation in jejunal biopsy sam- ples from 31 children with type 1 diabetes by immuno- histochemistry, in situ hybridization, and RT-PCR. We found enhanced expressions of HLA-DR, HLA-DP, and intercellular adhesion molecule-1 by immunohistochem- istry even on structurally normal intestine of patients with type 1 diabetes and no signs of celiac disease. In addition, the densities of IL-1– and IL-4 –positive cells detected by immunohistochemistry and IL-4 mRNA– expressing cells evaluated by in situ hybridization were increased in the lamina propria in patients with type 1 diabetes and normal mucosa. Instead, the densities of IL-2, -interferon (IFN-), and tumor necrosis factor –positive cells, the density of IFN- mRNA positive cells, and the amounts of IFN- mRNA detected by RT-PCR correlated with the degree of celiac disease in patients with type 1 diabetes. Our study supports the hypothesis that a link exists between the gut immune system and type 1 diabetes. Diabetes 52:2287–2295, 2003 A ccumulating data suggest that the gut immune system plays a role in the development of type 1 diabetes (1), an autoimmune disease that results from the destruction of insulin-secreting pancreatic islet -cells by autoreactive T-cells (2). In experimental studies, several indications of the involve- ment of gut immune system in the development of auto- immune diabetes have been established. First, diet modifies the development of autoimmune diabetes in BB rats and NOD mice (3–5). Second, the islet infiltrating T-cells express gut-associated homing receptor 7-inte- grin, and antibodies that block this receptor or its endo- thelial ligand mucosal addressin cell adhesion molecule-1 inhibit the development of autoimmune diabetes in NOD mice (6 – 8). Third, mesenterial lymphocytes from a young NOD mouse transfer autoimmune diabetes to the recipi- ents, indicating that diabetogenic T-cells are present in the gut-associated immune system (9). Finally, feeding auto- antigen may induce the development of autoreactive cyto- toxic lymphocytes and acceleration of autoimmune diabetes (10,11). In humans, a link between the gut immune system and type 1 diabetes has also been suggested: enhanced im- mune responses to several cow milk proteins have been reported in serologic studies on patients with newly diagnosed type 1 diabetes (12–14). We have also shown that GAD-specific T-cells in patients with type 1 diabetes express gut-associated homing receptor 47-integrin (15). Accordingly, T-cells derived from human diabetic pancreas have been demonstrated to show mucosal hom- ing properties (16). Furthermore, the association between type 1 diabetes and celiac disease (CD) has been recognized for some years (17,18), the prevalence of CD among children and adults with type 1 diabetes being as high as 2– 8% (19 –24). Both diseases are associated with the HLA class II alleles DQB1*0201 and DQA1*0501 (HLA-DQ2) (25), which may partly explain the association of diseases, but recently it has been proposed that long-term exposure to gluten could induce type 1 diabetes (26). In a previous study, we found markers of immune activation even in structurally normal small intestine of patients with type 1 diabetes (27). The expression of HLA class II antigens in the villous epithelium and the density of 47-expressing cells in the lamina propria were increased in patients when compared with control subjects. However, it is not clear how an inflammation in the gut is linked to the process that destroys islet -cells, and to date very few studies have been performed in the small intestine of patients with type 1 diabetes. Our aim was to characterize the immune activation in jejunal biopsy specimens from pediatric patients with type 1 diabetes. We investigated the protein expression of interleukin-1 (IL-1), IL-2, IL-4, -interferon (IFN-), and tumor necrosis factor- (TNF-) in jejunal biopsies from 21 pediatric patients with type 1 diabetes. We also inves- tigated the lymphocytes, measuring their activation as well as the expression of HLA class II antigens on the epithe- lium. Furthermore, in the same samples, we analyzed the mRNA expression of IL-4 and IFN- by in situ hybridiza- tion. The specific amounts of IL-2, IL-4, IFN-, TNF-, chemokine receptor-4 (CCR-4), and CCR-5 mRNA were From the 1 Hospital for Children and Adolescents, University of Helsinki, Helsinki, Finland; the 2 Department of Molecular Medicine, National Public Health Institute, Helsinki, Finland; the 3 Division of Pediatrics, Department of Molecular and Clinical Medicine, Faculty of Health Sciences, University of Linko ¨ ping, Linko ¨ ping, Sweden; and the 4 Department of Virology, University of Turku, Finland. Address correspondence and reprint requests to Mia Westerholm-Ormio, Hospital for Children and Adolescents, Research Laboratory, Biomedicum Helsinki, B433b, P.O. Box 63, FIN-00014 University of Helsinki, Finland. E-mail address: mia.westerholm-ormio@hus.fi. Received for publication 31 October 2002 and accepted in revised form 22 May 2003. AEC, carbatzole; CCR, chemokine receptor; CD, celiac disease; EMA, endomysium antibodies; ICAM-1, intercellular adhesion molecule-1; IEL, intraepithelial lymphocyte; IFN-, -interferon; IL, interleukin; mAb, monoclo- nal antibody; TCR, T-cell receptor; TNF-, tumor necrosis factor ; tTGA, tissue transglutaminase antibodies. © 2003 by the American Diabetes Association. DIABETES, VOL. 52, SEPTEMBER 2003 2287