Genes overexpressed in pancreatic islets of patients with new-onset type 1 diabetes are potential candidates for novel disease-related autoantigens. RT-PCR-based subtractive hybridization was used on islets from a patient who died at the onset of type 1 diabetes, and it identified a type 1 diabetes-related cDNA encoding hepatocarcinoma-intestine-pancreas/pancreatic-associated protein (HIP/PAP). This protein belongs to the family of Reg proteins implicated in islet regeneration; its gene contains a putative interleukin-6 (IL-6) response element. Islets from healthy cadaveric human donors released HIP/PAP protein into the culture medium, and this release was enhanced by the addition of IL-6. The expression pattern of mouse homologues of HIP/PAP was determined in pancreata of prediabetic and diabetic NOD mice. Both groups showed positive immunostaining for HIP/PAP in islets and ductal epithelium. To test whether HIP/PAP is a target of islet-directed autoimmunity, we measured splenic T-cell responses against HIP/PAP in NOD mice. Spontaneous proliferation was detected after 4 weeks. Lymphocytes from islet infiltrates and pancreatic lymph nodes from 7- to 10-week-old NOD mice were used to establish an HIP/PAP-specific I-Ag7-restricted T-cell line, termed WY1, that also responded to mouse islets. WY1 cells homed to islets of NOD-SCID mice and adoptively transferred disease when coinjected with purified CD8+ cells from diabetic NOD mice. Our conclusion was that differential cloning of Reg from islets of a type 1 diabetic patient and the response of Reg to the cytokine IL-6 suggests that HIP/PAP becomes overexpressed in human diabetic islets because of the local inflammatory response. HIP/PAP acts as a T-cell autoantigen in NOD mice. Therefore, autoimmunity to HIP/PAP might create a vicious cycle, accelerating the immune process leading to diabetes.
Address correspondence and reprint requests to Robert S. Sherwin, Department of Internal Medicine, Yale University, New Haven, CT 06520-8020. E-mail:.
Received for publication 14 March 2001 and accepted in revised form 25 October 2001.
AEC, 3-amino-9-ethylcarbazol; Bruff’s/FCS, Bruff’s medium supplemented with 3% FCS; CRD, carbohydrate recognition domain; FACS, fluorescence-activated cell sorter; FITC, fluorescein isothiocyanate; HIP-CRD, CRD of HIP/PAP; HIP/PAP, hepatocarcinoma-intestine-pancreas/pancreatic-associated protein; HRP, horseradish peroxidase; IL-6, interleukin-6; PBS-T, PBS with 0.05% Tween 20; PE, phycoerythrin; SSC, sodium chloride-sodium citrate.