The Roles Of Daintain/Allograft Inflammatory Factor-1(AIF-1) In The Pathogenesis Of Type 1 Diabetes

Type 1 diabetes is a multifactorial autoimmune disease, which is characterised by Tcell mediated destruction of the beta cells in the pancreas. The early stages of the diseaseprocess are termed insulitis, the infiltration of the pancreatic islets by mononuclearimmune cells, including macrophages, dendritic cells, and T cells. Beta-cell death in thecourse of insulitis is probably caused by direct contact with activated macrophages andT-cells, and/or exposure to soluble mediators secreted by these cells, including cytokines,nitric oxide (NO), and oxygen free radicals. Although the molecular pathways for theinitiation, perpetuation, and eventual destruction of the beta cell in the disease areunknown, accumulating experimental evidence implicates cytokines as key mediators.In the mid 1990s, Dr Chen isolated and characterized a polypeptide from porcineintestines and named it as "daintain". Contemporaneously, Utans et al identified a novelmacrophage factor, allograft inflammatory factor-1(AIF-1), in rat cardiac allografls withchronic rejection. Daintain is identical to AIF-1 in amino acid sequence, therefore we callthe polypeptide as daintain/AIF-1. As an inflammation maker in macrophages, thiscytokine has rapidly gained interest from a fast growing group of scientists. Our perviousstudy demonstated there was an accumulative daintain/AIF-1 immunostraining in thepancreas of BB rats when they were suffering from insulitis. To further investigate theroles of daintain/AIF-1 in type 1 diabetes, the following work has been done, including:1. daintain/AIF-1 purification and identification. Daintain/AIF-1 was purified fromporcine intestine with a sephadex G-25 (fine) column chromatography, DEAE anionexchange column chromatography and reverse-phase HPLC purification.2. monoclonal antibodies (mabs) against daintain/AIF-1 and insulin production. Thepurified daintain/AIF-1 and insulin were then used to immunize Balb/c mice as theimmunogen. Both hybridization technique and enzyme linked immunosorbent assay(ELISA) were performed to generate cell lines which secreted mabs against the twopeptides. Isotypes, titer and affinity of the mabs were identified.3. daintain/AIF-1 participated in the pathogenesis of type 1 diabetes. Using the mabagainst daintain/AIF-1, daintain/AIF-1 was found as a circulating protein in the blood ofBalb/c and NOD mice with immunoprecipitation followed by western blotting. Theconcentration of daintain/AIF-1 in the plasma of NOD mice was distinctly higher compared with the same aged Balb/c mice, which suggested daintain/AIF-1 wasassociated with type 1 diabetes. Furthermore there was still daintain/AIF-1immunostraining in the pancreas of BB rats when they were suffering from type 1 diabetes,but no beta cells in pancreas. These data implicated that daintain/AIF-1 was involved inbeta cell death in type 1 diabetes.4. the roles and action mechanism of daintain/AIF-1 in type 1 diabetes. Whendaintain/AIF-1 was intravenously injected into NOD mice, it promoted white blood cellproliferation, increased the concentration of blood glucose, and decreased theconcentration of insulin in the blood and pancreas, aggravated diabetes in NOD mice. Invitro, daintain/AIF-1 inhibited insulin secretion from islets of Langerhans, and damagedthe primary pancreas islet cells. Daintain/AIF-1 was labeled with FITC, and fluorescencewas detected on pancreas islet cells by total internal reflection fluorescence microscope(TIRFM) and flow cytometer. Daintain/AIF-1-interacting protein was identified ascyatathionine-beta-synthase (CBS) in the mice pancreas by peptide mass fingerprinting.5. As a circulating protein in the blood, daintain/AIF-1 impaired plasma superoxidedismutase activity, enhanced concentrations of C-reactive protein and fibrinogen,accompany with higher blood viscosity and faster blood coagulation. The serum from themice administrated with daintain/AIF-1 oxidized more dithiothreitol (DTT). Furthermore,AIF-1 interacting protein was separated by an AIF-1/histidine fusion protein nickelaffinity assay. MALDI-TOF mass analysis identified hemoglobin subunit beta-1 as anAIF-1 interacting protein. This interaction was verified by westem blotting. This was afunctional interaction, because AIF-1 induced cytolysis of erythrocytes, and further boundwith hemoglobin subunit beta-1 resulting in heme release in vitro. Oxidative stress,inflammation, free heme and blood coagulation contribute to the vesicular complicationsin diabetes. These data suggested that daintain/AIF-1 associated with the vesicularcomplications in diabetes.In conclusion, dainain/AIF-1 as an inflammatory factor taken critical roles in theinitiation and progress of type 1 diabetes.