Error bars refer to the standard deviations for the 3 samples in each group. Although recent studies have underscored a central role for IL-23 and Th-17 cells WS-383 in experimental allergic encephalomyelitis and collagen-induced arthritis,32,33 previous work showed that IL-12 administration could exacerbate diabetes in NOD mice, in part through the stimulation of IFN–secreting Th1 cells that infiltrate the islets.34,35 Although p40 and IFN-Cdeficient NOD mice display minimal alterations in the frequency or kinetics of diabetes development,36C38 the progression to diabetes in several models is associated with increased expression of IFN-Cinduced genes.39C41 Thus, IFN- might play an important role, although other pathways may contribute to disease development in the absence of this cytokine. To determine whether IFN- is involved WS-383 in the diabetes in GM-CSF/IL-3Cdeficient mice, Tetracosactide Acetate we introgressed an IFN-Cnull allele and thereby generated triply cytokine-deficient mice on the C57Bl/6 background (Figure 5). in impaired glucose homeostasis.1 T1D clusters in families and is frequently associated with other autoimmune disorders, suggesting that an underlying genetic susceptibility compromises tolerance to multiple normal tissues. Nonobese diabetic (NOD) mice are widely used as a model for T1D because they display many similar aspects of disease pathogenesis and harbor a general predisposition to autoimmunity, which is modulated by genetic background.2 In NOD mice, the development of diabetes proceeds from an initial phase of insulitis, characterized by T and B cell infiltrates in the absence of -cell damage, to an aggressive stage in which cells are destroyed and glucose homeostasis is disrupted. Extensive linkage analysis of families with T1D and NOD mice yielded more than 20 genetic susceptibility loci.3 Among these, the major histocompatibility (MHC) class II locus exerts the most potent influence on disease development. Several non-MHC-related genes have also been implicated, including insulin, CTLA-4, IL-2, CD25, the protein tyrosine phosphatase WS-383 PTPN22, and the membrane transporter NRAMP-1. Nonetheless, multiple additional loci remain to be identified, although characterization of these gene products has been hampered by the large number of immune defects associated with disease and a limited understanding of the key pathogenic mechanisms. Antigen-presenting cells are thought to play an important role in the development of diabetes.4 Dendritic cells and macrophages contribute to the maintenance of tolerance through central deletion of autoreactive thymocytes and the induction of recessive and dominant modes of suppression in the periphery.5 Among the phenotypic abnormalities observed in patients with T1D and NOD mice are the impaired responses of hematopoietic cells to granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3).6C19 Alterations in the number and/or function of dendritic cells, macrophages, and granulocytes derived from cultures of hematopoietic precursors in GM-CSF and IL-3 have been described, but the contribution of these cytokine defects to altered antigen presenting cell function in vivo and the pathogenesis of diabetes remains unclear. We previously established roles for GM-CSF and IL-3 in the maintenance of immune homeostasis through promoting the efficient phagocytosis of apoptotic cells by macrophages.20 Consistent with other strains of mice that display an impaired uptake of dying cells,21 aged GM-CSF and, to a greater extent, GM-CSF/IL-3 doubly deficient mice developed a systemic lupus erythematous (SLE)-like disorder characterized by anti-double-stranded DNA antibodies and immune complex-mediated glomerulonephritis. Here we report that aged GM-CSF/IL-3Cdeficient mice also develop insulitis, destruction of insulin-producing cells, and compromised glucose homeostasis. Similar to patients with T1D and NOD mice, disease pathogenesis in this model involves p40 and CTLA-4, suggesting that functional defects in GM-CSF and IL-3 contribute to autoimmune diabetes. Methods Mice Mice deficient in GM-CSF,22 IL-3,23 GM-CSF/IL-3,24 interferon- (IFN-),25 and GM-CSF/IL-3/IFN-20 were backcrossed at least 9 generations onto the C57Bl/6 strain and housed under specific pathogen-free conditions. Genotypes were confirmed by polymerase chain reaction (PCR), as described previously.20 All mouse experiments were conducted under a protocol approved by the Association for Assessment and Accreditation of Laboratory Animal Care-accredited Dana-Farber Cancer Institute Institutional Animal Care and Use Committee (IACUC). Pathology Pancreases were fixed in 10% buffered formalin, embedded in paraffin, cut in 5-m sections, and stained with hematoxylin and eosin. Islets were examined in 7 to 11 fields per specimen WS-383 at magnification 100. Inflammation was evaluated as peri-insulitis and insulitis. Peri-insulitis was noted when an aggregate of lymphocytes surrounded the islet. The inflammatory infiltrates were graded as 1-3+; 1+ represented an infiltrate of less than 10 cells, 2+ represented an infiltrate of 10-50 cells, and 3+ represented an infiltrate greater than 50 mononuclear cells. Insulitis was noted when lymphocytes were present within the islets. Each islet was evaluated for necrosis as evidenced by marked nuclear pallor and loss of nuclear WS-383 content with vacuolization of the cytoplasm and ghost-like remnants of cells or marked.
