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In previous studies we have shown that sulfatide (galactosylceramide-3-O-sulfate), in various species, is present in the insulin-producing cells in pancreatic islets of Langerhans. In this study the synthesis of sulfatide in the islets has been investigated by pulse chase labeling at varying glucose levels and in the presence or absence of the glycosphingolipid synthesis inhibitory agents, Brefeldin A, fumonisin B1 and chloroquine and the distribution of sulfatide by immune-electronmicroscopy. The data showed that (1) sulfatide was produced in islets of Langerhans, (2) the main pathway for synthesis was through recycling involving partial degradation in the lysosome, and that (3) high glucose levels, although not primarily reflected in an increased synthesis of sulfatide, lead to an increased expression of mRNA for the UDP-galactose:ceramide galactosyltransferase, producing the immediate precursor of sulfatide. Furthermore, mass spectrometry analyses revealed a high proportion of short chain fatty acids, C16:0 (50%) and no hydroxylated forms and thus special physicochemical properties, indicating important differences between pancreatic and brain/neural sulfatide. Immune electron microscopy revealed an intracellular expression of sulfatide in the secretory granules, the Golgi network and the lysosomes of the islets. These results indicate that sulfatide follows the same intracellular route as insulin and suggest a functional association between these molecules. We have raised the hypothesis that sulfatide possibly plays a role in the trafficking of insulin in the islets of Langerhans in rat pancreas.

Sulfatide-reactive CD1d-restricted natural killer T (NKT) lymphocytes belong to the type II NKT cell subset with diverse TCRs, and have been found to regulate experimental autoimmune encephalomyelitis, tumor immunity, and experimental hepatitis in murine models. NKT cells can be activated by self-lipids presented by CD1d, manifested as autoreactivity. The identity of most of these self-lipids remains unknown. By isolating lipids from a CD1d-expressing, highly stimulatory antigen presenting cell, we identified isoforms of β-glucosylceramide (GlcCer), with sphingosine and fatty acid chain lengths of C24:0 and C16:0, that activated a sulfatide-reactive type II NKT cell hybridoma. A screen of structurally related glycosphingolipids demonstrated β-galactosylceramide (GalCer) as another ligand, and further, that the lysoforms were the most potent isoform of the glycosphingolipid ligands, followed by isoforms with a long fatty acid chain of C24. Thus, the same type II NKT cell was activated by several ligands, namely sulfatide, GlcCer, and GalCer. However, CD1d-dependent reactivity to antigen presenting cells lacking all GlcCer-based glycosphingolipids, or all glycosphingolipids, was maintained. This suggests that other endogenous, nonglycosphingolipid, lipid ligands contribute to steady-state autoreactivity by type II NKT cells. antigen presenting molecule CD1d, expressed predominantly by hematopoietic cells. Upon activation, NKT cells display innatelike features such as rapid production of large amounts of both inflammatory and anti-inflammatory cytokines, including IFN-γ and IL-4, and they enhance DC maturation. Through these features, they are important in the initiation of innate immunity and also in the direction and regulation of the adaptive immune response resulting in the modulation C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu 2852 Sara Rhost et al. Eur. J. Immunol. 2012. 42: 2851-2860 of tumor immunity, autoimmunity, and microbial infections [1][2][3]. NKT cells can be activated both by microbial and endogenous lipids. The latter may be upregulated in antigen presenting cells (APCs) upon their activation, or be derived from other cells of the body and presented by APCs [4][5][6]. Sulfatide was among the first self-glycosphingolipids demonstrated to stimulate T lymphocytes [7]. Sulfatide is found in large amounts in the central nervous system, mainly located in the myelin, a target of the autoimmune process during multiple sclerosis. Indeed, the frequencies of sulfatide-reactive T cells were increased in multiple sclerosis patients [7], and sulfatide-reactive CD1d-restricted NKT cells accumulated in the CNS of mice during experimental autoimmune encephalomyelitis [8], a model for the human disease, suggesting that the cells were engaged in the disease process. This notion was supported by studies showing that sulfatide injection protected CD1d-sufficient mice from the disease, consistent with the hypothesis that that sulfatide-reactive NKT cells could suppress the autoimmune process ...

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