Exacerbated Experimental Autoimmune Encephalomyelitis in Ceramide Synthase 6 Knockout Mice is Associated with Enhanced Activation/Migration of Neutrophils (P2.195)

OBJECTIVE: Determining the role of C16-Ceramide in regulating the cellular immune response in EAE and MS BACKGROUND: The cellular component of the autoimmune cascade is an essential step in the neuropathogenesis of MS and mediated by the presence of chemokines on endothelial cells and expression of corresponding chemokine leukozyte-receptors. CCL2 and CCL5 mediate migration of leukocytes into CNS lesions and CXCL1/CXCL2 expression by macrophages recruit neutrophils to sites of inflammation . Blockade of CXCR2 or CCR1 with specific antibodies during EAE ameliorates the disease course. Hence, expression of chemokine-receptors on leukocytes is crucial for their infiltration of the CNS and therefore dictates the course of EAE and MS. Ceramides are involved in important cellular processes (apoptosis, inflammation) depending upon cell, receptortypes and downstream targets. DESIGN/METHODS: MOG-EAE was induced in CerS6KO- and bone marrow chimeric mice. Spinal cord segments were immunohistochemically analyzed for mRNA, protein and sphingolipids. Neutrophils and monocytes were isolated from murine total bone-marrow cells/PBMCs; mRNA from white blood cells, neutrophils and monocytes. Bone marrow cells were incubated with various stimuli and mRNA and sphingolipid concentrations were determined. Quantitative PCR was used to determine mRNA levels, LC-MS/MS to determine sphingolipid levels, protein-electrophoresis and electroblotting to assess protein concentrations. FACS- analyses, in vitro cell adhesion- and migration assays were performed. The release of NO was assessed. Human samples were assessed for comparison. RESULTS: We were able to demonstrate that the lack of CerS6/C16-Cer exacerbates EAE pathology. Our data indicate that CerS6/C16-Cer are upregulated by G-CSF or IFN-γ/TNF-α to inhibit migration and activation of neutrophils, respectively. We identified G-CSF as stimulus for C16-Cer/CerS6 expression. CONCLUSIONS: For the first time -so we know- our results assign C16-Ceramide a clinically significant role in feedback mechanisms limiting immune response. Our findings may contribute significantly to the development of targeted, individualized therapeutic approaches in MS. Disclosure: Dr. Mayer has received personal compensation for activities with Novartis and Merck & Company. Dr. Eberle has nothing to disclose. Dr. Ebel has nothing to disclose. Dr. Barthelmes has nothing to disclose. Dr. Tafferner has nothing to disclose. Dr. Ferreiros Bouzas has nothing to disclose. Dr. Ulshoefer has nothing to disclose. Dr. Henke has nothing to disclose. Dr. Maenner de Bazo has received personal compensation for activities with Genzyme. Dr. Foerch has received personal compensation for activities with Genzyme, Biogen Idec, and Merck & Co., Inc. Dr. Weigert has nothing to disclose. Dr. Grosch has nothing to disclose. Dr. Geisslinger has nothing to disclose. Dr. Willecke has nothing to disclose. Dr. Schiffmann has nothing to disclose.