The Modulatory Effects of Vitamin D on the Expression of IL-12 and TGF-ß in the Spinal Cord and Serum of Mice with Experimental Autoimmune Encephalomyelitis.

BACKGROUND & OBJECTIVE: The immunoregulatory effects of transforming growth factor (TGF)-ßand interleukin-12 (IL-12) and immunomodulatory actions of vitamin D (VD) were reported in several studies. This study aims to evaluate VD effects on IL-12 and TGF-ß expression in experimental autoimmune encephalomyelitis (EAE). METHODS: EAE was induced in three groups of C57BL/6 mice by immunization with MOG and administered intra-peritoneally 200 ngVD, PBS or olive oil (OO) from day +3 to +30. One group was also considered as healthy control group. At day 31, cytokines expression in the spinal cord and their serum levels were determined using real time-PCR and ELISA, respectively. RESULTS: IL-12 gene expression and its serum levels in PBS-injected- or OO-administrated EAE groups were significantly higher than healthy group. IL-12 gene expression in EAE group treated with VD was significantly decreased compared to PBS-injected- or OO-administrated EAE groups (P<0.001 and P<0.02, respectively). Serum IL-12 levels were significantly reduced in VD-treated EAE mice compared to PBS-injected- or OO-administrated EAE groups (P<0.004 and P<0.05, respectively). No significant difference was observed between PBS-injected-EAE, OO-administrated EAE and healthy groups regarding the expression of TGF-ß. In EAE mice treated with VD, TGF-ß expression was significantly higher than healthy group and PBS-injected EAE mice (P<0.01 and P<0.04, respectively). VD-treated EAE group had also higher serum TGF-ß levels than PBS-injected-, OO-administrated and healthy groups (P<0.05). CONCLUSION: VD modulates the expression of IL-12 and TGF-ß in spinal cord and serum of EAE mice.

Isolation and phenotyping of normal mouse liver dendritic cells by an improved method.

INTRODUCTION: Dendritic cells (DCs) are bone marrow-derived cells, which migrate to lymphoid and non-lymphoid organs via blood. Liver DCs are believed to play an important role in the regulation of hepatic allograft acceptance. However, because of inherent difficulties in isolating adequate numbers of DCs from liver, limited information is available on the phenotype and functions of liver DCs. To address this issue, we isolated DCs from normal C57BL/6 mouse liver using a modified procedure and described their immunophenotypic characteristics. MATERIALS AND METHODS: Non-parenchymal cells (NPCs) were obtained by collagenase digestion of perfused liver fragments and density gradient centrifugation (14.5% nycodenz column). After overnight (18 hr) incubation of the NPCs, enrichment for transiently adherent, low- density cells on 13% nycodenz gradients permitted the recovery of low numbers of cells (approximately 1.2-1.5 x 10(5) per liver), many of which displayed distinct DCs morphology (abundant cytoplasm with prominent projections and irregularly shaped nuclei). RESULTS: Flowcytometric analysis revealed that most of these cells were recognized by anti-CD11c (60-70%). The results obtained from double staining with PE and FITC conjugated monoclonal antibodies indicated that these cells were CD11c(+)/MHC-II(+) (53%), CD11c(+)/CD86(+) (53.5%), CD11c(+)/ CD8α(+) (36%) and CD11c(+)/CD11b(+) (45%). CONCLUSION: These findings indicate that the purity of DCs isolated by nycodenz gradient is higher than other reported methods. Considering the similar ratio of lymphoid (CD11c(+)/CD8α(+)) and myeloid (CD11c(+)/CD11b(+)) DCs in the liver, and the known role of lymphoid DCs in tolerance induction, it seems that this subpopulation of DCs is not the main reason of liver tolerogenecity. Therefore, other factors such as the immaturity of liver DCs or the effect of liver microenvironment on these cells, etc. may explain the acceptance of hepatic allograft.