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PTU-036 The immunomodulatory effects of vitamin d on low-density cells and monocyte-derived dendritic cells from human peripheral blood in vitro
  1. A N Milestone,
  2. D Bernado-Ordiz,
  3. N R English,
  4. E R Mann,
  5. A L Hart,
  6. S C Knight,
  7. H O Al-Hassi
  1. Antigen Presentation Research Group, Imperial College London, London, UK

Abstract

Introduction Emerging evidence suggests Vitamin D (VD) possesses a wide range of immunomodulatory properties. Epidemiological evidence also implicates VD insufficiency in Inflammatory Bowel Disease (IBD).1 Dendritic Cells (DC) are conductors of the immune system, uniquely capable of primary antigen presentation to naïve T cells, determining either immune response or tolerance. Studies on laboratory-generated monocyte-derived dendritic cells (MoDC) demonstrate 1,25-dihydroxyvitaminD3 (1,25(OH)2D3), the biologically active form of VD, induces an immature and tolerogenic DC phenotype.2 In vivo studies on murine models of IBD suggest therapeutic potential for VD.3

Methods We determined the immunomodulatory effects of 1,25(OH)2D3 on maturation and functional phenotype of blood DC, in particular DC-enriched Low-Density Cells (LDC). Peripheral blood mononuclear cells (PBMC) were isolated on Ficoll gradients from blood of healthy volunteers. LDC enriched for DC were isolated on a NycoPrep gradient from non-adherent cells after overnight PBMC culture. MoDC were obtained from magnetically sorted CD14+ monocytes of PBMC, incubated for 5 days with IL-4 and GM-CSF. LDC and MoDC were cultured for another 24 h in complete medium with and without 1,25(OH)2D3 and/or LPS. LDC and MoDC maturation was determined by flow cytometry using monoclonal antibodies to maturation markers including CD14, CD40, HLA-DR and ILT-3, which is an inhibitory receptor and a marker of DC immaturity. MoDC functional phenotype was determined by phagocytotic capacity (FITC-Dextran uptake) which is reduced in mature DC. Cell morphology was also assessed by electron microscopy (EM).

Results VD-conditioned LDC demonstrated an immature phenotype compared with controls with significantly increased expression of monocyte marker CD14 (<0.01, n=10) and ILT-3 (p<0.02, n=10), but significantly decreased CD40 (p<0.001, n=14) and HLA-DR (p<0.03, n=7), even when co-cultured with the antigen LPS for 4 or 24 h. Furthermore, FITC-Dextran uptake by VD-conditioned MoDC was enhanced dose dependently, correlating with increased CD14 expression, suggesting reversion to a monocyte-like state (p<0.001). EM revealed a greater proportion of morphologically immature LDC cells in the VD groups compared with controls.

Conclusion 1,25(OH)2D3 promotes an immature phenotype in both LDC and MoDC in vitro inducing a tolerogenic-type phenotype and monocyte-like state. These immunomodulatory effects suggest a potential therapeutic role for VD in IBD possibly by promotion of oral tolerance.

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