Jozef Laureys

Prevention of autoimmune diabetes in NOD mice by 1,25 dihydroxyvitamin D3

Summary1,25 dihydroxyvitamin D3, the active form of vitamin D, has immunomodulatory properties in vitro and in vivo. We report that treatment with 1,25 dihydroxyvitamin D3 (5 μg/kg on alternate days) prevents the development of clinical diabetes in NOD mice, an animal model of human autoimmune diabetes. Diabetes incidence in female NOD mice at the age of 200 days was reduced to 8% in the 1,25 dihydroxyvitamin D treated group vs 56% in the control group (p<0.0001). In parallel, treatment with 1,25 dihydroxyvitamin D3 resulted in a complete normalisation of the capacity to induce suppressor mechanisms in an autologous MLR, which is severely depressed in control NOD mice. The existence of such suppressor cells was confirmed in transfer experiments, whereby cotransfer of splenocytes from 1,25 dihydroxyvitamin D3 treated NOD mice prevented diabetes transfer by splenocytes from diabetic NOD mice into irradiated, 6–8-week-old male NOD mice. Other known immune defects of the NOD mice, such as defective natural killer cell killing of YAC-1 targets and defective thymocyte activation by anti-CD3 were not corrected. The pharmacological doses of 1,25 dihydroxyvitamin D3 were universally well tolerated as reflected by a normal weight gain of the mice. Serum calcium was increased (2.5±0.2 vs 2.2±0.2 mmol/l in the control group, P<0.005), whereas osteocalcin levels nearly doubled and bone calcium content was halved. These findings show that 1,25 dihydroxyvitamin D3 can prevent diabetes in NOD mice, probably through the correction of their defective suppressor function.

Identification and immune regulation of 25-hydroxyvitamin D-1-alpha-hydroxylase in murine macrophages

Receptors for 1,25(OH)2vitaminD3 are found in most immune cells and important immunological effects have been described in vitro, reflected by its capacity to prevent autoimmunity and to prolong graft survival. The aim of this study was to examine the presence and nature of the enzyme responsible for final activation of the molecule, 1‐α‐hydroxylase, in murine macrophages and to analyse its regulation and possible role in the immune system. Peritoneal macrophages from C57Bl/6 mice were incubated with lipopolysaccharide (LPS; 100 μg/ml), interferon‐gamma (IFN‐γ; 500 U/ml) or a combination of both. By quantitative reverse transcriptase‐polymerase chain reaction, using primers based on the murine renal cDNA sequence, low levels of 1‐α‐hydroxylase mRNA were detected in freshly isolated cells (18 ± 7 × 10−6 copies/β‐actin copies). Analysis of the cDNA sequence of the gene revealed identical coding sequences for the macrophage and renal enzymes. mRNA levels rose three‐fold with LPS (NS), but a six‐fold increase was seen after IFN‐γ stimulation (P < 0·05). Combining LPS and IFN‐γ did not result in a major additional increase, but addition of cyclosporin A further increased levels 2·5‐fold both in IFN‐γ‐ and combination‐stimulated cells (P < 0·05). Time course analysis revealed that up‐regulation of 1‐α‐hydroxylase was a late phenomenon, preceded by the up‐regulation of activating macrophage products such as IL‐1 and tumour necrosis factor‐alpha. Finally, a defect in 1‐α‐hydroxylase up‐regulation by immune stimuli was found in autoimmune non‐obese diabetic mice. In conclusion, we propose that the up‐regulation of 1‐α‐hydroxylase in activated macrophages, resulting in the synthesis of 1,25(OH)2D3, might be a negative feedback loop in inflammation. A defect in this system might be an additional element in tipping the balance towards autoimmunity.

In Vitro and In Vivo Analysis of the Immune System of Vitamin D Receptor Knockout Mice

Immune cells carry receptors for 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3; vitamin D receptor (VDR)] and individuals with severe vitamin D deficiency have immune abnormalities. The aim of this study was to investigate the role of vitamin D in the immune system by studying VDR‐knockout (VDR‐KO) mice. VDR‐KO mice had the same metabolic phenotype as rachitic animals with severe hypocalcemia. Leukocytosis, lymphocyte subset composition in different immune organs, and splenocyte proliferation to several stimuli were normal, except for a lower response to anti‐CD3 stimulation (simulation index [SI] of 13 ± 4 vs. 24 ± 9 in wild‐type mice; p < 0.01). Macrophage chemotaxis was impaired (41 ± 19% vs. 60 ± 18% in wild‐type mice; p < 0.01) but phagocytosis and killing were normal. In vivo rejection of allogeneic (31 ± 12 days vs. 45 ± 26 days of survival in wild‐type mice, NS) or xenogeneic (10 ± 2 days vs. 16 ± 9 days of survival in wild‐type mice, NS) islet grafts was comparable with wild‐type mice. Surprisingly, VDR‐KO mice were protected from low‐dose streptozotocin‐induced diabetes mellitus (LDSDM; 5% vs. 65% in wild‐type mice; p < 0.001). Correcting hypocalcemia by use of lactose‐rich or polyunsaturated fat‐rich diets fully restored the immune abnormalities in vitro and the sensitivity to diabetes in vivo. On the other hand, treatment with 1,25(OH)2D3 protected wild‐type mice against diabetes but did not protect normocalcemic VDR‐KO mice. We conclude that immune defects observed in VDR‐KO mice are an indirect consequence of VDR disruption because they can be restored by calcium homeostasis normalization. This study proves that although 1,25(OH)2D3 is a pharmacologic and probably a physiological immunomodulator, its immune function is redundant. Moreover, we confirm the essential role of calcium in the immune system.