SUMMARYReceptors for 1,25(OH) 2 vitaminD 3 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-a -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 m g/ml), interferon-gamma (IFN-g; 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-a -hydroxylase mRNA were detected in freshly isolated cells (18^7 Â 10 26 copies/b -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-g stimulation (P , 0´05). Combining LPS and IFN-g did not result in a major additional increase, but addition of cyclosporin A further increased levels 2´5-fold both in IFN-g-and combination-stimulated cells (P , 0´05). Time course analysis revealed that upregulation of 1-a -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-ahydroxylase up-regulation by immune stimuli was found in autoimmune non-obese diabetic mice. In conclusion, we propose that the up-regulation of 1-a -hydroxylase in activated macrophages, resulting in the synthesis of 1,25(OH) 2 D 3 , might be a negative feedback loop in inflammation. A defect in this system might be an additional element in tipping the balance towards autoimmunity.

1,25 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 micrograms/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 dihydroxy-vitamin 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.

Prevention of type 1 diabetes in NOD mice by 1,25-dihydroxyvitamin D 3 [1␣,25(OH) 2 D 3 ] is accompanied by a T-helper (Th) 1/Th2 cytokine shift in the pancreas. The aim of this study was to investigate whether this immune shift also occurs outside of the pancreas and whether it is limited to autoantigen-specific immune responses. NOD mice treated with 1␣,25(OH) 2 D 3 (5 µg/kg every 2 days) or control vehicle were immunized with GAD65 (p524-543) or ovalbumin (OVA) in the rear footpads. First, we examined T-cell proliferation and cytokine production (via enzyme-linked immunosorbent assay) of draining lymph node cells in vitro with or without peptide rechallenge. Although no differences in proliferation were measured between control and 1␣,25(OH) 2 D 3 -treated mice after in vitro GAD65 rechallenge, a marked shift in cytokine secretion profile was seen in 1␣,25(OH) 2 D 3 -treated mice: interleukin-4 was increased (37 ± 5 vs. 21 ± 12 pg/ml in controls, P < 0.005), whereas ␥-interferon levels were decreased (6 ± 3 vs. 9 ± 3 ng/ml in controls, P < 0.05). This shift was absent in OVA-primed mice. Second, we measured cytokine profiles by reverse transcriptase-polymerase chain reaction in popliteal lymph nodes at different time points after priming with GAD65 or OVA in vivo. A marked Th1/Th2 shift occurred in 1␣,25(OH) 2 D 3 -treated mice after in vivo priming with GAD65. Again, this shift was absent after OVA immunization. Finally, we measured cytokine profiles after rechallenge with a panel of autoantigens (GAD65, heat shock protein 65, insulin Bchain) and control antigens (OVA, keyhole limpet hemocyanine, myelin proteolipid protein, tetanus toxin) and confirmed the Th1/ Th2 shift in autoantigen-injected mice but not in control antigen-injected mice. In conclusion, the immune deviation induced by 1␣,25(OH) 2 D 3 in NOD mice can also be induced in the peripheral immune system but is limited to pancreatic autoantigens.