Jos Depovere

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.

Prevention of autoimmune destruction of syngeneic islet grafts in spontaneously diabetic nonobese diabetic mice by a combination of a vitamin D3 analog and cyclosporine.

BACKGROUND Type 1 diabetes is characterized by the presence of an autoimmune memory, responsible for the destruction of even syngeneic islet grafts. This recurrence of autoimmunity is partly responsible for the need of extensive immunosuppression in pancreas and islet transplantation in type 1 diabetic patients. The aim of the study was to evaluate the capacity of a 20-epi-analog of vitamin D3, KH1060, both alone and in combination with cyclosporine (CsA) to prevent diabetes recurrence in syngeneic islet grafts in nonobese diabetic (NOD) mice. METHODS Spontaneously diabetic NOD mice grafted with syngeneic islets (n=500) under the kidney capsule were treated with KH1060, CsA, or a combination of both drugs from the day before transplantation until recurrence or 60 days after transplantation. RESULTS Vehicle-treated mice showed a recurrence of diabetes in 100% of cases (n=17) within 4 weeks. Treatment with high doses of CsA (15 mg/kg/day) or KH1060 (1 microg/kg/2 days) significantly prolonged islet survival (60 days and 50 days, respectively, versus 9.5 days in controls; P<0.001 and P<0.0001). Mice treated with subtherapeutical doses of both drugs combined (KH1060 0.5 microg/kg/2 days + CsA 7.5 mg/kg/day) had significant prolongation of graft survival (48 days; P<0.001) and more importantly, four of five mice that were still normoglycemic 60 days after transplantation showed no recurrence after discontinuation of all treatment. Histology of the grafts of control and combination-treated mice demonstrated that graft infiltration and islet destruction were less severe in grafts of combination-treated mice. Cytokine mRNA analysis in the grafts 6 days after transplantation revealed a clear suppression of interleukin-12 and T helper 1 cytokines and higher levels of interleukin-4 in combination-treated mice. CONCLUSIONS KH1060, an analog of 1,25(OH)2D3, delays autoimmune disease recurrence after syngeneic islet transplantation in NOD mice, both alone and especially in combination with CsA, possibly restoring tolerance to beta cells in 30% of cases.

Islet xenograft destruction in the hu‐PBL‐severe combined immunodeficient (SCID) mouse necessitates anti‐CD3 preactivation of human immune cells

Introduction of the hu‐PBL‐SCID mouse model has yielded a potentially useful tool for research in transplantation. The aim of this study was to define the conditions necessary for a reconstituted human immune system to destroy in a consistent manner rat islet xenografts in the alloxan‐diabetic hu‐PBL‐SCID mouse. We examined different time points of hu‐PBL reconstitution, different transplantation sites of the islets and several hu‐PBL reconstitution protocols. Major differences in graft destruction were observed between the different hu‐PBL reconstitution protocols, irrespective of timing of hu‐PBL reconstitution or site of transplantation. Although preactivation of hu‐PBL did not improve the level of hu‐PBL chimerism, histological and immunohistochemical analysis of the grafts revealed a severe human lymphocytic infiltration and β cell destruction only in the grafts of mice receiving preactivated hu‐PBL. This β cell injury resulted in impaired glucose tolerance, with in some animals recurrence of hyperglycaemia, and decreased insulin and C‐peptide levels after glucose stimulation. Therefore, we conclude that activation of hu‐PBL prior to transfer is essential in achieving xenograft infiltration and destruction in hu‐PBL‐SCID mice. The need for immune manipulation suggests that interactions between hu‐PBL and xenografts in this model may be hampered by incompatibilities in cross‐species adhesion and/or activation signals.