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

Receptors for 1,25(OH)(2)vitaminD(3) are found in most immune cells and imp ortant 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 responsibl e for final activation of the molecule, 1-alpha-hydroxylase, in murine macr ophages and to analyse its regulation and possible role in the immune syste m. Peritoneal macrophages from C57Bl/6 mice were incubated with lipopolysac charide (LPS; 100 mu g/ml), interferon-gamma (IFN-gamma; 500 U/ml) or a com bination of both. By quantitative reverse transcriptase-polymerase chain re action, using primers based on the murine renal cDNA sequence, low levels o f 1-alpha-hydroxylase mRNA were detected in freshly isolated cells (18 +/- 7 x 10(-6) copies/beta-actin copies). Analysis of the cDNA sequence of the gene revealed identical coding sequences for the macrophage and renal enzym es. mRNA levels rose three-fold with LPS (NS), but a six-fold increase was seen after IFN-gamma stimulation (P < 0.05). Combining LPS and IFN-gamma di d not result in a major additional increase, but addition of cyclosporin A further increased levels 2.5-fold both in IFN-gamma- and combination-stimul ated cells (P < 0.05). Time course analysis revealed that up-regulation of 1-alpha-hydroxylase was a late phenomenon, preceded by the up-regulation of activating macrophage products such as IL-1 and tumour necrosis factor-alp ha. Finally, a defect in 1-alpha-hydroxylase up-regulation by immune stimul i was found in autoimmune non-obese diabetic mice. In conclusion, we propos e that the up-regulation of 1-alpha-hydroxylase in activated macrophages, r esulting in the synthesis of 1,25(OH)(2)D-3, might be a negative feedback l oop in inflammation. A defect in this system might be an additional element in tipping the balance towards autoimmunity.