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

Abstract

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-alpha-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 microg/ml), interferon-gamma (IFN-gamma; 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-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 enzymes. 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 did not result in a major additional increase, but addition of cyclosporin A further increased levels 2.5-fold both in IFN-gamma- and combination-stimulated 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-alpha. Finally, a defect in 1-alpha-hydroxylase up-regulation by immune stimuli was found in autoimmune non-obese diabetic mice. In conclusion, we propose that the up-regulation of 1-alpha-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.

Fig. 1

mRNA expression of 1-α-hydroxylase in murine kidney and macrophages, analysed by nested reverse transcriptase-polymerase chain reaction (RT-PCR). Total RNA was extracted from murine kidney and fresh macrophages taken from C57Bl/6 mice. cDNA was synthesized and nested PCR performed as described in Materials and Methods. Four overlapping PCR fragments, spanning the coding sequence of the 1-α-hydroxylase gene, were amplified: 1, bases 18–486; 2, bases 413–968; 3, bases 878–1483; 4, bases 1403–1913 (co-ordinates from [17]). PCR products were separated by electrophoresis, by loading 25 μl on an ethidium bromide-stained 1·5% agarose gel. MW, 100-bp molecular weight marker (Life Technologies, Gaithersburg, MD).

Fig. 2

1-α-hydroxylase mRNA levels in peritoneal murine C57Bl/6 macrophages (a) and a murine macrophage cell line (P388.D1) (b). 1-α-hydroxylase mRNA levels are expressed relative to the β-actin polymerase chain reaction product amplified from the same sample ((1-α-hydroxylase copies/β-actin copies) ×10⁶). mRNA levels of freshly isolated and stimulated (incubated for 48 h with lipopolysaccharide (LPS; 100 μg/ml), IFN-γ (500 U/ml) or a combination of both) cells are shown (a). Similar results are found in the murine macrophage cell line, P388.D1 (b). Means of four to seven separate experiments are shown (data ±s.e.m.). *P < 0·05 versus fresh; **P < 0·01 versus fresh.

Fig. 3

Differential regulation of 1-α-hydroxylase in murine macrophages compared with renal tubular cells: effect of 1,25(OH)₂D₃. Fresh peritoneal macrophages harvested from C57Bl/6 mice, P388.D1 cells (murine macrophage cell line) and cultured renal tubular cells were analysed after incubation under identical conditions with IFN-γ (500 U/ml) in the presence of 1,25(OH)₂D₃, at a concentration of 10⁻⁸ m. In view of the difference in absolute 1-α-hydroxylase mRNA levels in the different cell types, relative levels are expressed. ▪, mRNA levels in the presence of 1,25(OH)₂D₃; □, the situations without 1,25(OH)₂D₃. Means of three to seven experiments are shown (data ±s.e.m.). *P < 0·05.

Fig. 4

Time course of cytokine and 1-α-hydroxylase transcription in activated murine macrophages. Peritoneal macrophages were stimulated as described in Materials and Methods with lipopolysaccharide (100 μg/ml, ○) or IFN-γ (500 U/ml, •). Cells were harvested at different time points (6, 24 and 48 h), whereafter 1-α-hydroxylase (a) and cytokine mRNA (tumour necrosis factor-alpha (TNF-α; (b)), IL-1 (c), inducible nitric oxide synthase (iNOS) (d), IL-12 (e)) levels were measured by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). mRNA levels are expressed relative to the β-actin PCR product amplified from the same sample ((1-α-hydroxylase or cytokine copies/β-actin copies) × 10⁶). Means of four to seven separate experiments are shown (data ±s.e.m.). *P < 0·05 versus fresh; **P < 0·03 versus fresh.

Fig. 5

Defect in 1-α-hydroxylase transcription in activated macrophages from autoimmune non-obese diabetic (NOD) mice. Levels of 1-α-hydroxylase mRNA from freshly isolated and stimulated (incubated for 48 h with lipopolysaccharide (LPS; 100 μg/ml), IFN-γ (500 U/ml) or a combination of both) peritoneal macrophages isolated from C57Bl/6 (•) and NOD (○) mice are shown. Each circle represents a separate experiment. Note the defect in 1-α-hydroxylase transcription in NOD mice in all treatment groups (NS in all treatment groups versus fresh NOD macrophages; *P < 0·05 versus C57Bl/6 macrophages).

Fig. 6

Effect of cyclosporin A (CsA) on 1-α-hydroxylase mRNA levels in activated macrophages of C57Bl/6 and non-obese diabetic (NOD) mice. Levels of 1-α-hydroxylase mRNA from 48 h IFN-γ (500 U/ml) and lipopolysaccharide (LPS; 100 μg/ml) + IFN-γ (500 U/ml)-stimulated C57Bl/6 (▪) and NOD (□) peritoneal macrophages in the absence or presence of CsA (100 ng/ml) are shown. Means of four to seven separate experiments are shown (data ±s.e.m.). Note that CsA further stimulated 1-α-hydroxylase transcription. *P < 0·05; **P < 0·01 versus macrophages in the absence of CsA.