Overbergh L, Decallonne B, Valckx D, Verstuyf A, Depovere J, Laureys J, Rutgeerts O, Saint-Arnaud R, Bouillon R, Mathieu C
Laboratorium voor experimentele geneeskunde en endocrinologie (LEGENDO), Catholic University of Leuven, Belgium.
Clin Exp Immunol. 2000 Apr;120(1):139-46. doi: 10.1046/j.1365-2249.2000.01204.x.
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.
1,25(OH)₂维生素D₃的受体存在于大多数免疫细胞中，体外实验已证实其具有重要的免疫效应，这体现在它能够预防自身免疫并延长移植物存活时间。本研究的目的是检测小鼠巨噬细胞中负责该分子最终活化的酶——1-α-羟化酶的存在及性质，并分析其调控机制及其在免疫系统中的可能作用。将C57Bl/6小鼠的腹腔巨噬细胞与脂多糖（LPS；100μg/ml）、γ-干扰素（IFN-γ；500U/ml）或两者的组合进行孵育。通过定量逆转录聚合酶链反应，使用基于小鼠肾脏cDNA序列的引物，在新鲜分离的细胞中检测到低水平的1-α-羟化酶mRNA（18±7×10⁻⁶拷贝/β-肌动蛋白拷贝）。对该基因的cDNA序列分析显示，巨噬细胞和肾脏中的酶具有相同的编码序列。LPS刺激后mRNA水平增加了三倍（无统计学意义），但IFN-γ刺激后增加了六倍（P<0.05）。联合使用LPS和IFN-γ并没有导致进一步的显著增加，但添加环孢素A在IFN-γ刺激组和联合刺激组中均使水平进一步增加了2.5倍（P<0.05）。时间进程分析表明，1-α-羟化酶的上调是一种晚期现象，在活化巨噬细胞产物如IL-1和肿瘤坏死因子-α上调之后。最后，在自身免疫性非肥胖糖尿病小鼠中发现免疫刺激导致的1-α-羟化酶上调存在缺陷。总之，我们认为活化巨噬细胞中1-α-羟化酶的上调导致1,25(OH)₂D₃的合成，这可能是炎症中的一种负反馈环。该系统中的缺陷可能是使平衡向自身免疫倾斜的一个额外因素。
