BACKGROUND & AIMS:
INTRODUCTION:1alpha,25-dihydroxyvitamin D(3)[1alpha,25(OH)(2)D(3)], the biologically active metabolite of vitamin D3, acts through an intracellular vitamin D receptor (VDR) and has several immunostimulatory effects. Animal studies have shown that production of some matrix metalloproteinases (MMPs) may be upregulated in rat chondrocytes by administration of 1alpha,25(OH)(2)D(3); and cell cultures have suggested that 1alpha,25(OH)(2)D(3) may affect chondrocytic function. Discoordinate regulation by vitamin D of MMP-1 and MMP-9 in human mononuclear phagocytes has also been reported. These data suggest that vitamin D may regulate MMP expression in tissues where VDRs are expressed. Production of 1alpha,25(OH)(2)D(3) within synovial fluids of arthritic joints has been shown and VDRs have been found in rheumatoid synovial tissues and at sites of cartilage erosion. The physiological function of 1alpha,25(OH)(2)D(3) at these sites remains obscure. MMPs play a major role in cartilage breakdown in the rheumatoid joint and are produced locally by several cell types under strict control by regulatory factors. As 1alpha,25(OH)(2)D(3) modulates the production of specific MMPs and is produced within the rheumatoid joint, the present study investigates its effects on MMP and prostaglandin E(2) (PGE(2)) production in two cell types known to express chondrolytic enzymes.
AIMS:To investigate VDR expression in rheumatoid tissues and to examine the effects of 1alpha,25-dihydroxyvitamin D(3) on cultured rheumatoid synovial fibroblasts (RSFs) and human articular chondrocytes (HACs) with respect to MMP and PGE(2) production.
METHODS:Rheumatoid synovial tissues were obtained from arthroplasty procedures on patients with late-stage rheumatoid arthritis; normal articular cartilage was obtained from lower limb amputations. Samples were embedded in paraffin, and examined for presence of VDRs by immunolocalisation using a biotinylated antibody and alkaline-phosphatase-conjugated avidin-biotin complex system. Cultured synovial fibroblasts and chrondrocytes were treated with either 1alpha,25(OH)(2)D(3) or interleukin (IL)-1beta, or both. Conditioned medium was assayed for MMP and PGE(2) by enzyme-linked immunosorbent assay (ELISA), and the results were normalised relative to control values.
RESULTS:The rheumatoid synovial tissue specimens (n=18) immunostained for VDRs showed positive staining but at variable distributions and in no observable pattern. VDR-positive cells were also observed in association with some cartilage-pannus junctions (the rheumatoid lesion). MMP production by RSFs in monolayer culture was not affected by treatment with 1alpha,25(OH)(2)D(3) alone, but when added simultaneously with IL-1beta the stimulation by IL-1beta was reduced from expected levels by up to 50%. In contrast, 1alpha,25(OH)(2)D(3) had a slight stimulatory effect on basal production of MMPs 1 and 3 by monolayer cultures of HACs, but stimulation of MMP-1 by IL-1beta was not affected by the simultaneous addition of 1alpha,25(OH)(2)D(3) whilst MMP-3 production was enhanced (Table 1). The production of PGE(2) by RSFs was unaffected by 1alpha,25(OH)(2)D(3) addition, but when added concomitantly with IL-1beta the expected IL-1beta-stimulated increase was reduced to almost basal levels. In contrast, IL-1beta stimulation of PGE(2) in HACs was not affected by the simultaneous addition of 1alpha,25(OH)(2)D(3)(Table 2). Pretreatment of RSFs with 1alpha,25(OH)(2)D(3) for 1h made no significant difference to IL-1beta-induced stimulation of PGE(2), but incubated for 16h suppressed the expected increase in PGE(2) to control values. This effect was also noted when 1alpha,25(OH)(2)D(3) was removed after the 16h and the IL-1beta added alone. Thus it appears that 1alpha,25(OH)(2)D(3) does not interfere with the IL-1beta receptor, but reduces the capacity of RSFs to elaborate PGE(2) after IL-1beta induction. Cells within the rheumatoid lesion which expressed VDR were fibroblasts, macrophages, lymphocytes and endothelial cells. These cells are thought to be involved in the degradative processes associated with rheumatoid arthritis (RA), thus providing evidence of a functional role of 1apha,25(OH)(2)D(3) in RA. MMPs may play important roles in the chondrolytic processes of the rheumatoid lesion and are known to be produced by both fibroblasts and chondrocytes. The 1alpha,25(OH)(2)D(3) had little effect of basal MMP production by RSFs, although more pronounced differences were noted when IL-1beta-stimulated cells were treated with 1alpha,25(OH)(2)D(3), with the RSF and HAC showing quite disparate responses. These opposite effects may be relevant to the processes of joint destruction, especially cartilage loss, as the ability of 1alpha,25(OH)(2)D(3) to potentiate MMP-1 and MMP-3 expression by 'activated' chondrocytes might facilitate intrinsic cartilage chondrolysis in vivo. By contrast, the MMP-suppressive effects observed for 1alpha,25(OH)2D3 treatment of 'activated' synovial fibroblasts might reduce extrinsic chondrolysis and also matrix degradation within the synovial tissue. Prostaglandins have a role in the immune response and inflammatory processes associated with RA. The 1alpha,25(OH)2D3 had little effect on basal PGE2 production by RSF, but the enhanced PGE2 production observed following IL-1beta stimulation of these cells was markedly suppressed by the concomitant addition of 1alpha,25(OH)2D3. As with MMP production, there are disparate effects of 1alpha,25(OH)2D3 on IL-1beta stimulated PGE2 production by the two cell types; 1alpha,25(OH)2D3 added concomitantly with IL-1beta had no effect on PGE2 production by HACs. In summary, the presence of VDRs in the rheumatoid lesion demonstrates that 1alpha,25(OH)2D3 may have a functional role in the joint disease process. 1 alpha,25(OH)2D3 does not appear to directly affect MMP or PGE2 production but does modulate cytokine-induced production.
背景与目标:
简介:1alpha,25-dihydroxyvitamin D(3)[1alpha,25(OH)(2)D(3)],维生素D3的生物活性代谢物,通过细胞内维生素D受体(VDR)发挥作用,并具有多种免疫刺激作用。动物研究表明,通过施用1alpha,25(OH)(2)D(3),可在大鼠软骨细胞中上调某些基质金属蛋白酶(MMP)的产量;细胞培养表明1alpha,25(OH)(2)D(3)可能影响软骨细胞功能。还已经报道了维生素D对人单核吞噬细胞中MMP-1和MMP-9的不协调调控。这些数据表明维生素D可能调节表达VDR的组织中MMP的表达。已显示在关节炎关节的滑液内产生1alpha,25(OH)(2)D(3),并且在类风湿滑膜组织和软骨侵蚀部位发现了VDR。在这些站点的1alpha,25(OH)(2)D(3)的生理功能仍然不清楚。 MMP在类风湿关节软骨分解中起主要作用,在调节因子的严格控制下,MMP由几种细胞类型局部产生。由于1alpha,25(OH)(2)D(3)调节特定MMP的产生并在类风湿关节内产生,因此本研究调查了其对MMP和前列腺素E(2)(PGE(2))产生的影响。两种已知表达软骨分解酶的细胞类型。
目的:研究类风湿组织中的VDR表达并检查1α,25-二羟基维生素D(3)对类风湿性滑膜成纤维细胞(RSF)和人关节软骨细胞(HAC)产生MMP和PGE(2)的影响。
方法:对晚期风湿性关节炎患者行关节置换术,获取类风湿滑膜组织。正常的关节软骨是从下肢截肢获得的。将样品包埋在石蜡中,并使用生物素化抗体和碱性磷酸酶偶联的抗生物素蛋白-生物素复合物系统通过免疫定位检查VDR的存在。培养的滑膜成纤维细胞和软骨细胞分别用1alpha,25(OH)(2)D(3)或白介素(IL)-1beta或两者处理。通过酶联免疫吸附测定(ELISA)测定条件培养基中的MMP和PGE(2),并将结果相对于对照值进行标准化。
结果:对风湿性滑膜组织标本(n = 18)进行了VDR免疫染色,染色呈阳性,但分布可变,没有可观察的模式。还观察到VDR阳性细胞与一些软骨-盘nu连接处(类风湿病变)相关。 RSF在单层培养物中产生的MMP不受单独用1alpha,25(OH)(2)D(3)处理的影响,但是当与IL-1beta同时添加时,IL-1beta的刺激作用从预期水平降低了多达50%。相比之下,1alpha,25(OH)(2)D(3)对HACs的单层培养对MMPs 1和3的基础产生有轻微的刺激作用,但IL-1beta对MMP-1的刺激不受该作用的影响。同时添加1alpha,25(OH)(2)D(3),同时提高MMP-3的产生(表1)。 RSF产生的PGE(2)不受1alpha,25(OH)(2)D(3)的添加的影响,但是当与IL-1beta一起添加时,预期的IL-1beta刺激的增加减少到几乎基本水平。相反,同时添加1alpha,25(OH)(2)D(3)不会影响HACs中PGE(2)的IL-1beta刺激(表2)。用1alpha,25(OH)(2)D(3)预处理RSF 1h对IL-1beta诱导的PGE(2)刺激无显着影响,但孵育16h抑制了预期的PGE(2)增加控制值。当在16h后单独加入1alpha,25(OH)(2)D(3)和单独添加IL-1beta时,也注意到了这种效果。因此,似乎1alpha,25(OH)(2)D(3)不会干扰IL-1beta受体,但会降低RSF在IL-1beta诱导后修饰PGE(2)的能力。表达VDR的类风湿病变内的细胞是成纤维细胞,巨噬细胞,淋巴细胞和内皮细胞。这些细胞被认为参与了类风湿关节炎(RA)的降解过程,从而提供了1apha,25(OH)(2)D(3)在RA中的功能作用的证据。 MMPs在类风湿病变的软骨分解过程中可能起重要作用,并且已知由成纤维细胞和软骨细胞产生。 1alpha,25(OH)(2)D(3)对RSF产生基础MMP的影响很小,尽管当用1alpha,25(OH)(2)D处理IL-1beta刺激的细胞时,注意到了更明显的差异。 (3),RSF和HAC表现出截然不同的响应。这些相反的作用可能与关节破坏特别是软骨丧失的过程有关,因为1alpha,25(OH)(2)D(3)可能通过“活化”软骨细胞增强MMP-1和MMP-3的表达。促进体内固有软骨软骨分解。相比之下,观察到的1α,25(OH)2D3治疗“激活的”滑膜成纤维细胞的MMP抑制作用可能会减少外在软骨分解以及滑膜组织内的基质降解。前列腺素在与RA相关的免疫反应和炎症过程中起作用。 1alpha,25(OH)2D3对RSF产生的基础PGE2的影响很小,但是伴随着1alpha,25(OH)2D3的加入,IL-1β刺激后观察到的PGE2产生的增加明显受到抑制。与产生MMP一样,两种细胞类型对1α,25(OH)2D3对IL-1beta刺激的PGE2产生的影响也不同。与IL-1beta一起添加的1alpha,25(OH)2D3对HACs产生PGE2没有影响。总之,类风湿病变中VDR的存在表明1alpha,25(OH)2D3可能在关节疾病过程中发挥功能性作用。 1 alpha,25(OH)2D3似乎不直接影响MMP或PGE2的产生,但可以调节细胞因子诱导的产生。