Martinesi Maria, Treves Cristina, d'Albasio Giuseppe, Bagnoli Siro, Bonanomi Andrea G, Stio Maria
Department of Biochemical Sciences, University of Florence, Florence, Italy.
Inflamm Bowel Dis. 2008 May;14(5):597-604. doi: 10.1002/ibd.20354.
Lymphocytes are crucial in the pathogenesis of inflammatory bowel disease (IBD) and are an important target for drug development. Our aim was to verify whether 2 vitamin D derivatives, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and EB 1089, could induce cell apoptosis and affect cell-cell interaction by regulating adhesion molecule levels.
Peripheral blood mononuclear cell (PBMC) proliferation was studied by [3H]thymidine incorporation and apoptosis was determined using an enzyme-linked immunosorbent assay (ELISA) kit. (Poly(ADP-ribose)polymerase (PARP) cleavage, caspase-3, and ICAM-1 protein levels were determined by Western blot analysis.
Our results indicate that 1,25(OH)2D3 or EB 1089 or anti-TNF-alpha (infliximab) induce apoptosis in PBMC obtained from healthy subjects. In IBD patients apoptosis is induced by vitamin D derivatives and by anti-TNF-alpha only in CD patients. Caspase-3 activation and PARP cleavage are registered when PBMC were treated with vitamin D derivatives. ICAM-1 levels remarkably increase when PBMC was incubated with lipopolysaccharide (LPS) or TNF-alpha. The treatment with the vitamin D derivatives, alone or in combination with LPS or TNF-alpha, significantly decreases ICAM-1 levels both in healthy subjects and IBD patients. In HUVEC cocultured with PBMC, previously incubated with LPS or TNF-alpha associated with 1,25(OH)2D3, ICAM-1 levels decrease both in healthy subjects and IBD patients.
1,25(OH)2D3 and EB 1089 inhibit PBMC proliferation, induce apoptosis in PBMC of healthy subjects and IBD patients, and affect ICAM-1 expression on PBMC and on HUVEC cocultured with PBMC, suggesting that the ICAM-1 downregulation could provide a new target for controlling the recruitment of leukocytes at the sites of inflammation in IBD.
淋巴细胞在炎症性肠病（IBD）的发病机制中起关键作用，是药物研发的重要靶点。我们的目的是验证两种维生素D衍生物，即1,25 - 二羟基维生素D3 [1,25(OH)2D3] 和EB 1089，是否能通过调节黏附分子水平诱导细胞凋亡并影响细胞间相互作用。
通过[3H]胸苷掺入法研究外周血单个核细胞（PBMC）增殖，并使用酶联免疫吸附测定（ELISA）试剂盒测定细胞凋亡。通过蛋白质印迹分析测定聚（ADP - 核糖）聚合酶（PARP）裂解、半胱天冬酶 - 3和细胞间黏附分子 - 1（ICAM - 1）蛋白水平。
我们的结果表明，1,25(OH)2D3或EB 1089或抗TNF - α（英夫利昔单抗）可诱导健康受试者PBMC凋亡。在IBD患者中，维生素D衍生物和抗TNF - α仅在克罗恩病（CD）患者中诱导凋亡。当用维生素D衍生物处理PBMC时，可检测到半胱天冬酶 - 3激活和PARP裂解。当PBMC与脂多糖（LPS）或TNF - α孵育时，ICAM - 1水平显著升高。单独使用维生素D衍生物或与LPS或TNF - α联合处理，均可显著降低健康受试者和IBD患者的ICAM - 1水平。在与先前用LPS或TNF - α联合1,25(OH)2D3孵育的PBMC共培养的人脐静脉内皮细胞（HUVEC）中，健康受试者和IBD患者的ICAM - 1水平均降低。
1,25(OH)2D3和EB 1089抑制PBMC增殖，诱导健康受试者和IBD患者PBMC凋亡，并影响PBMC以及与PBMC共培养的HUVEC上ICAM - 1的表达，提示ICAM - 1下调可能为控制IBD炎症部位白细胞募集提供新靶点。
