Panichi V, De Pietro S, Andreini B, Bianchi A M, Migliori M, Taccola D, Giovannini L, Tetta C, Palla R
Internal Medicine Department and Neuroscience Department, Pharmacology Section, University of Pisa, Italy.
Kidney Int. 1998 Nov;54(5):1463-9. doi: 10.1046/j.1523-1755.1998.00152.x.
Calcitriol modulates in vivoand in vitro cytokine production: A role for intracellular calcium. Background. Several immunomodulatory properties of calcitriol are currently known, however, only little information is available regarding the in vivo and in vitro effects of calcitriol on cytokine production in chronic renal failure. Methods. To study the in vitro effect of calcitriol on lipopolysaccharide (LPS)-induced cytokine production, peripheral blood mononuclear cells (PBMC, 2.5 ml/ml) from 12 chronic dialytic (HD), 15 undialyzed chronic renal failure (CRF) patients and 10 normal subjects (N) were incubated at 37 degrees for 12 hours with 100 ng of LPS (E. coli and P. maltofilia). Increasing doses of calcitriol from 10-10 to 10-9 M were added and cell associated TNF-alpha and IL-1beta were determined by immunoreactive tests after three freeze-thaw cycles. The intradialytic TNF-alpha and IL-1beta production were evaluated in vivo in 12 HD patients before and after three months of intravenous calcitriol treatment (6 microgram/week). Intracellular calcium [Ca++]i was determined on PBMC with a cytofluorimetric assay using FLUO-3 AM as the indicator. Results. In vitro, TNF-alpha increased from 3.6 +/- 1.9 pg/cell to 1797 +/- 337 in N, from 4.5 +/- 1.7 to 1724 +/- 232 in CRF and from 3.4 +/- 2.3 to 1244 +/- 553 in HD after the LPS stimulus. The production of TNF-alpha was inhibited by calcitriol in a dose-dependent manner [LPS + Vit.D3 100 ng, 2.9 +/- 2.1 in N, 3.7 +/- 1.9 in CRF and 3.4 +/- 1.7 in HD; LPS + Vit.D3 50 ng, 263 +/- 296 (N), 6.73 +/- 11 (CRF), 38 +/- 28 (HD); LPS + Vit.D3 25 ng = 873 +/- 583 (N), 325 +/- 483 (CRF), 588 +/- 507 (HD); LPS + Vit.D3 12.5 ng, 954 +/- 483 (N), 912 +/- 510 (CRF), 875 +/- 527 (HD)]. Comparable data were observed on IL-1beta production. In vivo, the intradialytic TNF-alpha increase (from 8.5 +/- 2.3 to 19 +/- 5.6 pg/2.5 x 106 cell) during hemodialysis was markedly reduced after calcitriol therapy (from 6.6 +/- 3.1 to 11 +/- 4.7). [Ca++]i decreased from 105 +/- 25 to 72 +/- 18 nM (P < 0.05) and a positive correlation between cytokine levels and [Ca++]i was found (r = 0.79; P < 0.001). Conclusions. The in vitro increase of cell-associated cytokine after LPS challenge was inhibited by calcitriol in a dose-dependent manner. These data suggest a possible in vivo modulatory effect of calcitriol therapy on cytokine production in hemodialysis.
细胞内钙的作用。背景。目前已知骨化三醇具有多种免疫调节特性，然而，关于骨化三醇在慢性肾衰竭中对细胞因子产生的体内和体外作用的信息却很少。方法。为研究骨化三醇对脂多糖（LPS）诱导的细胞因子产生的体外作用，将来自12例慢性透析（HD）患者、15例未透析的慢性肾衰竭（CRF）患者和10名正常受试者（N）的外周血单个核细胞（PBMC，2.5 ml/ml）与100 ng LPS（大肠杆菌和嗜麦芽窄食单胞菌）在37℃孵育12小时。加入从10⁻¹⁰到10⁻⁹ M递增剂量的骨化三醇，在三次冻融循环后通过免疫反应试验测定细胞相关的肿瘤坏死因子-α（TNF-α）和白细胞介素-1β（IL-1β）。在12例HD患者静脉注射骨化三醇治疗（6微克/周）三个月前后，对透析期间的TNF-α和IL-1β产生进行体内评估。使用FLUO-3 AM作为指示剂，通过细胞荧光测定法测定PBMC中的细胞内钙[Ca²⁺]i。结果。体外实验中，LPS刺激后，正常受试者的TNF-α从3.6±1.9 pg/细胞增加到1797±337，CRF患者从4.5±1.7增加到1724±232，HD患者从3.4±2.3增加到1244±553。骨化三醇以剂量依赖的方式抑制TNF-α的产生[LPS + 维生素D3 100 ng，正常受试者中为2.9±2.1，CRF患者中为3.7±1.9，HD患者中为3.4±1.7；LPS + 维生素D3 50 ng，（正常受试者）为263±296，（CRF患者）为6.73±11，（HD患者）为38±28；LPS + 维生素D3 25 ng = 873±583（正常受试者），325±483（CRF患者），588±507（HD患者）；LPS + 维生素D3 12.5 ng，954±483（正常受试者），912±510（CRF患者），875±527（HD患者）]。在IL-1β产生方面观察到类似的数据。在体内，骨化三醇治疗后，血液透析期间透析内TNF-α的增加（从8.5±2.3增加到19±5.6 pg/2.5×10⁶细胞）明显减少（从6.6±3.1减少到11±4.7）。[Ca²⁺]i从105±25降至72±18 nM（P < 0.05），并且发现细胞因子水平与[Ca²⁺]i之间存在正相关（r = 0.79；P < 0.001）。结论。LPS刺激后体外细胞相关细胞因子的增加被骨化三醇以剂量依赖的方式抑制。这些数据表明骨化三醇治疗对血液透析中细胞因子产生可能具有体内调节作用。
