Herwig Ralf, Erlbacher Katharina, Ibrahimagic Amela, Kacar Mehtap, Brajshori Naime, Beqiri Petrit, Greilberger Joachim
Laboratories PD Dr. R. Herwig, 80337 Munich, Germany.
Heimerer-College, 10000 Pristina, Kosovo.
Biomedicines. 2022 Jul 25;10(8):1785. doi: 10.3390/biomedicines10081785.
Background: Vitamin D3 complexed to deglycosylated vitamin D binding protein (VitD-dgVDBP) is a water-soluble vitamin D dimeric compound (VitD-dgVDBP). It is not clear how VitD-dgVDBP affects circulating monocytes, macrophages, other immune cell systems, including phagocytosis and apoptosis, and the generation of reactive oxygen species (ROS) compared to dgVDBP. Methods: Flow cytometry was used to measure superoxide anion radical (O2*−) levels and macrophage activity in the presence of VitD-dgVDBP or dgVDBP. VitD-dgVDBP was incubated with normal human lymphocytes (nPBMCs), and several clusters of determination (CDs) were estimated. dgVDBP and VitD-dgVDBP apoptosis was estimated on malignant prostatic cells. Results: The macrophage activity was 2.8-fold higher using VitD-dgVDBP (19.8·106 counts) compared to dgVDBP (7.0·106 counts), but O2*− production was 1.8-fold lower in favor of VitD-dgVDBP (355·103 counts) compared to dgVDBP (630·106 counts). The calculated ratio of the radical/macrophage activity was 5-fold lower compared to that of dgVDBP. Only VitD-dgVDBP activated caspase-3 (8%), caspase-9 (13%), and cytochrome-C (11%) on prostatic cancer cells. PE-Cy7-labeled VitD-dgVDBP was found to bind to cytotoxic suppressor cells, monocytes/macrophages, dendritic and natural killer cells (CD8+), and helper cells (CD4+). After 12 h of co-incubation of nPBMCs with VitD-dgVDBP, significant activation and expression were measured for CD16++/CD16 (0.6 ± 0.1% vs. 0.4 ± 0.1%, p < 0.05), CD45k+ (96.0 ± 6.0% vs. 84.7 ± 9.5%, p < 0.05), CD85k+ (24.3 ± 13.2% vs. 3.8 ± 3.2%, p < 0.05), and CD85k+/CD123+ (46.8 ± 8.1% vs. 3.5 ± 3.7%, p < 0.001) compared to the control experiment. No significant difference was found using CD3+, CD4+, CD8+, CD4/CD8, CD4/CD8, CD16+, CD16++, CD14+, or CD123+. A significant decline in CD14+/CD16+ was obtained in the presence of VitD-dgVDBP (0.7 ± 0.2% vs. 3.1 ± 1.7%; p < 0.01). Conclusion: The newly developed water-soluble VitD3 form VitD-dgVDBP affected cytotoxic suppressor cells by activating the low radical-dependent CD16 pathway and seemed to induce apoptosis in malignant prostatic cells.
与去糖基化维生素D结合蛋白（VitD-dgVDBP）复合的维生素D3是一种水溶性维生素D二聚体化合物（VitD-dgVDBP）。与dgVDBP相比，VitD-dgVDBP如何影响循环单核细胞、巨噬细胞及其他免疫细胞系统，包括吞噬作用、凋亡以及活性氧（ROS）的产生尚不清楚。方法：使用流式细胞术测量在存在VitD-dgVDBP或dgVDBP的情况下超氧阴离子自由基（O2*−）水平和巨噬细胞活性。将VitD-dgVDBP与正常人淋巴细胞（nPBMCs）孵育，并评估多个测定簇（CDs）。在恶性前列腺细胞上评估dgVDBP和VitD-dgVDBP的凋亡情况。结果：与dgVDBP（7.0·106计数）相比，使用VitD-dgVDBP（19.8·106计数）时巨噬细胞活性高2.8倍，但有利于VitD-dgVDBP（355·103计数）时O2*−产生比dgVDBP（630·106计数）低1.8倍。计算得出的自由基/巨噬细胞活性比值比dgVDBP低5倍。仅VitD-dgVDBP能激活前列腺癌细胞上的半胱天冬酶-3（8%）、半胱天冬酶-9（13%）和细胞色素-C（11%）。发现PE-Cy7标记的VitD-dgVDBP能与细胞毒性抑制细胞、单核细胞/巨噬细胞、树突状细胞和自然杀伤细胞（CD8+）以及辅助细胞（CD4+）结合。nPBMCs与VitD-dgVDBP共孵育12小时后，与对照实验相比，CD16++/CD16（0.6±0.1%对0.4±0.1%，p<0.05）、CD45k+（96.0±6.0%对84.7±9.5%，p<0.05）、CD85k+（24.3±13.2%对3.8±3.2%，p<0.05）和CD85k+/CD123+（46.8±8.1%对3.5±3.7%，p<0.001）有显著激活和表达。使用CD3+、CD4+、CD8+、CD4/CD8、CD4/CD8、CD16+、CD16++、CD14+或CD123+未发现显著差异。在存在VitD-dgVDBP的情况下，CD14+/CD16+显著下降（0.7±0.2%对3.1±1.7%；p<0.01）。结论：新开发的水溶性维生素D3形式VitD-dgVDBP通过激活低自由基依赖性CD16途径影响细胞毒性抑制细胞，并且似乎能诱导恶性前列腺细胞凋亡。
