Manosudprasit Aggasit, Kantarci Alpdogan, Hasturk Hatice, Stephens Danielle, Van Dyke Thomas E
Department of Applied Oral Sciences, The Forsyth Institute, Cambridge, Massachusetts, USA
Department of Applied Oral Sciences, The Forsyth Institute, Cambridge, Massachusetts, USA.
J Leukoc Biol. 2017 Dec;102(6):1431-1440. doi: 10.1189/jlb.4A0416-209RR. Epub 2017 Oct 11.
The purpose of this study was to test the hypothesis that peripheral blood neutrophils (PMN) exhibit delayed spontaneous apoptosis in individuals with diabetes mellitus type 2 (T2DM) and that the delay is exacerbated further among people who coexpress chronic periodontitis (CP). Seventy-three individuals were enrolled, including those with T2DM ( = 16), CP ( = 15), T2DM + CP ( = 21), and healthy volunteers ( = 21). PMN apoptosis was determined by flow cytometry using TUNEL and Annexin V assays. The activity of caspase-3, -8, and -9 was measured by colorimetric assay. PMN surface death receptor quantification was performed by flow cytometry staining with fluorescence-conjugated anti-CD120a (TNFR1) and anti-CD95 [Fas receptor (FasR)] antibody. Analysis of inflammatory markers in serum samples was performed using multiplexed sandwich immunoassays. In healthy volunteers and individuals with T2DM, CP, and T2DM + CP, spontaneous PMN apoptosis observed at 12 h reached 85.3 ± 3.1, 67.3 ± 3.9, 62.9 ± 3.5 and 62.5 ± 5.4%, respectively ( < 0.05). Caspase-3 activity was significantly reduced in individuals with T2DM and T2DM + CP ( < 0.05) when compared with healthy volunteers. Caspase-8 activity was also significantly decreased in CP and T2DM + CP ( < 0.05), associated with reduced cell-surface FasR, TNFRs, and Fas ligand (FasL) serum levels. Glucose alone was not observed to impact PMN apoptosis; simultaneous incubation with the receptor for advanced glycation endproducts (RAGE) agonist S100B induced significant PMN apoptosis ( < 0.05). These data support the premise that the inhibition of PMN apoptosis in individuals with T2DM occurs through an advanced glycation endproducts/RAGE ligand/receptor-mediated interaction.
2型糖尿病（T2DM）患者外周血中性粒细胞（PMN）表现出延迟的自发凋亡，并且在同时患有慢性牙周炎（CP）的人群中这种延迟会进一步加剧。共招募了73名个体，包括T2DM患者（n = 16）、CP患者（n = 15）、T2DM + CP患者（n = 21）和健康志愿者（n = 21）。使用TUNEL和膜联蛋白V测定法通过流式细胞术测定PMN凋亡。通过比色法测定半胱天冬酶-3、-8和-9的活性。通过用荧光共轭抗CD120a（TNFR1）和抗CD95 [Fas受体（FasR）]抗体进行流式细胞术染色来进行PMN表面死亡受体定量。使用多重夹心免疫测定法对血清样本中的炎症标志物进行分析。在健康志愿者以及T2DM、CP和T2DM + CP患者中，在12小时观察到的自发PMN凋亡分别达到85.3±3.1%、67.3±3.9%、62.9±3.5%和62.5±,5.4%（P < 0.05）。与健康志愿者相比，T2DM和T2DM + CP患者的半胱天冬酶-3活性显著降低（P < 0.05）。CP和T2DM + CP患者的半胱天冬酶-8活性也显著降低（P < 0.05），这与细胞表面FasR、TNFRs和Fas配体（FasL）血清水平降低有关。单独的葡萄糖未观察到会影响PMN凋亡；与晚期糖基化终产物受体（RAGE）激动剂S100B同时孵育可诱导显著的PMN凋亡（P < 0.05）。这些数据支持了以下前提：T2DM患者中PMN凋亡的抑制是通过晚期糖基化终产物/RAGE配体/受体介导的相互作用发生的。
