Objective: To investigate the regulatory effect of interferon-α (IFN-α) on the apoptosis and killing function of CD56dimCD57+ natural killer (NK) cells in systemic lupus erythematosus (SLE) patients, and to explore the specific mechanism.
Methods: A total of sixty-four newly treated SLE patients and sixteen healthy controls (HC) enrolled in the Second Hospital of Dalian Medical University were selected as the research subjects. And the gene expression levels of molecules related to NK cell-killing function were detected by real-time quantitative polymerase chain reaction. CD56dimCD57+ NK cells were co-cultured with the K562 cells, and the apoptotic K562 cells were labeled with Annexin-Ⅴ and 7-amino-actinomycin D. Peripheral blood mononuclear cells were treated with 20, 40, and 80 μmol/L hydrogen peroxide (H2O2), and treated without H2O2 as control, the expression level of perforin (PRF) was detected by flow cytometry. The concentration of IFN-α in serum was determined by enzyme linked immunosorbent assay. The expression levels of IFN-α receptors (IFNAR) on the surface of CD56dimCD57+ NK cells were detected by flow cytometry, and were represented by mean fluorescence intensity (MFI). CD56dimCD57+ NK cells were treated with 1 000 U/mL IFN-α for 24, 48 and 72 h, and no IFN-α treatment was used as the control, the apoptosis and the expression levels of mitochondrial reactive oxygen species (mtROS) were measured by flow cytometry and represented by MFI.
Results: Compared with HC(n=3), the expression levels of PRF1 gene in peripheral blood NK cells of the SLE patients (n=3) were decreased (1.24±0.41 vs. 0.57±0.12, P=0.05). Compared with HC(n=5), the ability of peripheral blood CD56dimCD57+ NK cells in the SLE patients (n=5) to kill K562 cells was significantly decreased (58.61%±10.60% vs. 36.74%±6.27%, P < 0.01). Compared with the control (n=5, 97.51%±1.67%), different concentrations of H2O2 treatment significantly down-regulated the PRF expression levels of CD56dimCD57+ NK cells in a dose-dependent manner, the 20 μmol/L H2O2 PRF was 83.23%±8.48% (n=5, P < 0.05), the 40 μmol/L H2O2 PRF was 79.53%±8.56% (n=5, P < 0.01), the 80 μmol/L H2O2 PRF was 76.67%±7.16% (n=5, P < 0.01). Compared to HC (n=16), the serum IFN-α levels were significantly increased in the SLE patients (n=45) with moderate to high systemic lupus erythematosus disease activity index (SLEDAI≥10) [(55.07±50.36) ng/L vs. (328.2±276.3) ng/L, P < 0.001]. Meanwhile, compared with HC (n=6), IFNAR1 expression in peripheral blood CD56dimCD57+ NK cells of the SLE patients (n=6) were increased (MFI: 292.7±91.9 vs. 483.2±160.3, P < 0.05), and compared with HC (n=6), IFNAR2 expression in peripheral blood CD56dimCD57+ NK cells of the SLE patients (n=7) were increased (MFI: 643.5±113.7 vs. 919.0±246.9, P < 0.05). Compared with control (n=6), the stimulation of IFN-α (n=6) significantly promoted the apoptosis of CD56dimCD57+ NK cells (20.48%±7.01% vs. 37.82%±5.84%, P < 0.05). In addition, compared with the control (n=4, MFI: 1 049±174.5), stimulation of CD56dimCD57+ NK cells with IFN-α at different times significantly promoted the production of mtROS in a time-dependent manner, 48 h MFI was 3 437±1 472 (n=4, P < 0.05), 72 h MFI was 6 495±1 089 (n=4, P < 0.000 1), but there was no significant difference at 24 h of stimulation.
Conclusion: High serum IFN-α level in SLE patients may induce apoptosis by promoting mtROS production and inhibit perforin expression, which can down-regulate CD56dimCD57+ NK killing function.
目的: 探究干扰素-α (interferon-α, IFN-α)对系统性红斑狼疮(systemic lupus erythematosus, SLE)患者外周血CD56dimCD57+自然杀伤(natural killer, NK)细胞凋亡和杀伤功能的调控作用及具体机制。
方法: 选取大连医科大学附属第二医院就诊的64例初治SLE患者和16例健康人(healthy control, HC)为研究对象，采用实时荧光定量聚合酶链式反应检测NK细胞杀伤功能相关分子的基因表达水平；将CD56dimCD57+NK细胞与K562细胞共培养后，采用膜联蛋白和7氨基放线菌素标记凋亡的K562细胞；用浓度分别为20、40、80 μmol/L的过氧化氢(hydrogen peroxide, H2O2) 处理外周血单个核细胞，并以不使用H2O2处理作为对照组，采用流式细胞术检测穿孔素(per-forin, PRF)表达水平；采用酶联免疫吸附试验测定血清中IFN-α的浓度；采用流式细胞术检测CD56dimCD57+NK细胞表面IFN-α受体(interferon-α receptor, IFNAR)表达水平，并用平均荧光强度(mean fluorescence intensity, MFI)表示；用浓度为1 000 U/mL的IFN-α处理CD56dimCD57+NK细胞24、48、72 h，并以不使用IFN-α处理作为对照组，采用流式细胞术检测细胞凋亡及线粒体活性氧(mitochondrial reactive oxygen species, mtROS)表达水平，并用MFI表示。
结果: 与HC(n=3)相比，SLE患者(n=3)外周血总NK细胞的PRF1基因表达水平降低(1.24±0.41 vs. 0.57±0.12, P=0.05)。与HC(n=5)相比，SLE患者(n=5)外周血CD56dimCD57+NK细胞杀伤K562细胞的能力明显下降(58.61%±10.60% vs. 36.74%±6.27%, P < 0.01)。与对照组(n=5, 97.51%±1.67%)相比，不同浓度的H2O2处理可显著下调CD56dimCD57+NK细胞的PRF表达水平且呈剂量依赖性，浓度为20 μmol/L的H2O2组PRF为83.23%±8.48% (n=5, P < 0.05)，浓度为40 μmol/L的H2O2组PRF为79.53%±8.56% (n=5, P < 0.01)，浓度为80 μmol/L的H2O2组PRF为76.67%±7.16% (n=5，P < 0.01)。与HC (n=16)相比，系统性红斑狼疮疾病活动评分(systemic lupus erythematosus disease activity index, SLEDAI) 为中高疾病活动度(SLEDAI≥10)的SLE患者(n=45)血清IFN-α水平显著增高[(55.07±50.36) ng/L vs. (328.2±276.3) ng/L, P < 0.001]。同时与HC (n=6)相比，SLE患者(n=6)外周血CD56dimCD57+NK细胞IFNAR1表达增高(MFI: 292.7±91.9 vs. 483.2±160.3, P < 0.05)，与HC (n=6)相比，SLE患者(n=7)外周血CD56dimCD57+NK细胞IFNAR2表达增高(MFI: 643.5±113.7 vs. 919.0±246.9, P < 0.05)。与对照组(n=6)相比，IFN-α刺激(n=6)可显著促进CD56dimCD57+NK细胞凋亡(20.48%±7.01% vs. 37.82%±5.84%, P < 0.05)。同时，与对照组(n=4, MFI: 1 049±174.5)相比，不同时间下用IFN-α刺激CD56dimCD57+NK细胞可显著促进mtROS的产生，且呈时间依赖性，48 h的MFI为3 437±1 472 (n=4, P < 0.05)，72 h的MFI为6 495±1 089 (n=4, P < 0.000 1)，而刺激24 h时差异无统计学意义。
结论: SLE患者血清中高水平的IFN-α可能通过促进mtROS产生诱导细胞凋亡并抑制PRF表达，下调CD56dimCD57+NK杀伤功能。
Keywords: Interferon-α; Natural killer cells; Perforin; Reactive oxygen species; Systemic lupus erythematosus.