IL-18对原发性肝细胞癌患者自然杀伤样B细胞的体外调控作用

In vitro regulatory effects of IL-18 on natural killer-like B cells in patients with primary hepatocellular carcinoma

目的观察原发性肝细胞癌(hepatocellular carcinoma,HCC)患者外周血和肝脏浸润的自然杀伤样B(natural killer-like B,NKB)细胞变化,分析IL-18在体外对NKB细胞水平的影响和机制。方法入组43例HCC患者和21例健康对照者(normal control,NC),采集抗凝外周血,分离血浆和外周血单个核细胞(peripheral blood mononuclear cell,PBMC),16例接受手术治疗的HCC患者,取肿瘤组织和癌旁组织,分离肝脏内淋巴细胞(intrahepatic lymphocyte,IHL)。酶联免疫吸附试验检测血浆IL-12、IL-18和IL-18结合蛋白(IL-18 binding protein,IL-18BP)水平,流式细胞术检测PBMC和IHL中CD3-NKp46+CD19+NKB细胞比例和NKB细胞中IL-18+细胞比例。使用重组人IL-18(1 ng/ml和10 ng/ml)对PBMC和IHL进行刺激培养,检测NKB细胞比例和NKB细胞中IL-18+细胞比例变化,并检测培养上清中IL-18BP水平变化和NKB细胞中磷酸化核因子-κB(nuclear factor-κB,NF-κB)的变化。组间比较采用t检验、单因素方差分析或LSD-t检验。结果血浆IL-12水平在HCC组和NC组之间的差异无统计学意义(P=0.245),HCC组血浆IL-18水平低于NC组[(224.30±58.89)pg/ml vs(327.00±52.27)pg/ml,P<0.0001],HCC组血浆IL-18BP水平高于NC组[(4.42±0.97)ng/ml vs(0.92±0.18)ng/ml,P<0.0001]。HCC组外周血NKB细胞比例低于NC组[(2.68±1.23)%vs(8.88±2.95)%,P<0.0001],NKB细胞中IL-18+细胞比例亦低于NC组[(54.42±12.60)%vs(69.74±12.65)%,P<0.0001]。肿瘤组织IHL中NKB细胞比例低于癌旁组织[(2.89±0.86)%vs(4.66±1.17)%,P<0.0001],NKB细胞中IL-18+细胞比例亦低于癌旁组织[(51.50±13.18)%vs(62.13±9.24)%,P=0.013]。重组人IL-18刺激后培养上清中IL-18BP分泌水平降低(P<0.05)。1 ng/ml IL-18刺激对PBMC和IHL中NKB细胞比例、NKB细胞中IL-18+细胞比例、NKB细胞中磷酸化NF-κB水平均无影响(P>0.05),而10 ng/ml IL-18刺激不但提升NKB细胞比例和NKB细胞中IL-18+细胞比例,还可诱导NKB细胞中磷酸化NF-κB水平升高(P<0.01)。结论高浓度IL-18在体外可能通过抑制HCC患者IL-18BP表达促进NF-κB磷酸化,可能发挥正反馈作用,诱导NKB细胞活化和IL-18分泌。

Objective To investigate the changes in peripheral blood and liver-infiltrating natural killer-like B(NKB)cells in patients with primary hepatocellular carcinoma(HCC),and to assess the influence of IL-18 on NKB cells in vitro and the underlying mechanism.Methods Forty-three HCC patients and 21 normal controls(NC)were enrolled in the study.Peripheral blood samples were collected to isolate plasma and peripheral blood mononuclear cells(PBMC).Intrahepatic lymphocytes(IHL)were isolated from tumor tissues and para-tumor tissues obtained from 16 HCC patients who received surgery.IL-12,IL-18 and IL-18 binding protein(IL-18BP)levels in plasma were measured by enzyme linked immunosorbent assay.The percentages of CD3-NKp46+CD19+NKB cells and IL-18+NKB cells in PBMC and IHL were analyzed by flow cytometry.Changes in the percentages of NKB cells and IL-18+NKB cells were measured after stimulating PBMC and IHL with recombinant human IL-18(1 ng/ml and 10 ng/ml).Changes in IL-18BP levels in the culture supernatants and phosphorylated nuclear factor-κB(NF-κB)in NKB cells were also assessed.Student′s t test,one-way analysis of variance or LSD-t test was used for statistical analysis.Results There was no significant difference in plasma IL-12 level between HCC patients and NC(P=0.245).Compared with NC,HCC patients had decreased IL-18 level in plasma[(224.3±58.89)pg/ml vs(327.0±52.27)pg/ml,P<0.0001],but increased IL-18BP level[(4.421±0.97)ng/ml vs(0.92±0.18)ng/ml,P<0.0001].The percentages of peripheral blood NKB cells and IL-18+NKB cells were lower in HCC patients than in NC[(2.68±1.23)%vs(8.88±2.95)%and(54.42±12.60)%vs(69.74±12.65)%,both P<0.0001].The percentage of NKB cells in IHL was reduced in tumor tissues as compared with that in para-tumor tissues[(2.89±0.86)%vs(4.66±1.17)%,P<0.0001].Moreover,the percentage of IL-18+NKB cell was also down-regulated in tumor tissues as compared with that in para-tumor tissues[(51.50±13.18)%vs(62.13±9.24)%,P=0.013].Recombinant human IL-18 stimulation reduced the IL-18BP level in the culture supernatants(P<0.05).IL-18 stimulation at 1 ng/ml did not affect NKB cell percentage,IL-18+NKB cell percentage or NF-κB phosphorylation in NKB cells from PBMC or IHL(P>0.05),while 10 ng/ml of IL-18 not only elevated NKB cell percentage and IL-18+NKB cell percentage,but also promoted NF-κB phosphorylation in NKB cells(P<0.01).Conclusions In vitro stimulation with high concentration of IL-18 might promote NF-κB phosphorylation by inhibition of IL-18BP expression.This process might play a positive feedback role to induce the activation of NKB cells and IL-18 secretion.