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本研究探讨IL-27调节人外周血中性粒细胞中黏附分子Mac-1、趋化因子受体fMLP-R和促炎因子IL-1β表达的信号通路。采用Ficoll密度梯度离心及低渗裂解残存红细胞方法分离高纯度的外周血中性粒细胞。用RT-PCR方法检测中性粒细胞IL-27受体(WSX-1/TCCR和gp130)mRNA表达。分别应用IL-27及信号通路p38 MAPK、PI3K和ERK抑制剂SB203580、LY294002、U0126处理中性粒细胞,用实时定量RT-PCR检测作用前后fMLP-R和IL-1βmRNA水平变化;应用放射免疫法检测作用前后培养上清液中IL-1β的水平变化;流式细胞术检测作用前后Mac-1表达水平变化。结果表明:人外周血中性粒细胞协同表达IL-27受体的两个亚单位WSX-1/TCCR和gp130,IL-27下调中性粒细胞表面黏附分子Mac-1表达(p<0.05),SB203580阻断了IL-27对Mac-1的下调作用(p<0.05),而LY294002及U0126无阻断作用(p>0.05)。相反,IL-27上调中性粒细胞中fMLP-R和IL-1βmRNA的表达并能增加IL-1β释放(p<0.05),LY294002阻断了IL-27对fMLP-R和IL-1β的上调作用(p<0.05),而SB203580及U0126无阻断作用(p>0.05)。结论:IL-27与IL-27R相互作用,可能通过p38 MAPK和PI3K信号途径调节人外周血中性粒细胞中Mac-1、fMLP-R及IL-1β表达。
This study was aimed to investigate the signaling pathways by which IL-27 regulates the expression of adhesion molecules Mac-1, chemokine receptor fMLP-R and proinflammatory cytokine IL-1β in human peripheral blood neutrophils. Separation of high purity peripheral blood neutrophils by Ficoll density gradient centrifugation and hypotonic lysis of residual erythrocytes. The neutrophil IL-27 receptor (WSX-1 / TCCR and gp130) mRNA expression was detected by RT-PCR. Neutrophils were treated with IL-27 and p38 MAPK, PI3K and ERK inhibitors SB203580, LY294002 and U0126 respectively. The levels of fMLP-R and IL-1β mRNA were detected by real-time quantitative RT-PCR. Radioimmunoassay The levels of IL-1β in the culture supernatants before and after the test were detected. The expression of Mac-1 was detected by flow cytometry. The results showed that two subunits of IL-27 receptor (WSX-1 / TCCR, gp130 and IL-27) co-expressed by human peripheral blood neutrophils down-regulated the expression of neutrophil adhesion molecule Mac- , SB203580 blocked the downregulation of Mac-1 by IL-27 (p <0.05), but not by LY294002 and U0126 (p> 0.05). In contrast, IL-27 up-regulated the expression of fMLP-R and IL-1β mRNA in neutrophils and increased IL-1β release (p <0.05), while LY294002 blocked up-regulation of fMLP-R and IL-1β by IL-27 (P <0.05), while SB203580 and U0126 had no blocking effect (p> 0.05). Conclusion: IL-27 interacts with IL-27R and may regulate the expression of Mac-1, fMLP-R and IL-1β in human peripheral blood neutrophils through p38 MAPK and PI3K signaling pathways.