Synergistic Upregulation of Interleukin-8 Secretion from Pulmonary Epithelial Cells by Direct and Monocyte-Dependent Effects of Respiratory Syncytial Virus Infection

Respiratory syncytial virus (RSV) infection is the major cause of severe bronchiolitis in infants. Pathology of this infection is partly due to excessive proinflammatory leukocyte influx mediated by chemokines. Although direct infection of the respiratory epithelium by RSV may induce chemokine secretion, little is known about the role of cytokine networks. We investigated the effects of conditioned medium (CM) from RSV-infected monocytes (RSV-CM) on respiratory epithelial (A549) cell chemokine release. RSV-CM, but not control CM (both at a 1:5 dilution), stimulated interleukin-8 (IL-8) secretion from A549 cells within 2 h, and secretion increased over 72 h to 11,360 ± 1,090 pg/ml without affecting cell viability. In contrast, RSV-CM had only a small effect on RANTES secretion. RSV-CM interacted with direct RSV infection to synergistically amplify IL-8 secretion from respiratory epithelial cells (levels of secretion at 48 h were as follows: RSV-CM alone, 8,140 ± 2,160 pg/ml; RSV alone, 12,170 ± 300 pg/ml; RSV-CM plus RSV, 27,040 ± 5,260 pg/ml; P < 0.05). RSV-CM induced degradation of IκBα within 5 min but did not affect IκBβ. RSV-CM activated transient nuclear binding of NF-κB within 1 h, while activation of NF-IL6 was delayed until 8 h and was still detectable at 24 h. Promoter-reporter analysis demonstrated that NF-κB binding was essential and that NF-IL6 was important for IL-8 promoter activity in RSV-CM-activated cells. Blocking experiments revealed that the effects of RSV-CM depended on monocyte-derived IL-1 but that tumor necrosis factor alpha was not involved in this network. In summary, RSV infection of monocytes results in and amplifies direct RSV-mediated IL-8 secretion from respiratory epithelial cells by an NF-κB-dependent, NF-IL6-requiring mechanism.

呼吸道合胞病毒（Respiratory syncytial virus, RSV）感染是引发婴儿重症细支气管炎的首要病因。该感染的病理过程部分由趋化因子介导的促炎性白细胞过度浸润所导致。尽管RSV直接感染呼吸道上皮细胞可诱导趋化因子分泌，但目前对于细胞因子网络在此过程中的作用仍知之甚少。本研究探究了RSV感染单核细胞后的条件培养基（conditioned medium, CM，即RSV-CM）对呼吸道上皮（A549）细胞趋化因子释放的影响。以1:5比例稀释的RSV-CM（而非对照条件培养基）可在2小时内刺激A549细胞分泌白细胞介素8（interleukin-8, IL-8），且分泌水平在72小时内升至11360±1090 pg/ml，同时不会对细胞活力产生影响。与之相对，RSV-CM对RANTES的分泌仅存在微弱的促进作用。RSV-CM可与直接RSV感染产生协同效应，显著放大呼吸道上皮细胞的IL-8分泌（48小时时的分泌水平如下：单独RSV-CM组：8140±2160 pg/ml；单独RSV感染组：12170±300 pg/ml；RSV-CM联合RSV感染组：27040±5260 pg/ml；P<0.05）。RSV-CM可在5分钟内诱导IκBα降解，但不会对IκBβ的表达产生影响。RSV-CM可在1小时内激活NF-κB的瞬时核结合活性，而NF-IL6的激活则延迟至8小时，且在24小时时仍可被检测到。启动子-报告基因分析结果显示，NF-κB结合是必需的，且NF-IL6对于RSV-CM激活细胞中的IL-8启动子活性具有重要作用。阻断实验结果表明，RSV-CM的效应依赖于单核细胞来源的IL-1，而肿瘤坏死因子α并未参与该细胞因子网络。综上，单核细胞的RSV感染可通过依赖NF-κB且需要NF-IL6参与的机制，促进并放大呼吸道上皮细胞直接由RSV介导的IL-8分泌。