川崎病内皮细胞高表达IL-1β促进中性粒细胞活化和分泌Sema4D

High expression of IL-1β in coronary artery endothelial cells promotes neutrophil activation and Sema4D secretion in Kawasaki disease

目的探讨川崎病(KD)冠状动脉内皮细胞(HCAECs)条件培养基对中性粒细胞生物学功能的影响及机制。方法2022年12月在西安市儿童医院收集KD患儿和健康儿童混合血清,分别刺激HCAECs作为KD组和HC组,RT-PCR检测两组细胞IL-1β表达。HCAECs分为si-IL-1β组和si-NC组,分别转染IL-1βsiRNA和无效siRNA,然后加入KD血清刺激,RT-PCR检测HCAECs中IL-1βmRNA表达。收集si-IL-1β组和si-NC组细胞的培养上清液,经离心和滤菌后分别得到两组细胞的条件培养基,ELISA检测两组条件培养基中IL-1β浓度,Transwell实验检测条件培养基对中性粒细胞迁移能力的影响,流式细胞术检测条件培养基对中性粒细胞凋亡和Sema4D表达的影响。中性粒细胞分为TAPI-1预处理+si-NC条件培养基组、DMSO预处理+si-NC条件培养基组、TAPI-1预处理+si-IL-1β条件培养基组和DMSO预处理+si-IL-1β条件培养基组,用si-IL-1β组或si-NC组条件培养基分别刺激已用金属蛋白酶ADAM17的抑制剂TAPI-1或DMSO预处理的中性粒细胞,流式细胞术检测中性粒细胞Sema4D表达。结果RT-PCR显示,KD组较HC组HCAECs中IL-1β明显高表达(P<0.01);si-IL-1β组IL-1βmRNA表达较si-NC组明显降低(P<0.01)。与si-NC组比较,si-IL-1β组条件培养基IL-1β含量明显降低(P<0.01),条件培养基处理的中性粒细胞迁移能力明显下降(P<0.01),而中性粒细胞凋亡和Sema4D表达均增加(P<0.01)。与DMSO预处理+si-NC条件培养基组比较,TAPI-1预处理+si-NC条件培养基组Sema4D+中性粒细胞百分比明显升高(P<0.05),而TAPI-1预处理+si-IL-1β条件培养基组和DMSO预处理+si-IL-1β条件培养基组中性粒细胞Sema4D无明显差异(P>0.05)。结论川崎病内皮细胞高表达IL-1β促进中性粒细胞迁移并抑制中性粒细胞凋亡,而且可能通过激活ADAM17促进中性粒细胞分泌Sema4D。

Objective To investigate the effects and mechanisms of human coronary artery endothelial cell(HCAEC)conditioned medium on the biological functions of neutrophils in Kawasaki disease(KD).Methods Pooled serum were collected from KD patients and healthy children in December 2022 in Xi'an Children's Hospital to stimulate HCAECs,named as KD group and HC group.RT-PCR was performed to detect the expression of IL-1β in HCAECs in two groups.Additionally,HCAECs were divided into si-IL-1β group and si-NC group,and respectively transfected with IL-1β siRNA and invalid siRNA,and then stimulated with KD pooled serum.RT-PCR was used to measure the mRNA expression of IL-1β in si-IL-1β group and si-NC group.The supernatants in both si-IL-1β group and si-NC group were collected after centrifugation and filtration to obtain the conditioned medium.The concentration of IL-1β in the conditioned medium was measured by ELISA.Transwell experiment was performed to assess the effects of the conditioned medium on neutrophil migration.Flow cytometry was used to assess the effects of conditioned medium on neutrophil apoptosis and surface expression of Sema4D.Neutrophils were pretreated with the metalloproteinase ADAM17 inhibitor TAPI-1 or DMSO,and stimulated with the conditioned medium from si-IL-1β group or si-NC group,namely TAPI-1 pretreatment+si-NC conditioned medium group,DMSO pretreatment+si-NC conditioned medium group,TAPI-1 pretreatment+si-IL-1β conditioned medium group and DMSO pretreatment+si-IL-1β conditioned medium group.Flow cytometry was used to measure Sema4D expression in neutrophils.Results RT-PCR showed that the expression of IL-1β in HCAECs in KD group was significantly higher than that in HC group(P<0.01),and the IL-1β mRNA expression in si-IL-1β group was significantly lower than that in si-NC group(P<0.01).Compared to si-NC group,the IL-1βconcentration in conditioned medium was significantly decreased in si-IL-1β group(P<0.01),the migration ability of neutrophils was also significantly reduced(P<0.01),while the neutrophil apoptosis and the Sema4D expression were increased(P<0.01).Compared to DMSO pretreatment+si-NC conditioned medium group,the percentage of Sema4D+neutrophils was significantly increased in TAPI-1 pretreatment+si-NC conditioned medium group(P<0.05),while there was no significant difference in neutrophil Sema4D expression between TAPI-1 pretreatment+si-IL-1β conditioned medium group and DMSO pretreatment+si-IL-1β conditioned medium group(P>0.05).Conclusion Increased expression of IL-1β in endothelial cells promotes the neutrophil migration,and inhibits the neutrophil apoptosis,and may enhance the secretion of Sema4D by neutrophils through ADAM17 activation in Kawasaki disease.