Department of Surgery-Otorhinolaryngology, Head and Neck Surgery, The Queen Elizabeth Hospital, and the University of Adelaide, Adelaide, SA, Australia.
Int Forum Allergy Rhinol. 2019 Oct;9(10):1220-1226. doi: 10.1002/alr.22401. Epub 2019 Aug 12.
The neutrophil serine proteases neutrophil elastase (NE), cathepsin G (CG), and proteinase 3 (PR3) are implicated in the regulation of inflammatory conditions. Pseudomonas aeruginosa elastase (PE), also a serine protease, has been found to behave similarly to NE and has been proposed to assist the pathogen in evading the host immune response. The effect of serine proteases on human nasal epithelial barrier function requires further investigation to better understand the pathophysiology of inflammatory conditions.
Purified human neutrophil serine proteases and PE were applied to primary human nasal epithelial cells grown at air-liquid interface (HNEC-ALI) cultures from 6 patients. Barrier integrity and function was assessed via transepithelial electrical resistance (TER), permeability assays, immunofluorescence of Zona occludens-1 (ZO-1), and ciliary beat frequency (CBF) measurements. Cytotoxicity assays were employed to assess cell viability. Interleukin 6 (IL-6) and IL-8 enzyme-linked immunosorbent assay (ELISA) assessed cytokine release from HNEC-ALI.
The application of serine proteases showed detrimental effects on HNEC-ALI barrier integrity. Reduction in TER occurred with NE, CG, and PE with increased paracellular permeability with NE, CG, PR3, and PE. Discontinuous tight junctions with reduction in ZO-1 expression were identified using immunofluorescence. Neutrophil serine proteases were not toxic cells to the HNECs and had no detrimental effects on the CBF.
Serine proteases derived from neutrophils and from P. aeruginosa showed detrimental effects on the mucosal barrier integrity with increased permeability, allowing for potential bacterial invasion. This finding may further assist in understanding the pathophysiology present in chronic inflammatory airway diseases.
中性粒细胞丝氨酸蛋白酶弹性蛋白酶（NE）、组织蛋白酶 G（CG）和蛋白酶 3（PR3）参与炎症的调节。假单胞菌弹性蛋白酶（PE）也是一种丝氨酸蛋白酶，其行为类似于 NE，并被认为有助于病原体逃避宿主免疫反应。丝氨酸蛋白酶对人鼻腔上皮屏障功能的影响需要进一步研究，以更好地理解炎症状态的病理生理学。
从 6 名患者的气液界面（HNEC-ALI）培养物中提取纯化的人中性粒细胞丝氨酸蛋白酶和 PE，应用于人鼻上皮细胞（HNEC-ALI）。通过跨上皮电阻（TER）、通透性测定、紧密连接蛋白-1（ZO-1）免疫荧光和纤毛摆动频率（CBF）测量评估屏障完整性和功能。细胞毒性测定用于评估细胞活力。白细胞介素 6（IL-6）和白细胞介素 8（IL-8）酶联免疫吸附试验（ELISA）评估 HNEC-ALI 细胞因子的释放。
丝氨酸蛋白酶的应用对 HNEC-ALI 屏障完整性有不良影响。NE、CG 和 PE 的应用导致 TER 降低，NE、CG、PR3 和 PE 导致旁通透性增加。用免疫荧光法检测到紧密连接不连续，ZO-1 表达减少。中性粒细胞丝氨酸蛋白酶对 HNEC 无细胞毒性，对 CBF 无不良影响。
来自中性粒细胞和铜绿假单胞菌的丝氨酸蛋白酶对黏膜屏障完整性有不良影响，通透性增加，允许潜在的细菌入侵。这一发现可能有助于进一步了解慢性炎症性气道疾病中的病理生理学。
