Meakins-Christie Laboratories, McGill University, Montreal, QC, Canada; Department of Thoracic Medicine, Chang Gung Memorial Hospital, College of Medicine, Chang Gung University, Taipei, Taiwan.
Clin Exp Allergy. 2014 Nov;44(11):1347-60. doi: 10.1111/cea.12421.
Infiltration of fibrocytes (FC) in the airway smooth muscle is a feature of asthma, but the pathological significance is unknown.
We sought to explore whether FC modulate the phenotype of airway smooth muscle cells (ASMC) in asthmatic vs. control subjects.
Fibrocytes were isolated from CD14+ monocytes from asthmatic and normal subjects. Proliferation of ASMC of asthmatic or normal subjects was analysed by (3) H-thymidine incorporation, cell number counting and Ki-67 expression after treatment of ASMC with FC-conditioned medium (FCCM) or co-culture with FC. ASMC-associated cytokines/chemokines implicated in asthma (TGF-β1, eotaxin, IL-6 and IL-8) were measured in co-culture or transwell culture of ASMC + FC by ELISA. Immunofluorescence staining was performed to localize these cytokines in ASMC. Cytokine secretion was measured in the transwell culture of ASMC + FC, where NF-κB-p65 or ERK1/2 in ASMC was silenced by siRNA. Contractile phenotype of ASMC in transwell culture was assessed by immunoblotting of α-smooth muscle actin (α-SMA) and myosin light chain kinase (MLCK).
Fibrocytes did not affect ASMC proliferation and expression of TGF-β1, eotaxin, α-SMA and MLCK; however, ASMC production of IL-8 and IL-6 was increased in the co-culture and transwell culture by FC. ASMC treated with FCCM were immunopositive for IL-8/IL-6 and produced more IL-8/IL-6. Furthermore, siRNA silencing of NF-κB-p65 or ERK1/2 in transwell cultures of asthmatic ASMC with normal subject FC decreased IL-8 and IL-6 production.
Fibrocytes promoted IL-8 and IL-6 production by ASMC, demonstrating a proinflammatory role for FC and a possible mechanism of the inflammatory phenotype in asthma.
纤维细胞（FC）浸润气道平滑肌是哮喘的一个特征，但病理意义尚不清楚。
我们试图探讨 FC 是否调节哮喘和正常对照受试者气道平滑肌细胞（ASMC）的表型。
从哮喘和正常受试者的 CD14+单核细胞中分离纤维细胞。用 FC 条件培养基（FCCM）处理或与 FC 共培养后，分析哮喘或正常受试者的 ASMC 增殖，通过（3）H-胸腺嘧啶掺入、细胞计数和 Ki-67 表达进行分析。通过 ELISA 在 ASMC+FC 的共培养或 Transwell 培养中测量与哮喘相关的 ASMC 相关细胞因子/趋化因子（TGF-β1、嗜酸性粒细胞趋化因子、IL-6 和 IL-8）。通过免疫荧光染色定位这些细胞因子在 ASMC 中的位置。在 ASMC+FC 的 Transwell 培养中测量细胞因子分泌，其中 ASMC 中的 NF-κB-p65 或 ERK1/2 通过 siRNA 沉默。通过 Transwell 培养中免疫印迹α-平滑肌肌动蛋白（α-SMA）和肌球蛋白轻链激酶（MLCK）评估 ASMC 的收缩表型。
纤维细胞不影响 ASMC 的增殖和 TGF-β1、嗜酸性粒细胞趋化因子、α-SMA 和 MLCK 的表达；然而，FC 共培养和 Transwell 培养中 ASMC 产生的 IL-8 和 IL-6 增加。用 FCCM 处理的 ASMC 对 IL-8/IL-6 呈免疫阳性反应，并产生更多的 IL-8/IL-6。此外，在含有正常供体 FC 的哮喘 ASMC 的 Transwell 培养物中，NF-κB-p65 或 ERK1/2 的 siRNA 沉默降低了 IL-8 和 IL-6 的产生。
纤维细胞促进 ASMC 产生 IL-8 和 IL-6，表明 FC 具有促炎作用，可能是哮喘炎症表型的一种机制。
