Scleroderma-specific autoantibodies embedded in immune complexes mediate endothelial damage: an early event in the pathogenesis of systemic sclerosis

Background: Consistently with their diagnostic and prognostic value, autoantibodies specific for systemic sclerosis (SSc) embedded in immune complexes (ICs) elicited a pro-inflammatory and pro-fibrotic cascade in healthy skin fibroblasts, engaging Toll-like Receptors (TLRs) via their nucleic acid components. The objective of this study was to investigate the pathogenicity of SSc-ICs in endothelial cells.

Methods: ICs were purified from sera of SSc patients bearing different autoantibody specificities (antibodies against DNA topoisomerase I, centromeric proteins, RNA polymerase and Th/To), patients with systemic lupus erythematosus (SLE), primary anti-phospholipid syndrome (PAPS) or healthy controls (NHS) using polyethylen glycol precipitation. Human umbilical vein endothelial cells (HUVECs) were incubated with ICs, positive and negative controls. mRNA levels of endothelin-1 (et-1), collagenIα1 (colIa1), interferon (IFN)-α and IFN-β were investigated by Real-Time PCR; et-1 and il-6 mRNA levels were assessed after pre-treatment with bafilomycin. ICAM-1 expression was evaluated by cell-ELISA; secretion of IL-6, IL-8 and transforming growth factor (TGF)-β1 in culture supernatants was measured by ELISA. The expression of Fcg receptors (CD64, CD32 and CD16) was assessed in endothelial cells at FACS analysis. Intracellular signalling pathways culminating with NFkB, p38MAPK, SAPK-JNK and Akt were assessed by Western Blotting. Healthy skin fibroblasts were stimulated with supernatants from HUVEC incubated with ICs, and TGF-b1 secretion, mRNA levels of colIα1 and matrix metalloproteinase (mmp)-1, protein expression of a smooth muscle actin (a-SMA) and IL-6 were evaluated by Western Blotting; et-1 mRNA levels were assessed in fibroblasts pre-treated with IL-6 and TGF-b inhibitors and stimulated with ATA-ICs.

Results: All SSc stimulated IL-6 secretion; ACA-ICs and anti-Th/To-ICs increased ICAM-1 expression; all SSc-ICs but anti-Th/To-ICs augmented IL-8 levels; all SSc-ICs but ACA and ARA-ICs up-regulated et-1 and all SSc-ICs but ARA-ICs affected TGF-b1 secretion. colIa1, IFN-a and IFN-b mRNA levels were not affected by any SSc-IC. FcgRII (CD32) and FcgRIII (CD16) were not detectable on HUVECs, while FcgRI (CD64) was minimally expressed. A differential modulation of tlr expression was observed: tlr2, tlr3 and tlr4 were up-regulated by ATA-ICs and ACA-ICs, while anti-Th/To-ICs resulted in tlr9 up-regulation. Pre-treatment with bafilomycin did not affect the up-regulation of et-1 and il-6 induced by ATA-ICs, ACA-ICs and anti-Th/To-ICs; a 23% reduction in both genes was reported for ARA-ICs. All SSc-ICs activated p38MAPK and AKT, all SSc-ICs but ARA-ICs yielded the activation of NFkB; ATA-ICs and ACA-ICs increased the activation rate of both subunits of SAPK-JNK. When healthy skin fibroblasts were stimulated with supernatants from HUVECs incubated with SSc-ICs, TGF-b1 secretion, colIa1, a-SMA and IL-6 expression levels were significantly modulated. Pre-treatment with IL-6 and TGF-b inhibitors prevented et-1 up-regulation induced by ATA-ICs by 85% and 77% respectively.

Conclusions: These data provide the first demonstration of the pathogenicity of ICs from scleroderma patients with different autoantibodies on the endothelium. Endothelial activation induced by SSc-ICs ultimately led to a pro-fibrotic phenotype in healthy skin fibroblasts.

背景：鉴于自身抗体在诊断与预后评估中的价值，结合于免疫复合物（immune complexes, ICs）中的系统性硬化症（systemic sclerosis, SSc）特异性自身抗体，可通过其核酸成分激活Toll样受体（Toll-like Receptors, TLRs），在健康皮肤成纤维细胞中诱导促炎与促纤维化级联反应。本研究旨在探究系统性硬化症免疫复合物（SSc-ICs）在内皮细胞中的致病作用。

方法：采用聚乙二醇沉淀法，从携带不同特异性自身抗体（抗DNA拓扑异构酶I、着丝粒蛋白、RNA聚合酶及Th/To抗体）的系统性硬化症患者、系统性红斑狼疮（systemic lupus erythematosus, SLE）患者、原发性抗磷脂综合征（primary anti-phospholipid syndrome, PAPS）患者以及健康对照（healthy controls, NHS）的血清中纯化免疫复合物。将人脐静脉内皮细胞（human umbilical vein endothelial cells, HUVECs）与免疫复合物、阳性对照及阴性对照共同孵育。采用实时荧光定量PCR检测内皮素-1（endothelin-1, ET-1）、胶原蛋白Iα1（collagen Iα1, ColIα1）、干扰素α（interferon α, IFN-α）及干扰素β（interferon β, IFN-β）的mRNA表达水平；经巴弗洛霉素预处理后，检测ET-1与IL-6的mRNA表达水平。采用细胞ELISA检测细胞间黏附分子1（intercellular adhesion molecule 1, ICAM-1）的表达水平；采用ELISA检测培养上清中IL-6、IL-8及转化生长因子β1（transforming growth factor β1, TGF-β1）的分泌水平。采用流式细胞术（fluorescence-activated cell sorting, FACS）检测内皮细胞表面Fcγ受体（Fc gamma receptors, FcγRs；CD64、CD32及CD16）的表达水平。采用蛋白质免疫印迹检测以核因子κB（nuclear factor kappa B, NF-κB）、p38丝裂原活化蛋白激酶（p38 mitogen-activated protein kinase, p38MAPK）、应激活化蛋白激酶-JNK（stress-activated protein kinase-JNK, SAPK-JNK）及蛋白激酶B（protein kinase B, Akt）为终点的细胞内信号通路。将健康皮肤成纤维细胞用经免疫复合物孵育的人脐静脉内皮细胞培养上清进行刺激，采用蛋白质免疫印迹检测TGF-β1分泌水平、ColIα1与基质金属蛋白酶1（matrix metalloproteinase 1, MMP-1）的mRNA表达水平，以及α平滑肌肌动蛋白（alpha smooth muscle actin, α-SMA）与IL-6的蛋白表达水平；经IL-6与TGF-β抑制剂预处理后，检测经抗DNA拓扑异构酶I免疫复合物（ATA-ICs）刺激的成纤维细胞中ET-1的mRNA表达水平。

结果：所有系统性硬化症免疫复合物均可促进IL-6的分泌；抗着丝粒抗体免疫复合物（ACA-ICs）与抗Th/To抗体免疫复合物（anti-Th/To-ICs）可上调ICAM-1的表达；除抗Th/To抗体免疫复合物外，其余系统性硬化症免疫复合物均可升高IL-8水平；除抗Th/To抗体免疫复合物外，其余系统性硬化症免疫复合物均可上调ET-1的表达；除抗RNA聚合酶抗体免疫复合物（ARA-ICs）外，其余系统性硬化症免疫复合物均可影响TGF-β1的分泌。所有系统性硬化症免疫复合物均未对ColIα1、IFN-α及IFN-β的mRNA表达水平产生影响。人脐静脉内皮细胞表面未检测到FcγRII（CD32）与FcγRIII（CD16）的表达，仅微量表达FcγRI（CD64）。研究观察到不同Toll样受体（TLRs）的表达呈现差异性调控：ATA-ICs与ACA-ICs可上调TLR2、TLR3及TLR4的表达，而抗Th/To抗体免疫复合物可上调TLR9的表达。经巴弗洛霉素预处理后，ATA-ICs、ACA-ICs及抗Th/To抗体免疫复合物诱导的ET-1与IL-6表达上调未受影响；而ARA-ICs处理组中这两个基因的表达水平均下降了23%。所有系统性硬化症免疫复合物均可激活p38MAPK与Akt；除ARA-ICs外，其余系统性硬化症免疫复合物均可激活NF-κB；ATA-ICs与ACA-ICs可提升SAPK-JNK两个亚基的激活速率。当用经系统性硬化症免疫复合物孵育的人脐静脉内皮细胞培养上清刺激健康皮肤成纤维细胞时，TGF-β1的分泌水平、ColIα1、α-SMA及IL-6的表达水平均发生显著变化。经IL-6与TGF-β抑制剂预处理后，可分别阻断85%与77%的ATA-ICs诱导的ET-1表达上调。

结论：本研究数据首次证实了携带不同自身抗体的硬皮病患者免疫复合物在内皮细胞中的致病作用。系统性硬化症免疫复合物诱导的内皮细胞激活最终可在健康皮肤成纤维细胞中引发促纤维化表型。