Zhang X, Zhao J X, Sun L, Liu X Y
Department of Rheumatology and Immunology, Peking University Third Hospital, Beijing 100191, China; Department of Rheumatology, Peking Union Medical Colledge, Beijing 100730, China.
Department of Rheumatology and Immunology, Peking University Third Hospital, Beijing 100191, China.
Beijing Da Xue Xue Bao Yi Xue Ban. 2017 Aug 18;49(4):663-668.
It has been found that serum CXCL16 concentration in rheumatoid arthritis (RA) patients are significantly higher than those in osteoarthritis (OA) and normal subjects, and are positively correlated with disease activity and bone erosion. However, how is CXCL16 involved in the pathogenesis of RA is unclear. To evaluate the expression of CXCL16 and its receptor CXCR6 in fibroblast-like synoviocytes (FLS) of rheumatoid arthritis (RA) patients, and to explore the role of CXCL16 in the proliferation of RA-FLS.
FLS were isolated from knee synovial tissues obtained from 8 patients of RA, 7 osteoarthritis (OA) and 3 normal controls. The diagnosis of RA was in line with the 1987 American Rheumatology Association (ACR) RA classification criteria, osteoarthritis met the 1996 ACR revised knee osteoarthritis classification criteria. Control synovium were obtained from trauma caused knee joint injury in healthy individuals who required surgery. Human knee FLS were cultured by tissue explants adherent method.FLS between passages 3 and 5 were used in the experiment. Expression of CXCL16 and its receptor CXCR6 were performed in Western blot analysis. FLS proliferation following stimulation with TNF-α and different concentrations of CXCL16 was examined by cell counting kit-8 (CCK-8). Expression of phosphorylated AKT (pAKT) in RA-FLS stimulated by CXCL16 was quantified by Western blot. Different concentrations of recombinant human CXCL16 were added to the culture medium of RA-FLS. After 48 h culture, supernantants were collected, and TNF-α, IL-6, RANKL and MMP3 in culture supernatants of RA-FLS were determined by enzyme-linked immunosorbent assays (ELISA) operated following the kit instructions.
Expression of CXCL16 and CXCR6 in RA-FLS was significantly higher than that of OA and controls (P<0.05), but no significant difference was found between OA-FLS and control FLS. Proliferation of RA-FLS was markedly up-regulated after stimulation of CXCL16 (P <0.05). In the case of the CXCL16 stimulated OA-FLS and control FLS, the FLS proliferation remained basically unchanged. Expression of phosphorylated AKT in RA-FLS increased remarkably in condition of CXCL16 (50,100, 200 μg/L) stimulation. The levels of IL-6 and RANKL in culture supernatants of RA-FLS were obviously increased under CXCL16 (200 μ g/L) stimulation, while TNF-α and MMP-3 levels in the culture supernatants remained unchanged after CXCL16 (200 μg/L) stimulation.
This study shows that the expression of CXCL16 and its receptor was highly elevated in RA-FLS. Recombinant CXCL16 promoted RA-FLS proliferation and activation in vitro. All these indicate that CXCL16 play an important role in the pathogenesis of RA, anti-CXCL16 treatment may help to relieve inflammation and bone damage of RA patients. However, due to the limitations of this study, the role of CXCL16 and its receptors in RA-FLS remains to be elucidated by further research.
研究发现类风湿关节炎（RA）患者血清CXCL16浓度显著高于骨关节炎（OA）患者和正常受试者，且与疾病活动度及骨侵蚀呈正相关。然而，CXCL16如何参与RA发病机制尚不清楚。本研究旨在评估CXCL16及其受体CXCR6在类风湿关节炎（RA）患者成纤维样滑膜细胞（FLS）中的表达情况，并探讨CXCL16在RA-FLS增殖中的作用。
从8例RA患者、7例骨关节炎（OA）患者及3例正常对照者的膝关节滑膜组织中分离FLS。RA的诊断符合1987年美国风湿病学会（ACR）的RA分类标准，骨关节炎符合1996年ACR修订的膝关节骨关节炎分类标准。对照滑膜取自因外伤导致膝关节损伤且需手术治疗的健康个体。采用组织块贴壁法培养人膝关节FLS。实验选用第3至5代的FLS。通过蛋白质免疫印迹法检测CXCL16及其受体CXCR6的表达。用细胞计数试剂盒-8（CCK-8）检测肿瘤坏死因子-α（TNF-α）和不同浓度CXCL16刺激后RA-FLS的增殖情况。通过蛋白质免疫印迹法定量检测CXCL16刺激后RA-FLS中磷酸化AKT（pAKT）的表达。向RA-FLS培养基中加入不同浓度的重组人CXCL16。培养48小时后收集上清液，按照试剂盒说明书操作，采用酶联免疫吸附测定法（ELISA）检测RA-FLS培养上清液中的TNF-α、白细胞介素-6（IL-6）、核因子κB受体活化因子配体（RANKL）和基质金属蛋白酶3（MMP3）。
RA-FLS中CXCL16和CXCR6的表达显著高于OA-FLS和对照FLS（P<0.05），但OA-FLS与对照FLS之间无显著差异。CXCL16刺激后RA-FLS的增殖明显上调（P<0.05）。CXCL16刺激OA-FLS和对照FLS时，FLS增殖基本保持不变。CXCL16（50、100、200μg/L）刺激条件下，RA-FLS中磷酸化AKT的表达显著增加。CXCL16（200μg/L）刺激后，RA-FLS培养上清液中IL-6和RANKL水平明显升高，而TNF-α和MMP-3水平在CXCL16（200μg/L）刺激后保持不变。
本研究表明，CXCL16及其受体在RA-FLS中的表达显著升高。重组CXCL16在体外促进RA-FLS的增殖和活化。所有这些表明CXCL16在RA发病机制中起重要作用，抗CXCL16治疗可能有助于缓解RA患者的炎症和骨损伤。然而，由于本研究的局限性，CXCL16及其受体在RA-FLS中的作用仍有待进一步研究阐明。
