Alquraini Ali, Garguilo Steven, D'Souza Gerard, Zhang Ling X, Schmidt Tannin A, Jay Gregory D, Elsaid Khaled A
Department of Pharmaceutical Sciences, School of Pharmacy, MCPHS University, 179 Longwood Ave, Boston, MA, 02115, USA.
Department of Emergency Medicine, Rhode Island Hospital, Providence, RI, USA.
Arthritis Res Ther. 2015 Dec 4;17:353. doi: 10.1186/s13075-015-0877-x.
Lubricin/proteoglycan-4 (PRG4) is a mucinous glycoprotein secreted by synovial fibroblasts and superficial zone chondrocytes. PRG4 has a homeostatic multifaceted role in the joint. PRG4 intra-articular treatment retards progression of cartilage degeneration in pre-clinical posttraumatic osteoarthritis models. The objective of this study is to evaluate the binding of recombinant human PRG4 (rhPRG4) and native human PRG4 (nhPRG4) to toll-like receptors 2 and 4 (TLR2 and TLR4) and whether this interaction underpins a PRG4 anti-inflammatory role in synovial fluid (SF) from patients with osteoarthritis (OA) and rheumatoid arthritis (RA).
rhPRG4 and nhPRG4 binding to TLR2 and TLR4 was evaluated using a direct enzyme linked immunosorbent assay (ELISA). Association of rhPRG4 with TLR2 and TLR4 overexpressing human embryonic kidney (HEK) cells was studied by flow cytometry. Activation of TLR2 and TLR4 on HEK cells by agonists Pam3CSK4 and lipopolysaccharide (LPS) was studied in the absence or presence of nhPRG4 at 50, 100 and 150 μg/ml. Activation of TLR2 and TLR4 by OA SF and RA SF and the effect of nhPRG4 SF treatment on receptor activation was assessed. PRG4 was immunoprecipitated from pooled OA and RA SF. TLR2 and TLR4 activation by pooled OA and RA SF with or without PRG4 immunoprecipitation was compared.
rhPRG4 and nhPRG4 exhibited concentration-dependent binding to TLR2 and TLR4. rhPRG4 associated with TLR2- and TLR4-HEK cells in a time-dependent manner. Co-incubation of nhPRG4 (50, 100 and 150 μg/ml) and Pam3CSK4 or LPS reduced TLR2 or TLR4 activation compared to Pam3CSK4 or LPS alone (p <0.05). OA SF and RA SF activated TLR2 and TLR4 and nhPRG4 treatment reduced SF-induced receptor activation (p <0.001). PRG4 depletion by immunoprecipitation significantly increased TLR2 activation by OA SF and RA SF (p <0.001).
PRG4 binds to TLR2 and TLR4 and this binding mediates a novel anti-inflammatory role for PRG4.
润滑素/蛋白聚糖-4（PRG4）是一种由滑膜成纤维细胞和表层软骨细胞分泌的黏液糖蛋白。PRG4在关节中具有维持稳态的多方面作用。在临床前创伤后骨关节炎模型中，关节内注射PRG4可延缓软骨退变的进展。本研究的目的是评估重组人PRG4（rhPRG4）和天然人PRG4（nhPRG4）与Toll样受体2和4（TLR2和TLR4）的结合情况，以及这种相互作用是否是PRG4在骨关节炎（OA）和类风湿关节炎（RA）患者滑液（SF）中发挥抗炎作用的基础。
使用直接酶联免疫吸附测定（ELISA）评估rhPRG4和nhPRG4与TLR2和TLR4的结合。通过流式细胞术研究rhPRG4与过表达TLR2和TLR4的人胚肾（HEK）细胞的结合。在有无50、100和150μg/ml nhPRG4的情况下，研究激动剂Pam3CSK4和脂多糖（LPS）对HEK细胞上TLR2和TLR4的激活作用。评估OA滑液和RA滑液对TLR2和TLR4的激活作用以及nhPRG4滑液处理对受体激活的影响。从汇集的OA和RA滑液中免疫沉淀PRG4。比较有或无PRG4免疫沉淀的汇集OA和RA滑液对TLR2和TLR4的激活作用。
rhPRG4和nhPRG4与TLR2和TLR4的结合呈浓度依赖性。rhPRG4与TLR2-和TLR4-HEK细胞的结合呈时间依赖性。与单独使用Pam3CSK4或LPS相比，nhPRG4（50、100和150μg/ml）与Pam3CSK4或LPS共同孵育可降低TLR2或TLR4的激活（p<0.05）。OA滑液和RA滑液激活TLR2和TLR4，而nhPRG4处理可降低滑液诱导的受体激活（p<0.001）。通过免疫沉淀去除PRG4可显著增加OA滑液和RA滑液对TLR2的激活（p<0.001）。
PRG4与TLR2和TLR4结合，这种结合介导了PRG4的一种新的抗炎作用。
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