MECHANISMS OF ARTHRITIS SUPPRESSION BY TSG-6 (TNFIP6)

附件（由申请人提供）： 肿瘤坏死因子α诱导蛋白6（Tnfip 6）是TNF α刺激基因6（TSG-6）的产物，是响应促炎刺激而分泌的透明质酸（HA）结合蛋白，并且已经在类风湿性关节炎（伊拉）患者的滑液和组织中检测到。用重组Tnfip 6治疗小鼠已显示出抑制炎性白细胞浸润，并改善RA小鼠模型中的关节肿胀。tnfip 6还通过增强血清抗-α胰蛋白酶抑制剂（lal）的蛋白酶抑制剂活性提供针对软骨降解的保护。这些发现清楚地表明，Tnfip 6拮抗白细胞流入以及炎性组织损伤。然而，Tnfip 6在体内实现这些治疗效果的确切机制仍然未知。我们的初步体外研究表明，Tnfip 6与细胞表面HA受体CD 44相互作用，并调节白细胞在HA上的CD 44依赖性滚动。表达CD 44的细胞在HA覆盖的微血管内皮上滚动是白细胞募集过程中的关键步骤，先天性和适应性免疫系统的效应细胞通过该过程进入炎症部位。因此，体内通过Tnfip 6调节细胞滚动可能对白细胞外渗产生负面影响，导致炎症消退。 我们假设Tnfip 6，特别是当以药理学剂量给药时，通过与细胞表面CD 44的相互作用抑制白细胞流入发炎的滑膜关节。我们将通过对患有蛋白聚糖诱导的关节炎（PGIA）的BALB/c小鼠的滑膜微循环进行实时活体视频显微镜检查来测试这一假设，随后用重组Tnfip 6治疗（Aim 1）。我们将记录并分析踝关节滑膜微血管中白细胞与内皮细胞的滚动和粘附相互作用，踝关节远端关节在PGIA和RA中均患有炎症，但迄今为止尚未进行实时调查。将分别使用Tnfip 6和CD 44缺陷的BALB/c小鼠研究内源性Tnfip 6和CD 44用于下调炎性白细胞募集的需要（目的2和3）。为了验证体内方法，活体显微镜实验的结果将与关节炎的形态学特征相关。 Tnfip 6还参与转录调节的两个丝氨酸蛋白酶抑制剂，不同的IAL，在体外刺激的Tnfip 6缺陷细胞的基因表达谱所建议的。这些抗蛋白酶之一，分泌性白细胞蛋白酶抑制剂（Slpi），具有抗炎特性。我们将研究Tnfip 6和SIpi之间的调节关系，以及SIpi在关节炎抑制中的作用（目的4）。 总之，Tnfip 6是一种关节炎相关蛋白，由炎症过程诱导并拮抗炎症过程。阐明Tnfip 6的多种功能将有助于阐明该蛋白对减少关节炎症的负调控环的贡献。

ESCRIPTION (provided by applicant): Tumor necrosis factor a-induced protein 6 (Tnfip6), the product of TNFa-stimulated gene 6 (TSG-6), is a hyaluronan(HA)-binding protein that is secreted in response to pro-inflammatory stimuli, and has been detected in synovial fluid and tissue from patients with rheumatoid arthritis (IRA). Treatment of mice with recombinant Tnfip6 has been shown to inhibit inflammatory leukocyte infiltration, and to ameliorate joint swelling in murine models of RA. Tnfip6 also provides protection against cartilage degradation through potentiation of the protease inhibitor activity of serum anti-a trypsin inhibitor (lal). These findings clearly suggest that Tnfip6 antagonizes leukocyte influx as well as inflammatory tissue damage. However, the exact mechanisms by which these therapeutic effects of Tnfip6 are achieved in vivo, remain unknown. Our preliminary in vitro studies show that Tnfip6 interacts with the cell surface HA receptor, CD44, and modulates the CD44-dependent rolling of leukocytes on HA. Rolling of CD44-expressing cells on HA-covered microvascular endothelium is a critical step in the process of leukocyte recruitment by which effector cells of the innate and adaptive immune system gain entry into sites of inflammation. Modulation of cell rolling by Tnfip6 in vivo, therefore, could have a negative impact on leukocyte extravasation, leading to resolution of inflammation. We hypothesize that Tnfip6, in particular when administered in pharmacological doses, inhibits leukocyte influx into inflamed synovial joints through interactions with cell surface CD44. We will test this hypothesis by conducting real-time intravital videomicroscopy on the synovial microcirculation of BALB/c mice with proteoglycan-induced arthritis (PGIA), following treatment with recombinant Tnfip6 (Aim 1). We will record and analyze the rolling and adhesion interactions of leukocytes with endothelium in the synovial microvessels of the ankle, a distal joint which is afflicted with inflammation in both PGIA and RA, but has not been accessible to real-time investigations thus far. Requirement for endogenous Tnfip6 and CD44 for down-regulation of inflammatory leukocyte recruitment will be studied using BALB/c mice deficient in Tnfip6 and CD44, respectively (Aims 2 and 3). To validate the in vivo approach, the results of intravital microscopy experiments will be correlated with the morphological features of arthritis. Tnfip6 also participates in the transcriptional regulation of two serine protease inhibitors, distinct from lal, as suggested by the gene expression profile of in vitro-stimulated Tnfip6-deficient cells. One of these antiproteases, secretory leukocyte protease inhibitor (Slpi), has anti-inflammatory properties. We will investigate the regulatory relationship between Tnfip6 and Slpi, and the role of SIpi in arthritis suppression (Aim 4). In summary, Tnfip6 is an arthritis-associated protein, induced by, and antagonistic to, inflammatory processes, Elucidation of the multiple functions of Tnfip6 will help unravel the contribution of this protein to a negative regulatory loop that curtails joint inflammation.