MECHANISMS OF ARTHRITIS SUPPRESSION BY TSG-6 (TNFIP6)

EScription(由申请人提供)： 肿瘤坏死因子α诱导蛋白6(Tnfip6)是肿瘤坏死因子α刺激基因6(TSG-6)的产物，是一种透明质酸(HA)结合蛋白，在类风湿关节炎(IRA)患者的滑液和组织中检测到。重组Tnfip6对小鼠的治疗已被证明可以抑制炎性白细胞的渗透，并改善类风湿关节炎小鼠模型的关节肿胀。Tnfip6还通过增强血清抗α-胰蛋白酶抑制物(LAL)的酶抑制活性来提供对软骨退化的保护。这些发现清楚地表明，Tnfip6可以对抗白细胞内流和炎症组织损伤。然而，Tnfip6在体内实现这些治疗效果的确切机制仍不清楚。我们的初步体外研究表明，Tnfip6与细胞表面HA受体CD44相互作用，并调节依赖CD44的白细胞在HA上的滚动。CD44表达的细胞在HA覆盖的微血管内皮细胞上的滚动是白细胞募集过程中的关键步骤，天然免疫系统和获得性免疫系统的效应细胞通过这一过程进入炎症部位。因此，Tnfip6在体内对细胞滚动的调节可能会对白细胞外渗产生负面影响，从而导致炎症的消退。 我们推测，Tnfip6，特别是在药物剂量下，通过与细胞表面CD44的相互作用，抑制白细胞流入炎症的滑膜关节。我们将通过对患有蛋白多糖诱导的关节炎(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验证这一假设，这些小鼠在接受重组Tnfip6治疗后(目标1)。我们将记录和分析白细胞与踝关节滑膜微血管内皮细胞的滚动和黏附相互作用，踝关节是一个远端关节，在PGIA和RA中都受到炎症的困扰，但到目前为止还无法进行实时研究。内源性Tnfip6和CD44对下调炎性白细胞募集的需求将分别在缺乏Tnfip6和CD44的BALB/c小鼠中进行研究(目标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特征相关联。 根据体外刺激的Tnfip6缺陷细胞的基因表达谱，Tnfip6还参与了两种丝氨酸蛋白酶抑制剂的转录调节，这与LAL不同。分泌型白细胞蛋白水解酶抑制物(SLPI)是其中一种具有抗炎作用的抗蛋白酶。我们将研究Tnfip6和Spli之间的调节关系，以及Sipi在抑制关节炎中的作用(目标4)。 总之，Tnfip6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Tnfip6的多种功能将有助于揭示该蛋白在抑制关节炎症的负调控环路中的作用。

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.