Sulfotransferases in the Synthesis of L-Selectin Ligands

描述（由申请人提供）：淋巴细胞从血液中回家进入淋巴结的过程中淋巴细胞再循环。归巢是由淋巴细胞在淋巴结内高内皮小静脉（HEVs）上的滚动引起的。这一步骤由l -选择素介导，l -选择素是一种c型凝集素，可识别hev上的一组碳水化合物基配体。一种名为MECA-79的功能阻断单抗识别相同的复合物，称为pnas。配体是由GlcNAc-6-sulfate， sialyl Lewis x和Gal-6-sulfate修饰的唾液粘蛋白。我们研究了双敲除（DKO）小鼠，其中存在于hev中的两种glcnac -6- o -硫转移酶已失活。这些小鼠表现出75%的淋巴结归巢减少和hev中PNAd的完全消除（MECA-79染色的完全丧失）。我们将研究gal -6-硫酸盐修饰对DKO小鼠hev上“残留”配体活性的影响，以及glcnac -6- o -硫转移酶完好无损的小鼠（Aim 1）。类风湿性关节炎（RA）患者的关节组织中存在共表达glcnac -6- o-磺基转移酶的PNAd+血管。这一发现在三种机械上不同的小鼠炎性关节炎模型中得到了概括。我们已经鉴定了一种新的抗体，称为克隆40，与MECA-79具有非常相似的结合特性，但更适合用于动物研究。我们将利用DKO小鼠和克隆40在小鼠关节炎模型中研究硫转移酶及其硫酸化产物在疾病发病机制中的作用（目的2）。由于一些小鼠模型对有效的人类治疗方法（如抗TNF-1治疗）具有很强的预测性，我们的工作可能会导致治疗RA的新方法。我们还将研究绵羊哮喘模型，其中l -选择素似乎发挥了高度新颖的作用（目的3）。我们的实验表明，气道外渗的白细胞可以利用l -选择素与炎症气道中表达的硫酸粘蛋白配体发生反应。这种相互作用导致白细胞的激活，从而导致气道阻力和气道反应性增加，这是哮喘的两个标志。为了评估这一假设，我们将确定分离的气道粘蛋白是否可以通过与l -选择素结合激活中性粒细胞并引起支气管活性物质的分泌。最后，我们发现肺中的气道粘蛋白是siglece -8的配体，siglece -8是一种已知能结合硫酸和唾液化糖的受体。这种Siglec存在于嗜酸性粒细胞上，通过抗体人工交联可以诱导细胞凋亡。我们将确定这些粘蛋白是否是siglece -8的天然配体，用于在嗜酸性粒细胞上交联siglece -8，从而触发这些白细胞的凋亡（目的4）。这将为清除在过敏性疾病（如哮喘气道）中积累的嗜酸性粒细胞提供一种稳态控制机制。了解这一机制可能会带来抑制嗜酸性粒细胞反应的新药理学方法。

DESCRIPTION (provided by applicant): Lymphocytes home from the blood into lymph nodes during the process of lymphocyte recirculation. Homing is initiated by the rolling of lymphocytes on high endothelial venules (HEVs) within the lymph node. This step is mediated by L-selectin, a C-type lectin, which recognizes a set of carbohydrate-based ligands on HEVs. A function-blocking mAb called MECA-79 recognizes the same complex, referred to as PNAd. The ligands are sialomucins modified by GlcNAc-6-sulfate, sialyl Lewis x, and Gal-6-sulfate. We have studied double knockout (DKO) mice in which two GlcNAc-6-O-sulfotransferases that are present in HEVs have been inactivated. These mice exhibit a 75% reduction in homing to lymph nodes and the total elimination of PNAd (complete loss of MECA-79 staining) from HEVs. We will investigate the possibility that Gal-6-sulfate modifications contribute to the "residual" ligand activity on HEVs in DKO mice, as well in mice in which GlcNAc-6-O-sulfotransferases are intact (Aim 1). PNAd+ blood vessels, which co-express GlcNAc-6-O-sulfotransferase, are present in joint tissues of rheumatoid arthritis (RA) patients. This finding is recapitulated in three mechanistically-distinct models of inflammatory arthritis in mouse. We have characterized a new antibody, called Clone 40, with very similar binding properties as MECA-79 but more suitable for use in animal studies. We will employ the DKO mice, together with Clone 40, in the mouse models of arthritis to study the role of the sulfotransferases and their sulfated products in disease pathogenesis (Aim 2). Since some murine models have been very predictive of efficacious human therapeutics (e.g., anti TNF-1 therapy), our work may lead to new approaches for the treatment of RA. We will also investigate a sheep model of asthma in which L-selectin appears to play a highly novel role (Aim 3). Our experiments suggest that extravasated leukocytes in the airways can utilize L-selectin to react with sulfated mucin ligands, which are expressed in inflamed airways. This interaction leads to activation of the leukocytes, which results in an increase in airway resistance and airway responsiveness, two hallmarks of asthma. To evaluate this hypothesis, we will determine whether isolated airway mucins can activate neutrophils through binding to L-selectin and cause the secretion of broncho-active substances. Finally, we have discovered that airway mucins in lungs are ligands for Siglec-8, a receptor known to bind sulfated and sialylated sugars. This Siglec is present on eosinophils and can induce apoptosis when artificially cross-linked by antibodies. We will determine whether these mucins are natural ligands for Siglec-8, serving to crosslink Siglec-8 on eosinophils and thus triggering apoptosis of these leukocytes (Aim 4). This would provide a homeostatic control mechanism for removing eosinophils that accumulate in allergic diseases, such as in asthmatic airways. Understanding this mechanism may lead to new pharmacologic approaches for dampening eosinophil responses.