Sulfotransferases in the Synthesis of L-Selectin Ligands

描述（由申请方提供）：淋巴细胞在淋巴细胞再循环过程中从血液返回淋巴结。归巢是由淋巴结内高内皮微静脉（HEV）上淋巴细胞的滚动启动的。这一步是由L-选择素介导的，L-选择素是一种C型凝集素，它识别HEV上的一组基于碳水化合物的配体。一种称为MECA-79的功能阻断mAb识别相同的复合物，称为PNAd。配体是由GlcNAc-6-硫酸盐、唾液酸刘易斯x和Gal-6-硫酸盐修饰的唾液粘蛋白。我们研究了双敲除（DKO）小鼠，其中存在于HEV中的两种GlcNAc-6-O-磺基转移酶已被灭活。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染色完全丧失）。我们将研究Gal-6-硫酸修饰对DKO小鼠以及GlcNAc-6-O-磺基转移酶完整的小鼠中HEV上的“残留”配体活性有贡献的可能性（目的1）。PNAd+血管共表达GlcNAc-6-O-磺基转移酶，存在于类风湿性关节炎（RA）患者的关节组织中。这一发现在小鼠炎性关节炎的三种机械上不同的模型中得到了概括。我们已经表征了一种新的抗体，称为克隆40，具有与MECA-79非常相似的结合特性，但更适合用于动物研究。我们将在关节炎小鼠模型中使用DKO小鼠和克隆40，以研究磺基转移酶及其硫酸化产物在疾病发病机制中的作用（目的2）。由于一些鼠模型对有效的人治疗剂具有很强的预测性（例如，抗TNF-1治疗），我们的工作可能会导致新的方法来治疗RA。我们还将研究绵羊哮喘模型，其中L-选择素似乎发挥了非常新颖的作用（目的3）。我们的实验表明，在气道外渗的白细胞可以利用L-选择素与硫酸粘蛋白配体，这是在发炎的气道表达。这种相互作用导致白细胞活化，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，我们将确定是否孤立的气道粘蛋白可以激活中性粒细胞通过结合L-选择素，并导致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Siglec-8的配体，Siglec-8是一种已知结合硫酸化和唾液酸化糖的受体。这种Siglec存在于嗜酸性粒细胞上，并且当通过抗体人工交联时可以诱导细胞凋亡。我们将确定这些粘蛋白是否是Siglec-8的天然配体，用于交联嗜酸性粒细胞上的Siglec-8，从而触发这些白细胞的凋亡（目的4）。这将提供一种稳态控制机制，用于清除在过敏性疾病中（如哮喘气道中）积聚的嗜酸性粒细胞。了解这一机制可能会导致新的药理学方法来抑制嗜酸性粒细胞的反应。

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.