SELECTIN BINDING SITES ON LEUKOCYTES AND INFLAMED VENULES

该实验室最近的研究表明，这三个成员都与 选择素(LEC-CAM)家族与单核细胞-内皮细胞中的β2-整合素 类风湿性关节炎(RA)中的粘附性相互作用。单抗转P- 选择素稳定地抑制90%的黏附，而单抗对E-和L- 选择素的抑制率高达50%。除了LFA-1、MO1/MAC1和它们的单抗 常见的β2链抑制率为30-50%。这项工作构成了第一个直接 有证据表明选择素参与了类风湿关节炎患者的白细胞募集。这 一节包含三个相互关联的项目，探讨 单核细胞-微血管相互作用中的选择素。具体目标#1重点 单核细胞和细胞系中E-和P-选择素配体的研究。这个 表达与或含有两个碳水化合物表位的糖蛋白 在选择素结合部位(CSLEX-1和HECA-452)将被分离并 特色化的。它们与含有细胞的融合蛋白的相互作用 然后将建立E-选择素和P-选择素的结合域。在……里面 此外，还将使用转染体来确定后 翻译糖基化引入E-和P-选择素结合位点 L-选择素。Lowe博士(第1节)将转导全长cDNA编码 L-选择素导入几种能产生配体的CHO转染体中 E和P-选择素。一株转L-选择素的U937细胞株也将 测试过。然后检查转染体中的L-选择素 E-和P-选择素结合位点的表达。具体目标#2定义了 滑膜上单核细胞L-选择素和β2-整合素的结合部位 类风湿关节炎的微血管构筑。L的主要内皮细胞拮抗剂- 选择素被认为是唾液酸化、岩藻糖化的O-连接簇 和硫酸低聚糖。我们将使用IgG1-L-选择素嵌合体和 免疫金标记技术来确定这是或结构上的 相关的寡糖在滑膜小静脉上表达。此外, 微血管脱落术对两者附着性的影响 将测定单核细胞和免疫球蛋白1-L-选择素嵌合体。可能的 唾液酸化Lewis X结构与单核细胞L-选择素的相互作用 内源性E-选择素和P-选择素将使用冰冻切片进行探索 黏附试验。相关实验将确定Beta2- 初步研究中检测到的整合素依赖黏附反映了 与ICAM-1或滑膜小静脉上的内皮选择素结合。这个 β2-整合素的主要内皮对抗性受体是ICAM- 1.然而，这些受体也包含一个低亲和力的结合位点 P-选择素，lewisX表位(CD15)，是潜在的载体 唾液酸化的Lewis X结构。因此，β2整合素可以与 ICAM-1和选择素均表达于滑膜小静脉表面。特定目标 #3将评估选择素抑制剂的特异性和有效性 在第1节合成的抗人选择素。他们的活动将是 选择素特异性冰冻切片检测、流式细胞仪检测的比较 在一种允许评估白细胞与内皮细胞粘附性的试验中 在生理剪应力水平上。本部分的目标是 确定抑制剂的特异性，并确定哪些检测是 有可能预测体内的疗效。

Recent studies from this laboratory implicate all three members of the selectin (LEC-CAM) family and the beta2-integrins in monocyte-endothelial adhesive interactions in human rheumatoid arthritis (RA). Mabs to P- selectin consistently inhibit > 90% of adhesion while Mabs to E- and L- selectin inhibit up to 50%. In addition Mabs to LFA-1, Mo1/Mac1 and their common beta2-chain inhibit 30-50%. This work constitutes the first direct evidence that selectins participate in leukocyte recruitment in RA. This section contains three inter-related projects exploring the role of selectins in monocyte-microvascular interactions. Specific aim #1 focuses on the ligands for E- and P-selectin in monocytes and cell lines. the glycoproteins expressing two carbohydrate epitopes linked to or contained in the selectin binding sites (CSLEX-1 and HECA-452) will be isolated and characterized. Their interactions with fusion proteins containing the cell binding domains of E- and P-selectin will then be established. In addition, transfectants will be used to determine whether post- translational glycosylation introduces E- and P-selectin binding sites into L-selectin. Dr. Lowe (Section 1) will transfect full length cDNAs encoding L-selectin into several CHO-transfectants capable of generating ligands for E and P-selectins. A U937 line transfected with L-selectin will also be tested. L-selectin in the transfectants will then be examined for expression of E- and P-selectin binding sites. Specific aim #2 defines the binding sites for monocytic L-selectin and beta2-integrins on synovial microvasculature in RA. The principle endothelial counter-receptor for L- selectin is thought to be an O-linked cluster of sialylated, fucosylated and sulfated oligosaccharides. We will use an IgG1-L-selectin chimera and immunogold labelling techniques to determine whether this or structurally related oligosaccharide are expressed on synovial venules. In addition, the effect of microvascular desialylation on the attachment of both monocytes and the IgG1-L-selectin chimera will be determined. The possible interaction between sialylated Lewisx structures on monocytic L-selectin and endogenous E- and P-selectin will be explored using the frozen section adhesion assay. Related experiments will determine whether the beta2- integrin dependent adhesion detected in preliminary studies reflects binding to ICAM-1 or to the endothelial selectins on synovial venules. The principle endothelial counter-receptor for the beta2-integrins is the ICAM- 1. However, these receptors also contain a low affinity binding site for P-selectin, the Lewisx epitope (CD15), and are potential carriers of the sialylated Lewisx structure. Thus the beta2-integrins may interact with both ICAM-1 and selectins on the surface of synovial venules. Specific aim #3 will evaluate the specificity and potency of the selectin inhibitors synthesized in section 1 against human selectins. Their activities will be compared in selectin-specific frozen section assays, flow cytometric assays and in an assay which permits evaluation of leukocyte-endothelial adhesion at physiologic levels of shear-stress. The goal of this section is to determine the specificity of the inhibitors and identify assays which are likely to predict efficacy in vivo.