SELECTIN BINDING SITES ON LEUKOCYTES AND INFLAMED VENULES

该实验室最近的研究表明， 选择素（LEC-CAM）家族和β 2-整合素在单核细胞-内皮细胞 类风湿性关节炎（RA）中的粘附相互作用。 单克隆抗体对P- 选择素始终抑制> 90%的粘附，而单克隆抗体对E-和L- 选择素抑制高达50%。 除了针对LFA-1、Mo 1/Mac 1及其 普通β 2链抑制30- 50%。 这项工作构成了第一个直接 证据表明，选择素参与RA白细胞募集。 这 部分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 对单核细胞和细胞系中E-和P-选择素配体的影响。 的 表达两个糖表位的糖蛋白 将分离选择素结合位点（CSLEX-1和HECA-452）中的蛋白质， 表征了 它们与含有细胞的融合蛋白的相互作用 然后建立E-和P-选择素的结合结构域。 在 此外，转染子将用于确定是否后- 翻译糖基化将E-和P-选择素结合位点引入 L-选择素 Lowe博士（第1节）将检测全长cDNA编码 将L-选择素导入几种能够产生配体的CHO-转染子中， E和P-选择素。 还将用L-选择素转染的U937系进行转染。 测试. 然后检查转染子中的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β 2-整合素的结合位点 RA微血管 内皮细胞的L-受体 选择素被认为是唾液酸化的、岩藻糖基化的 和硫酸化低聚糖。 我们将使用IgG 1-L-选择素嵌合体， 免疫金标记技术，以确定是否这种或结构 相关的寡糖在滑液小静脉上表达。 此外，本发明还提供了一种方法， 微血管去唾液酸化对两者附着的影响 将测定单核细胞和IgG 1-L-选择素嵌合体。 可能的 单核细胞L-选择素上唾液酸化Lewisx结构相互作用 和内源性E-和P-选择素将使用冷冻切片进行探索 粘附测定 相关实验将确定β 2- 在初步研究中检测到的整合素依赖性粘附反映了 与ICAM-1或滑膜微静脉上的内皮选择素结合。 的 β 2-整联蛋白的主要内皮反受体是ICAM-1。 1. 然而，这些受体也含有低亲和力结合位点， P-选择素，Lewisx表位（CD 15），是潜在的载体， 唾液酸化Lewisx结构。 因此，β 2-整联蛋白可以与 ICAM-1和选择素均表达于滑膜微静脉表面。 具体目标 #3将评价选择素抑制剂的特异性和效力 在第1节中针对人选择素合成。 他们的活动将是 在选择素特异性冰冻切片测定、流式细胞术测定中比较， 并且在允许评价白细胞-内皮粘附的测定中 在生理水平的剪切应力下 本节的目标是 确定抑制剂的特异性，并确定 可能预测体内功效。

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.