Characterization of A Novel 65kDa E-selectin Ligand on G-CSF Mobilized Leukocytes

描述（由申请人提供）：粒细胞集落刺激因子（G-CSF）动员的外周血（MPB）已取代传统的骨髓采集，成为造血干细胞移植的首选细胞来源。虽然一般认为G-CSF是收获的一种更安全的替代方法，但越来越多的观察结果表明，由于白细胞与血管内皮的粘附增强，G-CSF给药会导致血管和炎症并发症。白细胞-内皮粘附相互作用的主要介质是E-选择素，其是一种可诱导的内皮分子，可结合mAb HECA-452识别的唾液酸岩藻糖基化碳水化合物抗原。我们推断，血管和炎症并发症可能是由于G-CSF诱导的E-选择素配体在循环白细胞上的表达。我们发现P-选择素糖蛋白配体-1（P-selectin glycoprotein ligand-1，PSGL-1）和造血细胞E-和L-选择素配体（Hematopoietic Cell E-and L-selectin Ligand，HCELL）是G-CSF MPB白细胞上的E-选择素配体。重要的是，我们的研究还揭示了一种新的HECA-452反应性~ 65 kDa高亲和力E-选择素配体（下文称为“~ 65 kDa蛋白”）在G-CSF MPB白细胞上表达，但不在天然白细胞上表达，也不在正常人骨髓细胞上表达。这种~ 65 kDa的蛋白质表现出有效的Ca 2+依赖性E-选择素配体活性，如通过（i）与流体相E-选择素结合和（ii）在生理剪切流条件下E-选择素转染的细胞的亲和结合所证明的。在体外用药代动力学相关浓度的G-CSF处理正常人骨髓细胞可直接诱导该~ 65 kDa蛋白在髓样细胞上表达。我们推测，这种~ 65 kDa的E-选择素配体有助于G-CSF给药后的血管和炎症并发症。本提案的具体目的是：（1）鉴定作为G-CSF MPB白细胞上的E-选择素配体的~ 65 kDa蛋白;和（2）鉴定体内和体外给予G-CSF后表达该糖蛋白的人白细胞系细胞亚群。这种新的E-选择素配体的鉴定和表达的表征应该提供对E-选择素配体的生物学的见解，并大大增加我们对G-CSF MPB白细胞-内皮细胞相互作用的理解。预期所获得的结果将导致预防或减轻G-CSF诱导的血管和炎症并发症的新方法

DESCRIPTION (provided by applicant): Granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood (MPB) has replaced traditional bone marrow harvest as the preferred source of cells for hematopoietic stem cell transplantation. Though generally considered to be a safer alternative to harvest, there are increasing observations that G-CSF administration causes vascular and inflammatory complications due to enhanced adhesion of leukocytes to vascular endothelium. A major mediator of leukocyte-endothelial adhesive interactions is E-selectin, an inducible endothelial molecule that binds sialofucosylated carbohydrate antigens recognized by the mAb HECA-452. We reasoned that vascular and inflammatory complications may result from G-CSF-induced expression of E-selectin ligands on circulating leukocytes. We have found that P-selectin glycoprotein ligand-1 (PSGL-1) and Hematopoietic Cell E-and L-selectin Ligand (HCELL) are E-selectin ligands on G- CSF MPB leukocytes. Importantly, our studies also reveal that a novel HECA-452-reactive ~65kDa high affinity E-selectin ligand (hereinafter called "~65kDa protein") is expressed on G-CSF MPB leukocytes but not on native leukocytes and not on normal human bone marrow cells. This ~65kDa protein exhibits potent Ca2+-dependent E-selectin ligand activity, as evidenced by (i) binding to fluid phase E-selectin and (ii) avid binding of E-selectin- transfected cells under physiological shear flow conditions. Treatment of normal human bone marrow cells with G-CSF at a pharmacokinetically-relevant concentration in vitro induces the expression of this ~65kDa protein directly on myeloid cells. We hypothesize that this ~65kDa E-selectin ligand contributes to vascular and inflammatory complications following G-CSF administration. The specific aims of this proposal are: (1) To identify the ~65kDa protein serving as an E-selectin ligand on G-CSF MPB leukocytes; and (2) To identify the subset(s) of human leukocyte-series cells that express this glycoprotein following G-CSF administration in vivo and in vitro. The identification and the characterization of expression of this novel E-selectin ligand should provide insights into the biology of E-selectin ligands and greatly increase our understanding of G-CSF MPB leukocyte-endothelial interactions. It is anticipated that the results obtained will lead to new approaches for preventing or alleviating G-CSF-induced vascular and inflammatory complications