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

描述(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。虽然普遍认为G-CSF是一种比GRAGE更安全的选择，但越来越多的观察表明，G-CSF的应用会由于白细胞与血管内皮细胞的粘附性增强而导致血管和炎症并发症。白细胞-内皮黏附相互作用的主要介体是E-选择素，这是一种可诱导的内皮分子，它结合由单抗HECA-452识别的唾液酸糖基化的碳水化合物抗原。我们推测，血管和炎症并发症可能是由于G-CSF诱导循环白细胞上E-选择素配体的表达所致。我们发现P-选择素糖蛋白配体-1(PSGL-1)和造血细胞E-和L-选择素配体(HCELL)是G-CSF MPB白细胞上的E-选择素配体。重要的是，我们的研究还发现，一种新的HECA-452反应性~65 kDa高亲和力E-选择素配体(以下简称~65 kDa蛋白)在G-CSF MPB白细胞上表达，而在天然白细胞和正常人骨髓细胞上不表达。这个~65 kDa的蛋白显示出强大的钙依赖的E-选择素配体活性，这从(I)与流体相的E-选择素结合和(Ii)在生理剪切流条件下E-选择素转基因细胞的亲和结合中得到证明。体外用药动学相关浓度的G-CSF处理正常人骨髓细胞，可直接在髓系细胞上诱导该~65 kDa蛋白的表达。我们推测这个~65 kDa的E-选择素配体与G-CSF应用后的血管和炎症并发症有关。本方案的具体目的是：(1)鉴定G-CSFMPB白细胞上作为E-选择素配体的~65 kDa蛋白；(2)鉴定体内和体外注射G-CSF后表达这一糖蛋白的人白细胞系细胞亚群(S)。这种新的E-选择素配体的鉴定和表达特性将有助于深入了解E-选择素配体的生物学特性，并极大地增加我们对G-CSF、MPB、白细胞与内皮细胞相互作用的理解。预计所获得的结果将导致预防或减轻G-CSF引起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