Data_Sheet_1_Human Peripheral Blood Eosinophils Express High Levels of the Purinergic Receptor P2X4.pdf

Extracellular nucleotides are important mediators of cell activation and trigger multiple responses via membrane receptors known as purinergic receptors (P2). P2X receptors are ligand-gated ion channels, activated by extracellular ATP. P2X4 is one of the most sensitive purinergic receptors, that is typically expressed by neurons, microglia, and some epithelial and endothelial cells. P2X4 mediates neuropathic pain via brain-derived neurotrophic factor and is also involved in inflammation in response to high ATP release. It is therefore involved in multiple inflammatory pathologies as well as neurodegenerative diseases. We have produced monoclonal antibodies (mAb) directed against this important human P2X4 receptor. Focusing on two mAbs, we showed that they also recognize mouse and rat P2X4. We demonstrated that these mAbs can be used in flow cytometry, immunoprecipitation, and immunohistochemistry, but not in Western blot assays, indicating that they target conformational epitopes. We also characterized the expression of P2X4 receptor on mouse and human peripheral blood lymphocytes (PBL). We showed that P2X4 is expressed at the surface of several leukocyte cell types, with the highest expression level on eosinophils, making them potentially sensitive to adenosine triphosphate (ATP). P2X4 is expressed by leucocytes, in human and mouse, with a significant gender difference, males having higher surface expression levels than females. Our findings reveal that PBL express significant levels of P2X4 receptor, and suggest an important role of this receptor in leukocyte activation by ATP, particularly in P2X4high expressing eosinophils.

细胞外核苷酸是重要的细胞活化介质，可通过名为嘌呤能受体（P2）的膜受体触发多种细胞应答反应。P2X受体属于配体门控离子通道，可被细胞外三磷酸腺苷（ATP）激活。P2X4是敏感性最高的嘌呤能受体之一，通常由神经元、小胶质细胞以及部分上皮细胞和内皮细胞表达。P2X4可通过脑源性神经营养因子介导神经病理性疼痛，同时也参与高ATP释放引发的炎症反应，因此该受体既参与多种炎症性病理过程，也与神经退行性疾病密切相关。
我们制备了针对这一重要人源P2X4受体的单克隆抗体（mAb）。针对其中两株单克隆抗体的研究证实，它们同样可识别小鼠和大鼠的P2X4受体。实验表明，这些单克隆抗体可应用于流式细胞术、免疫沉淀及免疫组织化学实验，但无法用于蛋白质免疫印迹（Western blot）实验，提示其靶向的为构象表位。
我们还对小鼠和人外周血淋巴细胞（PBL）表面的P2X4受体表达情况进行了表征。结果显示，多种白细胞亚型的细胞膜表面均表达P2X4受体，其中嗜酸性粒细胞的表达水平最高，这使其对三磷酸腺苷（ATP）具有潜在应答敏感性。在人和小鼠体内，白细胞均表达P2X4受体，且该受体的膜表面表达水平存在显著的性别差异：雄性个体的表达水平高于雌性。
本研究结果表明，外周血淋巴细胞可表达足量的P2X4受体，同时提示该受体在ATP介导的白细胞活化过程中发挥重要作用，尤其是在高表达P2X4的嗜酸性粒细胞中。