Sasaki Fumiyuki, Koga Tomoaki, Saeki Kazuko, Okuno Toshiaki, Kazuno Saiko, Fujimura Tsutomu, Ohkawa Yasuyuki, Yokomizo Takehiko
Department of Biochemistry, Juntendo University School of Medicine, Tokyo, Japan.
Laboratory of Proteomics and Biomolecular Science Research Support Center, Juntendo University Graduate School of Medicine, Tokyo, Japan.
PLoS One. 2017 Sep 18;12(9):e0185133. doi: 10.1371/journal.pone.0185133. eCollection 2017.
Leukotriene B4 (LTB4) receptor 1 (BLT1) is a G protein-coupled receptor expressed in various leukocyte subsets; however, the precise expression of mouse BLT1 (mBLT1) has not been reported because a mBLT1 monoclonal antibody (mAb) has not been available. In this study, we present the successful establishment of a hybridoma cell line (clone 7A8) that produces a high-affinity mAb for mBLT1 by direct immunization of BLT1-deficient mice with mBLT1-overexpressing cells. The specificity of clone 7A8 was confirmed using mBLT1-overexpressing cells and mouse peripheral blood leukocytes that endogenously express BLT1. Clone 7A8 did not cross-react with human BLT1 or other G protein-coupled receptors, including human chemokine (C-X-C motif) receptor 4. The 7A8 mAb binds to the second extracellular loop of mBLT1 and did not affect LTB4 binding or intracellular calcium mobilization by LTB4. The 7A8 mAb positively stained Gr-1-positive granulocytes, CD11b-positive granulocytes/monocytes, F4/80-positive monocytes, CCR2-high and CCR2-low monocyte subsets in the peripheral blood and a CD4-positive T cell subset, Th1 cells differentiated in vitro from naïve CD4-positive T cells. This mAb was able to detect Gr-1-positive granulocytes and monocytes in the spleens of naïve mice by immunohistochemistry. Finally, intraperitoneal administration of 7A8 mAb depleted granulocytes and monocytes in the peripheral blood. We have therefore succeeded in generating a high-affinity anti-mBLT1 mAb that is useful for analyzing mBLT1 expression in vitro and in vivo.
白三烯B4（LTB4）受体1（BLT1）是一种在多种白细胞亚群中表达的G蛋白偶联受体；然而，由于尚未获得小鼠BLT1（mBLT1）单克隆抗体（mAb），mBLT1的精确表达情况尚未见报道。在本研究中，我们展示了通过用mBLT1过表达细胞直接免疫BLT1缺陷小鼠，成功建立了一种杂交瘤细胞系（克隆7A8），该细胞系可产生针对mBLT1的高亲和力mAb。使用mBLT1过表达细胞和内源性表达BLT1的小鼠外周血白细胞证实了克隆7A8的特异性。克隆7A8与人BLT1或其他G蛋白偶联受体，包括人趋化因子（C-X-C基序）受体4无交叉反应。7A8 mAb与mBLT1的第二个细胞外环结合，且不影响LTB4的结合或LTB4介导的细胞内钙动员。7A8 mAb对外周血中Gr-1阳性粒细胞、CD11b阳性粒细胞/单核细胞、F4/80阳性单核细胞、CCR2高表达和CCR2低表达单核细胞亚群以及一个CD4阳性T细胞亚群（从初始CD4阳性T细胞体外分化而来的Th1细胞）呈阳性染色。通过免疫组织化学，该mAb能够检测到未免疫小鼠脾脏中的Gr-1阳性粒细胞和单核细胞。最后，腹腔注射7A8 mAb可使外周血中的粒细胞和单核细胞减少。因此，我们成功制备了一种高亲和力抗mBLT1 mAb，其可用于体外和体内分析mBLT1的表达。
