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Generation and characterization of an antagonistic monoclonal antibody against an extracellular domain of mouse DP2 (CRTH2/GPR44) receptors for prostaglandin D2

机译：对抗前列腺素D2的小鼠DP2（CRTH2 / GPR44）受体胞外域的拮抗性单克隆抗体的产生和表征

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Prostaglandin D2 (PGD2) is a lipid mediator involved in sleep regulation and inflammation. PGD2 interacts with 2 types of G protein-coupled receptors, DP1 and DP2/CRTH2 (chemoattractant receptor homologous molecule expressed on T helper type 2 cells)/GPR44 to show a variety of biological effects. DP1 activation leads to Gs-mediated elevation of the intracellular cAMP level, whereas activation of DP2 decreases this level via the Gi pathway; and it also induces G protein-independent, arrestin-mediated cellular responses. Activation of DP2 by PGD2 causes the progression of inflammation via the recruitment of lymphocytes by enhancing the production of Th2-cytokines. Here we developed monoclonal antibodies (MAbs) against the extracellular domain of mouse DP2 by immunization of DP2-null mutant mice with DP2-overexpressing BAF3, murine interleukin-3 dependent pro-B cells, to reduce the generation of antibodies against the host cells by immunization of mice. Moreover, we immunized DP2-KO mice to prevent immunological tolerance to mDP2 protein. After cell ELISA, immunocytochemical, and Western blot analyses, we successfully obtained a novel monoclonal antibody, MAb-1D8, that specifically recognized native mouse DP2, but neither human DP2 nor denatured mouse DP2, by binding to a particular 3D receptor conformation formed by the N-terminus and extracellular loop 1, 2, and 3 of DP2. This antibody inhibited the binding of 0.5 nM [3H]PGD2 to mouse DP2 (IC50 = 46.3 ± 18.6 nM), showed antagonistic activity toward 15(R)-15-methyl PGD2-induced inhibition of 300 nM forskolin-activated cAMP production (IC50 = 16.9 ± 2.6 nM), and gave positive results for immunohistochemical staining of DP2-expressing CD4+ Th2 lymphocytes that had accumulated in the kidney of unilateral ureteral obstruction model mice. This monoclonal antibody will be very useful for in vitro and in vivo studies on DP2-mediated diseases.
机译：前列腺素D2（PGD2）是一种脂质调节剂，参与睡眠调节和炎症。 PGD​​2与2种G蛋白偶联受体DP1和DP2 / CRTH2（在2型辅助性T细胞上表达的化学吸引受体同源分子）/ GPR44相互作用，显示出多种生物学效应。 DP1激活导致Gs介导的细胞内cAMP水平升高，而DP2激活通过Gi途径降低该水平。并且还诱导非G蛋白依赖性，抑制蛋白介导的细胞反应。 PGD​​2对DP2的激活通过增强Th2细胞因子的产生，通过募集淋巴细胞引起炎症的发展。在这里，我们通过用过量表达DP2的BAF3（鼠白细胞介素3依赖性pro-B细胞）免疫DP2空突变小鼠，开发了针对小鼠DP2胞外域的单克隆抗体（MAbs），以减少通过抗宿主细胞产生的抗体小鼠免疫。此外，我们免疫了DP2-KO小鼠，以防止对mDP2蛋白的免疫耐受。经过细胞ELISA，免疫细胞化学和Western印迹分析后，我们成功地获得了一种新的单克隆抗体MAb-1D8，该抗体通过结合由小鼠DP2形成的特定3D受体构象而特异性识别天然小鼠DP2，但既不识别人DP2，也不识别变性小鼠DP2。 DP2的N末端和细胞外环1、2和3。该抗体抑制0.5 nM [ 3 H] PGD2与小鼠DP2的结合（IC50 = 46.3±18.6 nM），对15（R）-15-甲基PGD2诱导的300抑制作用表现出拮抗活性。 nM forskolin激活的cAMP产生（IC50 = 16.9±2.6 nM），并为单侧输尿管梗阻模型小鼠肾脏中积累的表达DP2的CD4 + Th2淋巴细胞的免疫组织化学染色提供了阳性结果。这种单克隆抗体对于DP2介导的疾病的体外和体内研究将非常有用。

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