Landells L J, Szilagy C M, Jones N A, Banner K H, Allen J M, Doherty A, O'Connor B J, Spina D, Page C P
Sackler Institute of Pulmonary Pharmacology, Division of Pharmacology and Therapeutics, King's College London, 5th Floor, Hodgkin Building, Guy's Campus, London SE1 9RT.
Br J Pharmacol. 2001 Jul;133(5):722-9. doi: 10.1038/sj.bjp.0704120.
In the present study, for the first time, PDE4 subtypes were identified and semi-quantified in both CD4 and CD8 lymphocytes from healthy and asthmatic individuals. CD4 and CD8 lymphocytes from healthy and mild asymptomatic asthmatic subjects (receiving beta-agonist therapy only) were isolated from peripheral venous blood using appropriate antibody coated paramagnetic beads. PDE4 subtypes and beta-actin were identified by digoxigenin (DIG)-labelling reverse transcriptase-polymerase chain reaction and semi-quantified by DIG-detection enzyme-linked immunosorbance assay. In CD4 and CD8 lymphocytes PDE4A, PDE4B and PDE4D were detected, with no significant differences observed between healthy and asthmatic groups. In CD8 lymphocytes, enzyme subtype expression was lower and showed more intersubject variability. In functional studies investigating the effects of various PDE inhibitors on PHA-induced proliferation of mononuclear cells from healthy and asthmatic subjects, CDP840 (0.03 - 10 microM), rolipram (0.1 - 10 microM) and theophylline (10 microM - 1 mM) inhibited PHA-induced proliferation of mononuclear cells from healthy and asthmatic subjects in a concentration-dependent manner, although no significant difference was observed between the groups investigated. In additional studies, total monocyte cyclic AMP PDE activity was investigated in cells isolated from asthmatic subjects both prior to and 24 h after allergen challenge. Total monocyte cyclic AMP PDE activity remained unaffected following challenge of asthmatic subjects with either house dust mite or cat dander and was inhibited in a concentration-dependent manner by rolipram (0.01 - 100 microM) both before and after allergen challenge.
在本研究中，首次在健康个体和哮喘患者的CD4和CD8淋巴细胞中鉴定并半定量了磷酸二酯酶4（PDE4）亚型。使用适当的抗体包被顺磁珠从外周静脉血中分离出健康和轻度无症状哮喘患者（仅接受β-激动剂治疗）的CD4和CD8淋巴细胞。通过地高辛（DIG）标记逆转录聚合酶链反应鉴定PDE4亚型和β-肌动蛋白，并通过DIG检测酶联免疫吸附测定进行半定量。在CD4和CD8淋巴细胞中检测到PDE4A、PDE4B和PDE4D，健康组和哮喘组之间未观察到显著差异。在CD8淋巴细胞中，酶亚型表达较低，且个体间变异性更大。在研究各种PDE抑制剂对健康和哮喘患者外周血单核细胞由植物血凝素（PHA）诱导的增殖作用的功能研究中，CDP840（0.03 - 10 microM）、咯利普兰（0.1 - 10 microM）和茶碱（10 microM - 1 mM）以浓度依赖性方式抑制健康和哮喘患者外周血单核细胞由PHA诱导的增殖，尽管在所研究的组之间未观察到显著差异。在进一步的研究中，在变应原激发前和激发后24小时，对从哮喘患者分离的细胞中的总单核细胞环磷酸腺苷（cAMP）磷酸二酯酶活性进行了研究。用屋尘螨或猫毛屑激发哮喘患者后，总单核细胞cAMP磷酸二酯酶活性未受影响，并且在变应原激发前后，咯利普兰（0.01 - 100 microM）均以浓度依赖性方式抑制该活性。
