Noraz N, Gozlan J, Corbeil J, Brunner T, Spector S A
Department of Pediatrics, University of California San Diego, La Jolla 92093-0672, USA.
AIDS. 1997 Nov 15;11(14):1671-80. doi: 10.1097/00002030-199714000-00003.
To investigate the role of the Fas-Fas ligand (FasL) interaction in HIV-1-induced apoptosis of primary CD4+ T lymphocytes.
Activated CD4+ T lymphocytes are the main target of HIV, and T-cell activation leads to the expression of Fas-FasL and enhances HIV-mediated apoptosis. Phytohemagglutinin-activated primary CD4+ T cells were infected with HIV; the process of cell death was examined, and whether the dying and dead cells were the productively infected cells. The modulation of Fas and FasL expression and its role in HIV-induced cell death was also investigated.
The number of viable and dead cells was determined by trypan blue exclusion. Apoptosis was quantified using an enzyme-linked immunosorbent assay measuring the release of cytoplasmic histone-associated DNA fragments. The percentage of HIV-infected cells was determined by FACS analysis, and viral production was assessed by a p24 core antigen assay. The following three markers, HIV-gp-120, annexin-V and 7-AAD, were used to monitor the apoptotic process in HIV-negative and positive cells. Fas and FasL expression was analyzed at the RNA level by reverse transcription polymerase chain reaction and at the protein level by flow cytometry. The contribution of Fas-FasL interactions to apoptosis was examined by blocking experiments using the antagonist ZB4 anti-Fas antibody.
HIV-induced apoptosis in activated purified CD4+ T lymphocytes required infectious virus and was dose-dependent. Apoptosis in HIV-infected cultures was mostly confined to productively infected cells. The expression of Fas and FasL was not significantly modulated by infection and blocking Fas-FasL interactions did not reduce the extent of apoptosis.
HIV-induced apoptosis of activated CD4+ T cells in vitro is confined to productively infected cells and is not mediated by a Fas-FasL interaction.
研究Fas-Fas配体（FasL）相互作用在HIV-1诱导的原代CD4+ T淋巴细胞凋亡中的作用。
活化的CD4+ T淋巴细胞是HIV的主要靶细胞，T细胞活化导致Fas-FasL表达并增强HIV介导的凋亡。用植物血凝素活化的原代CD4+ T细胞感染HIV；检测细胞死亡过程，以及濒死和死亡细胞是否为高效感染细胞。还研究了Fas和FasL表达的调节及其在HIV诱导的细胞死亡中的作用。
用台盼蓝排斥法测定活细胞和死细胞数量。使用酶联免疫吸附测定法测量细胞质组蛋白相关DNA片段的释放来定量凋亡。通过流式细胞术分析确定HIV感染细胞的百分比，并通过p24核心抗原测定评估病毒产生。使用HIV-gp-120、膜联蛋白-V和7-氨基放线菌素D这三种标志物监测HIV阴性和阳性细胞中的凋亡过程。通过逆转录聚合酶链反应在RNA水平和通过流式细胞术在蛋白质水平分析Fas和FasL表达。使用拮抗剂ZB4抗Fas抗体通过阻断实验研究Fas-FasL相互作用对凋亡的作用。
HIV诱导活化的纯化CD4+ T淋巴细胞凋亡需要感染性病毒，且呈剂量依赖性。HIV感染培养物中的凋亡主要局限于高效感染细胞。感染对Fas和FasL的表达没有明显调节作用，阻断Fas-FasL相互作用也没有降低凋亡程度。
HIV体外诱导活化的CD4+ T细胞凋亡局限于高效感染细胞，且不是由Fas-FasL相互作用介导的。
