Implications of the release of high-mobility group box 1 protein from dying cells during human immunodeficiency virus type 1 infection in vitro
Implications of the release of high-mobility group box 1 protein from dying cells during human immunodeficiency virus type 1 infection in vitro
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DOI：
10.1099/vir.0.016915-0
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发表时间：
2010-07-01
影响因子：
3.8
通讯作者：
Sonnerborg, Anders
中科院分区：
文献类型：
--
作者：
Barqasho, Babilonia;Nowak, Piotr;Sonnerborg, Anders
在人类免疫缺陷病毒1型（HIV-1）感染过程中，血浆高迁移率组盒1蛋白（HMGB1）水平升高，该分子对病毒复制有影响。本研究探讨了体外HIV-1感染过程中细胞死亡和HMGB1释放的模式。用HIV-1分离物感染MT4细胞和原代CD4(+) T细胞，监测HMGB1释放与细胞病变效应（CPE）和凋亡的关系。Western blotting检测细胞HMGB1释放情况。对于MT4细胞，采用酶联免疫吸附斑点法（ELISPOT）测定坏死过程中的释放。乳酸脱氢酶（LDH）活性用商业测定法定量。流式细胞术检测感染水平和细胞凋亡水平。感染后48 h， MT4细胞感染率为>= 90% （p.i）。CPE首先在60 h观察，并与HMGB1释放、LDH活性和caspase-3 （C3）激活相关。72h后，ELISPOT法清晰检测到HMGB1斑点。Annexin V和C3染色显示细胞凋亡与hiv -1相关的细胞死亡密切相关。在24或40 h p.i.单剂量添加Z-VAD（一种caspase抑制剂）可减少caspase阳性细胞的数量和HMGB1的释放。原发CD4(+) T细胞感染96 h时感染率中位数为22%。相关CPE与LDH和HMGB1释放相对应。在hiv -1诱导的细胞死亡过程中，坏死和凋亡都有助于HMGB1的释放，HMGB1蛋白可以诱导外周单核血细胞释放肿瘤坏死因子α。这些数据表明，HMGB1的被动释放有助于HIV-1发病机制的过度免疫激活特征。
Plasma levels of high-mobility group box 1 protein (HMGB1) are elevated during the course of human immunodeficiency virus type 1 (HIV-1) infection and the molecule has an impact on virus replication. This study investigated the mode of cell death and release of HMGB1 during HIV-1 infection in vitro. MT4 cells and primary CD4(+) T cells were infected with HIV-1 isolates, and HMGB1 release was monitored in relation to cytopathic effects (CPE) and apoptosis. HMGB1 release from cells was analysed by Western blotting. For MT4 cells, an enzyme-linked immunosorbent spot (ELISPOT) assay was adapted to measure the release during necrosis. Lactate dehydrogenase (LDH) activity was quantified using a commercial assay. Flow cytometry was used to determine the level of infection and apoptosis. MT4 cells were >= 90 % infected at 48 h post-infection (p.i.). CPE was first observed at 60 h and correlated with release of HMGB1, LDH activity and caspase-3 (C3) activation. HMGB1 spots were clearly detected by ELISPOT assay at 72 h p.i. Annexin V and C3 staining showed that apoptosis was substantially involved in HIV-1-related cell death. Addition of Z-VAD (a caspase inhibitor) in a single dose at 24 or 40 h p.i. decreased both the number of caspase-positive cells and the release of HMGB1. Infection of primary CD4(+) T cells showed a 22% (median) infection rate at 96 h. Related CPE corresponded to LDH and HMGB1 release. Both necrosis and apoptosis contributed to HMGB1 liberation during HIV-1-induced cell death and the protein could induce tumour necrosis factor-alpha release from peripheral mononuclear blood cells. These data imply that passive HMGB1 release contributes to the excessive immune activation characteristic of HIV-1 pathogenesis.