EXPRESSION OF BCL 2 IN SIV INFECTED THYMIC TISSUE

BCL 2 在 SIV 感染的胸腺组织中的表达

• 批准号: 6591316
• 负责人: M ROSENZWEIG
• 金额: $ 11.11万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6591316
• 关键词: AIDS; animal tissue; communicable diseases; immunology; inflammation; lymphatic system; virus

Cell death due to apoptosis plays a critical role in the development of T cells in the thymus Several intracellular molecular mechanisms have been identified that regulate apoptosis in thymocytes, including bcl-2, bcl-x, bax, R-ras and fas Retroviral infection of the thymus with HIV-1 or SIV results in selective thymocyte depletion, and apoptosis has been shown to contribute to this increase in cell death The precise mechanism whereby lentiviruses induce apoptosis remains controversial Using a novel system of in vitro T cell differentiation, we developed a multiparameter flow cytometry assay to determine the role of fas, fas-ligand and bcl-2 in apoptosis of thymocytes as a result of SIV infection We detected a 3-fold increase of apoptosis (detected by Tdt staining) in in vitro cultures 48 post infection with SIV239 Furthermore, the increase in Tdt+ cells is accompanied by a decrease in bcl-2 (2- 3-fold) expression and an increase in surface fas expressio n (4-5 fo ld) as determined by flow cytometry Fas ligand was found to be increased in single positive thymocytes in vivo, but not in the in vitro culture system This data clearly demonstrates that both the bcl-2 and fas pathways are involved in SIV induced apoptosis of thymocytes and that lentiviral infection leads to thymocyte apoptosis Using FACS we demonstrated that SIV infection results in an initial 15-20% decrease of surface fas expression in CD4+CD8+ and CD4+CD8- cells, and not CD4-CD8+ cells However, by day 7 post-infection, we observed a profound reversal of this phenomenon, with a dramatic increase (6 fold) in fas expression in CD4+CD8+ and CD4+CD8- cells, as compared to uninfected cultures These studies have been extended to examine the effects of acute SIV-infection in vivo, with respect to alterations in the thymus Data from these studies demonstrated that the surface levels of fas on thymocytes from normal neonates is generally low (1-3% of all cells), and does not demonstrate any specific phenotypic distribution After SIV infection the level of surface fas changes modestly, increasing to a maximum of 8% of thymocytes of d14 post-infection This was not accompanied by any significant alteration in surface expression of fas-ligand This observation was in contrast to the fluctuations in bcl-2 levels Uninfected neonatal thymocytes express large amounts of bcl-2, consistent with the observations of others However, rapidly during the course of SIV infection the number of bcl-2 positive cells decreases, as well as the amount of bcl-2 per cell as detected by flow cytometry The lowest levels of bcl-2 occur coincident with the largest amount of apoptosis, detected by Tdt staining By day 21 of acute SIV infection, the levels of virus begin to decline and this is associated with the resumption of normal thymic function Specifically, by d21, the number of apoptotic cells is drastically reduced, and the number of bcl-2 cells begins to approach baseline levels The level of bcl-2 on a per cell basis recovers only by day 50 post-infection Based on these observations, it would appear that dysregulation of the bcl-2 pathway is the predominant mechanism associated with the apoptosis that accompanies retroviral infection of the thymus REFERENCES Wykrzykowska JJ, Rosenzweig M, Veazey RS, Simon MA, Halvorsen K, Desrosiers RC, Johnson RP, Lackner AA Early regeneration of thymic progenitors in rhesus macaques infected with simian immunodeficiency virus J Exp Med 1998, 187:1767-1778 Gardner JP, Rosenzweig M*, Marks DF, Harper D, Gaynor K, Fallon RJ, Wall DA, Johnson RP, Scadden DT T lymphopoietic capacity of cord blood-derived CD34+ progenitor cells Exp Hematol 1998; 26:991-999

凋亡引起的细胞死亡在 胸腺中T细胞的发展几个细胞内分子 已经确定了调节胸腺细胞凋亡的机制， 包括Bcl-2，Bcl-X，BAX，R-RAS和FAS逆转录病毒感染 具有HIV-1或SIV的胸腺会导致选择性胸腺细胞耗竭，并且 凋亡已被证明会导致细胞死亡的这种增加 慢病毒诱导凋亡仍然存在的确切机制 使用新型体外T细胞分化系统有争议的， 我们开发了多参数流式细胞仪测定法，以确定 Fas，Fas-crigand和Bcl-2在胸腺细胞凋亡中的作用 SIV感染的结果我们检测到凋亡增加了3倍 （通过TDT染色检测）在体外培养物48感染后感染后 SIV239此外，TDT+细胞的增加伴随着 Bcl-2（2- 3倍）表达的降低和表面增加 通过流式细胞术FAS配体确定的FAS Expressio n（4-5 FO LD） 发现在体内单个阳性胸腺细胞中有所增加，但 在体外培养系统中，该数据清楚地表明 Bcl-2和FAS途径都参与SIV诱导的凋亡 胸腺细胞和慢病毒感染导致胸腺细胞 使用FACS的凋亡我们证明SIV感染导致 CD4+ CD8+和 CD4+ CD8细胞，而不是CD4-CD8+细胞，但是到第7天 感染后，我们观察到了这种现象的深刻逆转， 在CD4+ CD8+和 与未感染的培养物相比，CD4+CD8-细胞这些研究具有 被扩展以检查体内急性SIV感染的影响， 关于这些研究的胸腺数据的改变 证明了正常的胸腺细胞上Fas的表面水平 新生儿通常很低（所有细胞的1-3％），而不是 证明SIV感染后任何特定的表型分布 表面FA的水平适度变化，增加到 感染后D14的胸腺细胞中有8％没有任何伴随 FAS-配体表面表达的显着改变这 观察与Bcl-2水平的波动相反 未感染的新生儿胸腺细胞表达大量的Bcl-2， 然而，与他人的观察一致，但是在 SIV感染过程Bcl-2阳性细胞的数量减少， 以及流式细胞仪检测到的每个细胞的Bcl-2量 BCl-2的最低水平发生在最大数量 凋亡，通过TDT染色在急性SIV感染的第21天染色， 病毒的水平开始下降，这与 特异性恢复正常胸腺功能，由D21数字恢复 凋亡细胞的大幅减少，Bcl-2的数量 细胞开始接近基线水平Bcl-2的水平 基于这些细胞基础仅在感染后第50天恢复 观察结果，Bcl-2途径的失调似乎 是与凋亡相关的主要机制 伴随胸腺参考的逆转录病毒感染Wykrzykowska JJ，Rosenzweig M，Veazey RS，Simon MA，Halvorsen K，Desrosiers RC， Johnson RP，Lackner AA胸腺祖细胞的早期再生 感染猿猴免疫缺陷病毒的恒河猕猴 1998，187：1767-1778 Gardner JP，Rosenzweig M*，Marks DF，Harper D， Gaynor K，Fallon RJ，Wall DA，Johnson RP，Scadden DT T淋巴管 脐带血的CD34+祖细胞Exp Hematol的能力 1998; 26：991-999