INDUCTION OF APOPTOSIS BY HIV-1 INFECTED MONOCYTIC CELLS

我们利用人类巨噬细胞杂交瘤系统来分析HIV-1/巨噬细胞的相互作用，特别关注HIV-1感染对APC功能的影响。我们在HIV-1感染的早期发现了许多缺陷，但在研究HIV-1慢性感染株时有一个有趣的发现。T细胞与这些细胞共培养后可诱导细胞凋亡。有趣的是，在CD-4和CD-8+T细胞以及B细胞中都观察到了凋亡。Gp120和FasL的表面表达可解释CD-4+T细胞的凋亡，而CD-8T细胞和B细胞则不能。我们不能确定诱导CD-4+T细胞和CD-4+T细胞以及B细胞凋亡的可溶性因素。为了确保我们的因子是新的，我们比较了HIV-1感染和未感染的细胞中不同的趋化因子和与凋亡相关的细胞因子，包括SDF-1、LTN、RANTES、IP-10、MCP-1-β、I-309、L32、IL-8、TNF-β、LT-β、TNF-α、伽马-干扰素、β-干扰素、转化生长因子-β3、转化生长因子-β2和转化生长因子-β1。我们对该因子的3个候选多肽进行了纯化和部分测序，并产生了一组多克隆和单克隆抗体，这些抗体在体外具有中和活性。使用这些抗体，我们在HIV-1感染患者的PBMC中检测到了这种因子，但在未感染的对照组中没有检测到。虽然我们鉴定了3个多肽，但可能还有其他具有活性的可溶性因子。我们将把这些初步观察扩展到完全测序和分离部分测序的多肽和其他促凋亡多肽的特定cDNA克隆，筛选不同的表达文库，如果失败，则通过消减杂交。在授权期的第二部分，我们将确定该因子生产的多样性，以及促凋亡作用是普遍的还是有限的。我们还将鉴定靶细胞上的受体(S)。我们将试图通过量化颈部患者的生物液(血清和脑脊液)和组织(淋巴和脑)中的水平来确定促凋亡因子在HIV-1感染患者中的作用。处于疾病的不同阶段。对这一因素的识别可能有助于解释艾滋病中通过细胞凋亡发生的一些细胞损失。

We have utilized a system of human macrophage hybridomas to analyze HIV-1/macrophage interactions with a specific focus upon the effects of HIV-1 infection on APC function. We identified numerous defects early in the course of HIV-1 infection but one intriguing finding was made when studying chronically HIV 1 infected lines. Co-culture of T cells with these cells resulted in the induction of apoptosis. Interestingly, apoptosis was noted in both CD-4 and CD-8+ T cells as well as B cells. The surface expression of gp120 and FasL could account for the apoptosis observed in the CD-4+ T cells but not in the CD-8 T cells or B cells. We could not identify soluble factors that induced apoptosis in CD-4+ T cells and CD-4+ T cells as well as B cells. To ensure that our factors were novel we compared and found no difference in RNA production between the HIV-1 infected and uninfected cells for different chemokines and cytokines associated with apoptosis including SDF-1, Ltn, Rantes, IP-10, MCP-1-beta, I-309, L32, IL-8, TNF-beta, LT-beta, TNF-alpha, gamma- IFN, beta-IFN, TGF-beta3, TGF-beta2, and TGF-beta1. We purified and partially sequenced 3 candidates peptides for this factor and generated a panel of polyclonal and monoclonal antibodies which neutralize activity in vitro. Using these antibodies we detected this factor in the PBMC of HIV-1 infected patients but not in uninfected controls. Although we characterized 3 peptides, there may be other soluble factors which have activity. We will extend these preliminary observations to fully sequence and isolate specific cDNA clones for the partially sequenced peptides and the other pro-apoptotic peptides screening different expression libraries and if this fails by subtractive hybridization. In the second part of the granting period we will determine the diversity of production of the factor and if the pro-apoptotic effect is generalized or restricted. We will also identify receptor(s) on target cells. We will attempt to define a role for the pro-apoptotic factor in HIV-1 infected patients by quantifying levels in biological fluids (serum and cerebral spinal fluid) and tissues (lymph node and brain) from necked patients. at different stages of disease. Identification of this factor may help explain some of the cell loss that occurs through apoptosis in AIDS.