INDUCTION OF APOPTOSIS BY HIV-1 INFECTED MONOCYTIC CELLS

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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.