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
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