The RGD-containing domain of exogenous HIV-1 Tat inhibits the engulfment of apoptotic bodies by dendritic cells

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Abstract
HIV-1 Tat can be released by infected cells and exert various extracellular functions on bystander cells, possibly contributing to immunodeficiency. In order to investigate whether exogenous Tat can affect antigen presentation, the effects of synthetic Tat on the function of dendritic cells displaying antigen presenting cell phenotype were studied. Cultured dendritic cells were challenged with apoptotic bodies and monitored for cell engulfment and free intracellular calcium ([Ca2+]i) increase. The effect of synthetic HIV-1 Tat and its RGD-containing domain (peptide 65–80) or basic domain (peptide 46–60) on both functions was investigated. Dendritic cells were obtained by culture of monocytes with granulocyte-macrophage colony-stimulating factor. Apoptosis was induced in Jurkat cells by sublethal irradiation. Engulfment of radiolabelled apoptotic bodies by dendritic cells was obtained by a 45 min co-incubation at 37°C. Non-ingested apoptotic bodies were removed and cell-associated radioactivity evaluated in a γ-counter after cell lysis. Single cell analysis of calcium fluxes was performed by video-microscopy and ratio-imaging, after cell staining with the fluorescent calcium chelator FURA-2. Apoptotic bodies were engulfed by dendritic cells: this process was accompanied by [Ca2+]i rise. Synthetic HIV-1 Tat inhibited both apoptotic body engulfment and [Ca2+]i increase. The same inhibition was obtained with the RGD-containing domain (peptide 65–80), but not with the basic domain (peptide 46–60) of Tat, suggesting the involvement of an integrin. This integrin is likely to be anb3, since RGD-containing peptides from vitronectin, but not from fibronectin, inhibited apoptotic body engulfment. Furthermore, both HIV-1 Tat and its 65–80 peptide blocked [Ca2+]i increase due to b3-integrin cross-linking. Our results support a role for HIV-1 Tat in decreasing the function of dendritic cells, possibly impairing antigen presentation.