| Abstract |
The human immunodeficiency virus (HIV) is involved in a number of sophisticated interactions with the human immune system. Asymptomatic infection with HIV is characterized by a chronic activation of the immune system, although the mechanism of this activation is not precisely known. There is mounting evidence that a number of HIV-1 proteins can deregulate the immune system leading to cell apoptosis. The early loss of immune functions in the course of HIV infection is attributed to the preferential loss of uninfected effector/memory (CD45RO+) CD4+ T lymphocytes. As the basic function of these cells is to interact with antigen presenting cells such as macrophages and secrete appropriate cytokines, we investigated the disruption of the antigen presentation process by viral components rather than viral infectivity. The envelope glycoprotein gp120 appears to play a key role in this interaction as it has the ability to react with both CD4 and the chemokine co-receptors CCR5 and CXCR4. The interaction of a host cell with the gp120 results in the induction of an increased sensitivity to apoptosis, which is mediated by the Fas system, as the expression of the anti-apoptotic Bcl-2 is suspended and Caspace 3, is activated. Despite reports of the involvement of the third hypervariable region (V3 loop) of gp120 - the most critical determinant for cellular tropism - in T helper cell dysfunction, the mechanism via which it contributes to the pathogenesis is still not clearly understood. By using primary cells isolated from healthy blood donors and composite liposome particles with exposed V3 lipopeptides from the semi-conserved principal neutralizing domain of V3 of LAI HIV-1 isolate and entrapped tetanus toxoid as a recall antigen (lipo-V3/TT liposomes), we have demonstrated that this special domain of V3 when it is presented on the surface of macrophages delivers a specific signal of activation-apoptosis to normal effector CD4+ T lymphocytes during the process of antigen presentation. Especially, the lipo-V3/TT liposomes are shown to induce a V3-specific response characterised by an early, enhanced proliferation of the above T cells when they are responding to their cognate antigen, followed by a sharp apoptosis. The phenomenon requires the presence of monocyte-derived macrophages and CD4+T cells, but it is qualitatively and quantitatively distinct from the normal soluble antigen-mediated antigen presenting cell -T cell interaction. Presence of the β-chemokine RANTES, an agonist of CCR5 co-receptor, in the culture medium inhibited the phenomenon, suggesting that V3 plays a co-stimulatory role that involves at least the chemokine receptor CCR5 pathway during the process of antigen presentation to T cells. The function of V3 is not therefore restricted to influencing viral entry but it appears that it can also alter the biological behaviour of the recipient host cell. Addressing the question of how a hypervariable region could induce such a selective reaction, we demonstrated that the magnitude of the activation phase was dependent on the number of basic amino acids present in the V3 peptide, an observation confirmed by using V3 peptides from selected subtype B HIV-1 isolates or V3 lipopeptides with appropriate basic amino acid substitutions. The relative position of the amino acids in the V3 peptide did not affect the biological phenomenon. Furthermore, performing surface plasmon resonance biosensor analysis, we also provided direct evidence of the influence of basic amino acids in the interaction between V3 and the amino terminal domain of CCR5. Sulfation of tyrosines in the CCR5 peptide was essential. Our results confirm gp120 modeling predictions and demonstrate simple molecular ionic interactions as capable of affecting key cell events, the wider biological implications of which need to be further explored. In addition to the above-mentioned mechanisms, we investigated the intracellular effects on the recipient host cell. By monitoring the mRNA levels of selected genes, participating in either activation-induced or passively induced apoptosis, we identified the involvement of the Fas pathway, which is possibly activated by the V3-mediated signalling. The above observations may be very important if they also occur in HIV-1 infection, as they may explain the selective and progressive depletion of non-infected effector CD4+ T cells. In this context we designed in vitro functional and infectivity assays in order to establish if HIV infected macrophages are able to induce a similar activation-apoptosis phenomenon to that observed with V3 liposomes, and whether the V3-based peptides or liposomes interfere with viral entry. As targets for the viral entry we used primary peripheral macrophages from healthy donors and the transfected with CCR5 T cell line PM1. The HIV-1 strains we used were the M-tropic or R5 strain Ba-L and the T cell line adapted or X4 virus NL4.3. The effects of V3 peptides in the infectivity of cells were measured as percentage of the reduction of the viral antigen p24 production. In comparison the proliferation results in presence or absence of infected APCs, basically followed similar kinetic motifs, although the total counts in the case of infected macrophages were significantly lower. Possible reasons for such a reduction could be the down-regulation of mature MHC class II production on the surface of macrophages induced by the infection. The apoptotic profiles of the responding CD4+/CD45RO+ T cell population in presence of infected macrophages were significantly increased, compared to that observed with unifected APCs, possibly induced by the higher amounts of macrophage-secreted FasL in the culture. The out coming results from the study of the infection of the PM1 cells or primary macrophages showed that the V3 peptides with the higher density of basic aminoacids in their composition could compete and inhibit very efficiently the co-receptor use by the Ba-L virus, even in a very low concentration. In contrast the presence of the above peptides affected not significantly the infection of PM1 cells by the X4 strain giving very low inhibition rates of the p24 production. This result indicates that the efficient binding of the virus to the CXCR4 receptor might not hide ionic interactions between the receptor and the inhibitor peptides. As increased sensitivity to apoptosis and depletion of uninfected CD4+ T lymphocytes are believed to be the main route of pathogenenesis for HIV-1 infection, in the present study we provided information that the variability in electrostatic charges of V3 may well modulate biological signals and cell events via interaction with CCR5, which could potentially have major implications in the management and treatment of AIDS.
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