HiV-I-INDUCED APOPTOSIS
Apoptosis is considered a main defense mechanism of the organism and generally falls under one of two major headings: intrinsic (mitochondrial) or extrinsic (receptor mediated); both signaling pathways converge at the level of specific proteases.23-25 In general, lymphocyte apoptotic propensity is determined largely by the expression of certain apoptotic proteins that include, but are not limited to, the following: CD95, CD178 (note that CD95 ligand has been renamed CD178), DR-4 and DR-5, and tumor necrosis factor receptor type-1 (TNF-R1), which may be regulated by antiapoptotic proteins such as Bcl-2 or Bcl-xL.
T cell activation results in the increased expression of CD178, which may bind to CD95, expressed on target cells. Activated cells expressing CD178 may kill target cells26,27 or, in an autocrine loop, activate their own CD95, thus committing suicide.23 The DR-4 and DR-5 bind to and are activated by TNF-related apoptosis-inducing ligand (TRAIL), whereas TNF-R, when activated, can signal for proliferation as well as apoptosis. All of these cellular receptors contain a cytosolic death domain that, when stimulated, may activate the caspase cascade.The majority of reports investigating HAART and HIV-induced apoptosis involve studies with the CD95/CD178 ligand mechanism. However, conflicting results on CD95-induced apoptosis have been reported in the literature, probably due to differences in study designs. In an early study, spontaneous apoptosis (SA) and CD95 expression in unstimulated peripheral blood mononuclear cells (PBMCs) were correlated with an advanced stage of HIV-1.28 The majority of apoptotic cells from negative controls and patients with CD4 counts above 100ZμL did not express detectable amounts of CD95, whereas patients with lower CD4 T cell counts (in an advanced disease stage) tended to have an elevated number of apoptotic cells, most of which were CD95+.
In a second prospective study of pretreated patients undergoing a change in therapy after virological failure, responders (defined as CD4 cell recovery >100 cellsZμL) were compared with nonresponders. After 3 months of therapy, there was a significant and durable decrease in spontaneous apoptosis in the CD4 population in the responders, whereas CD95-induced apoptosis significantly declined through the first month but quickly returned to study-baseline values by month 3 in nonresponders.29The existence of a so-called fratricide mechanism further complicates our understanding of this process. Badley et al. originally demonstrated in vitro that HIV-infected macrophages can provide a source of CD178,30 and they later proposed that CD95-induced apoptosis is dependent on two factors: CD95 expression on the target cell and CD178 susceptibility.31 In one study, the investigators studied CD95-mediated apoptosis in PBMCs and CD178 expression in tonsillar tissue at various time points before, during, and after HAART. Therapy reduced apoptosis in both lymphoid tissue and peripheral blood, and CD95 susceptibility in CD4 lymphocytes was reduced, whereas CD178 expression remained unchanged during HAART. Differences in susceptibility to apoptosis were also observed for infected macrophages and infected T lymphocytes. Pinti et al. used the established cell lines A301 and U937 and their respective HIV-infected clones ACH-2 and U1 to investigate the effects of apoptotic stimuli on survival and expression of CD95. The pre- monocytic cell line U1 was less susceptible to apoptosis with a diminished level of CD95 expression as compared with its noninfected parental cell line, whereas both infected and noninfected lymphocyte cultures were susceptible to apoptosis.32 From these data, it is tempting to speculate that HIV-1 facilitates its own survival by downregulating the expression of CD95 in monocytes and macrophages, which are known reservoirs for latent virus.
However, these studies need to be confirmed with primary cell cultures.Antiretroviral Drugs and Apoptosis
Because of the early implementation of NRTIs in the treatment of HIV-1 infection, the addition of protease inhibitors (PIs) and NNRTIs has greatly improved clinical outcomes. Other than improving the resistance profile of these multidrug combinations, it has also been proposed that some possess immunomodulatory and antiapoptotic properties (for a review, see Phenix et al.33). There are, however, differences in the mechanisms of action of individual drugs from the PI class. Independent of their role in HIV suppression, it has been demonstrated that both ritonavir and saquinavir modulate susceptibility to apoptosis in vitro and in vivo,34-36 presumably by effecting the cleavage of key caspase proteins. Chavan et al. clearly demonstrated in one in vivo study that nelfinavir (Nfv) contributes to the rescue of peripheral T lymphocytes from spontaneous apoptosis.37 Interestingly, in another study, Nfv was found to inhibit apoptosis in Jurkat T cells at minimal doses after only 1 h, which indicates that it acts through a mechanism independent of transcription or protein synthesis (including pro- or antiapoptotic proteins).38 Additional experiments revealed that Nfv inhibits apoptosis by preventing permeability transition pore complex (PTPC) opening, which maintains the mitochondrial transmembrane potential and prevents the release of apoptogenic factors.38 Indinavir (IDV), on the other hand, does not appear to have any direct effect on apoptosis but may prolong cell survival by inhibiting entry into the cell cycle.39
A decrease in mitochondrial membrane potential can occur in apoptotic cells, including activated T lymphocytes, even if this is not the rule for cells undergoing cell death.40 HIV PI, independent from any viral infection, can hinder lymphocyte apoptosis by influencing mitochondrial homeostasis. An interesting study was undertaken in both resting and activated human lymphocytes exposed to different apoptotic stimuli.41 T cell activation was found to be accompanied by a significant increase in mitochondrial membrane potential, or hyperpolarization, which was undetectable in resting cells.
PIs were able to block activation-associated mitochondria hyperpolarization, which, in turn, was paralleled by an impairment of cell cycle progression. Remarkably, PIs also prevented zidovudine-mediated mitochondrial toxicity. Cells from drug-naive HIV-positive patients behaved identically to activated T cells, displaying hyperpolarized mitochondria, whereas lymphocytes from HIV patients under HAART, which included HIV PIs, seemed to react as resting cells, indicating that the hyperpolarization state of mitochondria represents a prerequisite for the sensitization of lymphocytes to activation-induced cell death (AICD).Lymphocyte dysfunction
It was observed that CD4 dysfunction, which occurs early after infection with HIV-1 (even before any decline in absolute numbers of CD4 T cells), involves an early loss of HIV-1-specific responses in terms of both proliferation and IL-2 production.42-44 The inhibition of IL-2 secretion was linked to the induction of clonal anergy in T cells, possibly due to the development of a proximal signal transduction defect in the IL-2 gene.45 It is also well known that IL-2 is the main survival factor for T lymphocytes, and any impairment of its production or utilization can result in cell death. It was observed that IL-2, in a dose-dependent manner, prevents T cells from entering apoptosis induced by -irradiation, mitomycin C, or dexamethasone.46 Results from another study suggest that the balance between cytokine (IL-2) and serum-induced Bcl-2 expression and cytokine-induced Bax expression may determine whether a cell undergoes cytokine-induced apoptosis.47
Even in healthy uninfected individuals, regulation of CD8 and CD4 T cell responses seems fundamentally different. CD8 T cell memory is stable, whereas specific CD4 T cell memory gradually declines.48 In uninfected subjects, however, this CD4 T decay is not associated with loss of immunologic function but, rather, is linked to the reduced expression of Bcl-2 and Bcl-xL.48 Conditions precipitating CD4 T cell loss (i.e., HIV) might compromise immunity despite maintenance of specific CD8 memory.
As Pitcher et al.17 demonstrates, the “overall frequencies of functional HIV-1 specific CD4 memory cells are considerably diminished in a cohort of long term HAART-treated subjects, indicating that prolonged viral suppression is associated with a decline in functional HIV-1-specific CD4 T cell memory.”17 Interestingly, one study also indicated that CD4 cell rescue (increase in CD4 lymphocyte numbers) is dependent on the CD8 subset rather than directly related to inhibition of apoptosis in the same CD4 population.49 Hansjee et al.50 noted a strong inverse relationship between SA rates in PBMCs and CD8 with the recovery of CD4, indicating that there is a very strong interdependent balance between these two lymphocyte classes and lymphocyte dysfunction is somehow linked to apoptosis.From these studies, it may be inferred that viral replication leads to activation and apoptosis of both CD4 and CD8 T lymphocytes, albeit by different mechanisms, and the magnitude of the CD8 cytotoxic T lymphocyte (CTL) response may be diminished due to a lack of CD4 “help.” Controlling the virus with HAART therapy reduces viral load but does not completely restore the preinfection equilibrium of T cell subpopulations, and it does not seem to restore the functionality of the T cells, which persist, even though certain therapies (i.e., Nfv) protect the organism from apoptosis. Moreover, integrated deoxyribonucleic acid (DNA) provirus in latently infected resting memory CD4 T cells are transcriptionally inactive; thus, they escape both immune recognition and the effects of antiviral therapy,51 underscoring the need for alternative therapeutic strategies that may restore immune function and limit immune pathology associated with this exaggerated immune activation.