SOLUBLE FACTORS FROM MACROPHAGES/MICROGLIA EXPOSED TO HIV-1
It has been reported that neuronal apoptosis may result indirectly due to the secretion of neurotoxic factors from brain resident macrophages or microglia in response to HIV-1 infection, stimulation by viral proteins, or immune activation.
Apoptotic neurons in the basal ganglia of HIV-1-infected patients were observed to be more abundant in the vicinity of mutinucleated giant cells and HIV-1 p24 immunoreactive cells.91 Although HIV-1 infects both CD4+ T cells and cells of the macro- phage/microglia lineage, the results of a recent study underscore the importance of only the latter cell type in the secretion of neurotoxic host factors upon HIV-1 infection.101 Cell culture media from HIV-1-infected T cells induced high levels of neuronal apoptosis; however, depletion of HIV-1 virions from the media reversed this effect. Although virion-containing cell culture media from HIV-1- infected macrophages induced greater neuronal apoptosis than virion-depleted macrophage supernatants, significant cell death was still observed in the latter. These results suggest that upon HIV-1 infection, both macrophages and T cells secrete virus factors that mediate neuronal apoptosis. In contrast, neurotoxic host factors were secreted from HIV-1-infected macrophages but not T cells.HIV-1-infected brain mononuclear phagocytes are capable of producing a long list of potential neurotoxins, which include eicosanoids (e.g., arachidonic acid and platelet-activating factor [PAF]), proinflammatory cytokines (e.g., tumor necrosis factor-α ∣TNF-α∣ and interleukin-1β [IL-1β]), free radicals (e.g., nitric oxide and superoxide anion), and neurotoxic amines (e.g., NTox).119-121 In addition, immune activation of macrophages by HIV-1 gp120 induces the production of arachidonic acid, TNF-α and IL-1β,122 and the glutamate-like agonist L-cysteine,123 an endogenous neurotoxin that acts via excessive A-methyl-D-aspartate (NMDA) receptor activation.124,125 L-cysteine production by gp120-stimulated macrophages was reduced when the cells were pretreated with a TNF-α neutralizing antibody or an IL-1 receptor antagonist, demonstrating that production of this neurotoxin was partially mediated by the cytokines TNF-α and IL-1β.123 Importantly, TNF-α and IL-1β are elevated in the brain, spinal cord, and cerebrospinal fluid of AIDS patients.126,127 Similar to gp120, HIV-1 Tat was also shown to induce the production of neurotoxins by macrophages.
Injection of synthetic Tat peptide into mouse brain induced lesions in areas that were associated with expression of TNF-α, IL-1β, IL-6, and inducible nitric oxide synthase (iNOS) in macrophages/ microglia and astrocytes.128 Blockade of TNF-α resulted in decreased IL-1β and iNOS expression, along with a reduction in the volume of the brain lesions, underlying a central role of TNF-α in Tat-mediated neurotoxicity. TNF-α was also demonstrated to inhibit glutamate uptake by astro- cytes.129 Thus, Tat-induced TNF-α expression may also result in increased levels of extracellular glutamate, leading to hyperactivation of NMDA receptors and subsequent neuronal cell death. When expressed in primary human macrophages, HIV-1 Tat from HAD patients induced elevated release and activation of matrix metalloproteinase (MMP)-2 and MMP-7.130 Conditioned media from these cells induced neuronal death, which was inhibited by anti-MMP-2 or anti-MMP-7 antibodies but not by antibodies against MMP-9 or Tat. Interestingly, this phenomenon was not observed for Tat from nondemented HIV-1-infected patients, indicating that HIV-1 Tat also causes neuronal death through an indirect mechanism that is Tat sequence dependent and involves the induction of specific MMPs. HIV-1 Tat was also demonstrated to mediate neuronal cell death through enhancing the activity of glycogen synthase kinase (GSK)-3β,131 an enzyme shown to play a direct role in the regulation of neuronal apoptosis.132 Although Tat was shown to associate with GSK-3β, direct addition of Tat to purified GSK-3β had no effect on enzyme activity. Instead, it was suggested that HIV-1 Tat influences GSK-3β by an indirect mechanism, involving TNF-α- mediated induction of PAF, which, in turn, induces GSK-3β activity.131The above list of neurotoxins is by no means exhaustive, and the identities of potentially neurotoxic substances released by HIV-1-infected or HIV-1-activated macrophages are currently under intense investigation.
Recent findings from our laboratory identified a novel mechanism by which HIV-1 infection of the CNS may result in neuronal apoptosis, and this highlights the selective pathological proteolysis of tissue proteins by proteases in the brain.133 Although increased MMP expression and activity were associated with multiple neurological diseases,134 including those caused by HIV-1 and other related lentiviruses,135-137 the causality and neurotoxic mechanism remain unclear. We recently demonstrated that HIV-1 infection of human macrophages resulted in elevated levels of secreted pro- MMP-2, although macrophages infected with an HIV-1 Env-deleted luciferase vector pseudotyped by a vesicular stomatis virus envelope did not show increased MMP-2 activity. Thus, MMP-2 induction was specific to HIV-1 Env-mediated infection and not a nonspecific cellular response to viral envelope. Recombinant pro-MMP-2, but not a catalytically inactive mutant of MMP-2, induced cell death in LAN-2 neuronal cells in a dose-dependent manner. This neurotoxicity of pro-MMP-2 was blocked by treatment with the synthetic MMP inhibitor Prinomastat, confirming the neurotoxic effects of MMP-2 proteolytic activity. MMPs are now recognized as pivotal processing enzymes of a wide variety of bioactive mediators that control many cellular processes.138 Chemokines, such as stromal- derived factor (SDF)-1, are abundantly expressed in the CNS139 and constitute a recently recognized class of MMP substrates.140,141 MMP-2 efficiently cleaves SDF-1 to yield a truncated molecule that lacks CXCR4 agonist activity in chemotaxis assays and has a greater than 100-fold reduction in CXCR4 binding affinity.140 Unexpectedly, TUNEL analysis indicated that LAN-2 neuronal cells (but not other nonneuronal cell lines expressing CXCR4) treated with SDF-1(5-67), a synthetic analogue of the cleaved SDF-1, showed higher levels of neuronal apoptosis than in those treated with fulllength SDF-1.133 A marked reduction in neurotoxicity was found after pretreatment of the cells with an SDF-1-neutralizing antibody or with pertussis toxin, demonstrating the specificity of cleaved SDF- 1(5-67) as a novel cytotoxin selective for neurons acting through an as-yet-unidentified Gαi-coupled receptor. Cleaved SDF-1(5-67) and MMP-2 also proved to be neurotoxic in a rodent model that recapitulated several aspects of HIV-1 infection of the brain, including the nature and site of neuropathy with concurrent motor abnormalities.133 These findings emphasize the role of proteolysis of constitutively expressed proteins to yield novel molecules in the pathogenesis of neurodegenerative diseases, as also illustrated in Alzheimer’s and Huntington’s diseases.The proapoptotic transcription factor p53 was proposed as a candidate mediator of HIV-induced neuronal injury. HeLa cells expressing CD4 undergo p53-dependent apoptosis upon exposure to HIV-1 gp120,142,143 and there is evidence of p53 accumulation in CNS tissue from monkeys with SIV encephalitis (SIVE)144 and in neurons of HAD patients.145 In addition, p53 mediates neuronal apoptosis induced by excitotoxins.146,147 Using murine cerebrocortical cultures, Garden et al. recently demonstrated that neurotoxic concentrations of HIV-1 gp120 induced p53 accumulation in both neurons and microglia.148 Neurons from mice deficient in p53 were resistant to gp120- induced apoptosis, whereas microglia from p53-deficient mice lost the ability to mediate gp120- induced neuronal apoptosis, suggesting that p53-dependent signaling is required in both neurons and microglia for gp120-induced neuronal apoptosis.148
In addition to cells of the macrophage/microglia lineage, recent data from our group also demonstrate that neurotoxic factors are released by astrocytes upon exposure to HIV-1.149 Expression of the envelope protein of the brain-derived HIV-1 strain JRFL in primary astrocytes resulted in the production of soluble factors that were highly toxic to LAN-2 neuronal cells. As no detectable HIV-1 gp120 was present in the conditioned media from these astrocytes, other molecules released in response to expression of the HIV-1 envelope were responsible for the cytotoxicity of the astrocyte-conditioned media. Moreover, the inability to block this neurotoxic effect with antagonists for the NMDA and AMPA receptors indicated that these neurotoxins do not act via the glutamate receptors. Data from our group also demonstrated upregulation of proteinase-activated receptor 1 (PAR-1) expression in astrocytes during HIVE.150 The fact that mRNA expression levels for PAR-1 and its agonist, prothrombin, were greater in HIV-1-infected patients than in those individuals with multiple sclerosis suggests that this increase is not solely an effect of inflammation but is possibly related to a virus-induced event. In vitro experiments revealed that supernatants from astrocytic cells stimulated with selective agonists for PAR-1 induced neurotoxicity in human fetal neurons via an NMDA receptor-mediated mechanism.