HIV-infected Macrophages Research Articles

An energy-based three-dimensional segmentation approach for the quantitative interpretation of electron tomograms

Electron tomography allows for the determination of the three-dimensional structures of cells and tissues at resolutions significantly higher than that which is possible with optical microscopy. Electron tomograms contain, in principle, vast amounts of information on the locations and architectures of large numbers of subcellular assemblies and organelles. The development of reliable quantitative approaches for the analysis of features in tomograms is an important problem, and a challenging prospect due to the low signal-to-noise ratios that are inherent to biological electron microscopic images. This is, in part, a consequence of the tremendous complexity of biological specimens. We report on a new method for the automated segmentation of HIV particles and selected cellular compartments in electron tomograms recorded from fixed, plastic-embedded sections derived from HIV-infected human macrophages. Individual features in the tomogram are segmented using a novel robust algorithm that finds their boundaries as global minimal surfaces in a metric space defined by image features. The optimization is carried out in a transformed spherical domain with the center an interior point of the particle of interest, providing a proper setting for the fast and accurate minimization of the segmentation energy. This method provides tools for the semi-automated detection and statistical evaluation of HIV particles at different stages of assembly in the cells and presents opportunities for correlation with biochemical markers of HIV infection. The segmentation algorithm developed here forms the basis of the automated analysis of electron tomograms and will be especially useful given the rapid increases in the rate of data acquisition. It could also enable studies of much larger data sets, such as those which might be obtained from the tomographic analysis of HIV-infected cells from studies of large populations.

HLA-A2 down-regulation on primary human macrophages infected with an M-tropic EGFP-tagged HIV-1 reporter virus

Multiple mechanisms are used by the human immunodeficiency virus type 1 (HIV-1) to interfere with host-cell immune effector functions. The 27-kD Nef protein has been shown to down-modulate specific genes of the major histocompatibility complex class I (MHC-I) on the surface of infected primary T cells, facilitating their escape from lysis by cytolytic T lymphocytes. Macrophages, as the other major immune cell type targeted by the virus, also contribute to the transmission, persistence, and pathogenesis of HIV-1. Yet, whether Nef modulates MHC-I expression on HIV-infected primary macrophages remains unclear. Currently available infectious HIV-1 molecular clones, which express a reporter gene, only infect T cells and/or do not express Nef. To overcome these limitations, we generated macrophage-tropic green fluorescent protein (GFP)-tagged HIV-1 viruses, which express the complete viral genome, and used these to assess the expression of human leukocyte antigen (HLA)-A2 on the surface of productively infected macrophages. The reporter viral genomes were replication-competent and stable, as Nef, p24 antigen, and GFP expression could be detected by immunostaining of infected, monocyte-derived macrophages (MDM) after more than 2 months postinfection. Fluorescence-activated cell sorter analyses of infected macrophages and T cells revealed that although wild-type reporter virus infection induced a statistically significant decrease in the density of surface HLA-A2, down-regulation of HLA-A2 was not seen in cells infected with reporter viruses encoding a frameshift or a single point mutation in Nef at prolines 74P and P80. The impact of Nef on HLA-A2 surface expression in MDM was also confirmed by confocal microscopy. These results suggest that the mechanisms of HLA-A2 down-modulation are similar in primary T cells and macrophages.

The shifting patterns of HIV encephalitis neuropathology.

HIV infected macrophages infiltrate the nervous system early in the progression of HIV infection, leading to a complex set of neuropathological alterations including HIV encephalitis (HIVE), leukoencephalopathy and vacuolar myelopathy that in turn result in neurodegeneration of selective cellular populations and pathways involved in regulating cognitive and motor functioning. Rapid progress in the development of highly active antiretroviral therapy (HAART) has changed the patterns of HIV related neuropathology and neurological manifestations in the past 10 years. The prevalence of opportunistic infections and central nervous system (CNS) neoplasms has decreased, and some groups have proposed that the frequency of chronic forms of HIVE have been rising as the HAART-treated HIV population ages. Accordingly, clinical manifestations have shifted from severe dementia forms to more subtle minor cognitive impairment, leading to the suggestion of a classification of HIV associated neurological conditions into an inactive form, a chronic variety, and a 'transformed' variant. From a neuropathological point of view these variants might correspond to: a) aggressive forms with severe HIVE and white matter injury, b) extensive perivascular lymphocytic infiltration, c) 'burnt-out' forms of HIVE and d) aging-associated amyloid accumulation with Alzheimer's-like neuropathology. Factors contributing to the emergence of these variants of HIVE include the development of viral resistance, immune reconstitution, anti-retroviral drug toxicity and co-morbid factors (e.g., methamphetamine, HCV). More detailed characterization of these proposed variants of HIVE is important in order to better understand the pathogenesis of HIV-associated neurological damage and to design more effective treatments to protect the nervous system.

Oligomerization Is Required for HIV-1 Nef-Induced Activation of the Src Family Protein-Tyrosine Kinase, Hck

Hck is a member of the Src protein-tyrosine kinase family and is expressed strongly in macrophages, an important HIV target cell. Previous studies have shown that Nef, an HIV-1 accessory protein essential for AIDS progression, binds and activates Hck through its SH3 domain. Structural analysis suggests that Nef forms oligomers in vivo, which may bring multiple Hck molecules into close proximity and promote autophosphorylation. Using bimolecular GFP fluorescence complementation, we show for the first time that Nef oligomerizes in living cells and that the oligomers localize to the plasma membrane. To test the role of Nef oligomerization in Hck activation, we fused Nef to the hormone-binding domain of the estrogen receptor (Nef-ER), allowing us to control its dimerization with 4-hydroxytamoxifen (4-HT). In Rat-2 fibroblasts co-expressing Nef-ER and Hck, 4-HT treatment induced Nef-ER dimer and tetramer formation, leading to Hck kinase activation and cellular transformation. The number of transformed foci observed with Nef-ER plus Hck in the presence of 4-HT was markedly greater than that observed with wild-type Nef plus Hck, suggesting that enforced oligomerization enhances activation of Hck by Nef in vivo. Enhanced transformation correlated with increased Hck/Nef complex formation at the plasma membrane. In complementary experiments, we observed that a Nef mutant defective for Hck SH3 domain binding (Nef-PA) suppressed Hck kinase activation and transformation by the wild-type Hck/Nef complex. This effect correlated with the formation of a ternary complex between wild-type Nef, Nef-PA, and Hck, suggesting that Nef-PA suppresses Hck activation by blocking wild-type Nef oligomerization. Finally, Nef-ER induced Hck activation in a 4-HT-dependent manner in the macrophage precursor cell line TF-1, suggesting that oligomerization is essential for signaling through Hck in a cell background relevant to HIV infection. Together, these data demonstrate that Nef oligomerization is critical to the activation of Hck in vivo, and suggest that inhibitors of oligomerization may suppress Nef signaling through Hck in HIV-infected macrophages, slowing disease progression.

Conserved Residues in the HIV-1 Nef Hydrophobic Pocket are Essential for Recruitment and Activation of the Hck Tyrosine Kinase

The Nef protein of the primate lentiviruses human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) is essential for high-titer viral replication and acquired immune deficiency syndrome (AIDS) progression. Nef binds to the macrophage-specific Src family member Hck through its SH3 domain, resulting in constitutive kinase activation capable of transforming rodent fibroblasts. Nef-Hck interaction may be essential for M-tropic HIV replication and AIDS pathogenesis, identifying this virus-host protein complex as a rational target for anti-HIV drug discovery. Here, we investigated whether interaction with Hck is a common feature of Nef alleles from different strains of HIV-1. We compared the ability of four different laboratory HIV-1 Nef alleles (SF2, LAI, ELI, and Consensus) to induce Hck activation and transformation in our Rat-2 fibroblast model. While SF2, LAI, and Consensus Nef all bound and activated Hck, ELI Nef failed to bind to the Hck SH3 domain in vitro and did not cooperate with Hck in fibroblast transformation. Molecular modeling identified three residues in the core region of SF2 Nef (Ala83, His116, and Tyr120) which are substituted in ELI with Glu, Asn, and Ile, respectively. Two of these residues (Ala83 and Tyr120) form part of the hydrophobic pocket that contacts Ile 96 in the RT loop of the Hck SH3 domain in the Nef-SH3 crystal structure. Substitution of SF2 Nef Tyr120 with Ile completely abolished Hck recruitment and activation. In a complementary experiment, substitution of ELI Ile120 with Tyr partly restored ELI Nef-induced Hck activation and transformation in Rat-2 cells. Hck activation increased further by substitution of ELI Glu83 with Ala and Asn116 with His, suggestive of a supportive role for these residues in Hck binding. This study provides the first biological evidence that the HIV-1 Nef hydrophobic pocket is critical to Hck recruitment and activation in vivo. Targeting the Nef hydrophobic pocket with a small molecule may be sufficient to disrupt Nef signaling through Hck in HIV-infected macrophages, slowing disease progression.

Tuftsin-AZT conjugate: potential macrophage targeting for AIDS therapy

The IgG-derived immunomodulating peptide tuftsin, Thr-Lys-Pro-Arg, is recognized by specific receptors on phagocytic cells, notably macrophages, and is capable of targeting proteins and peptides to these sites. Aiming to target 3'-azido-3'-deoxythymidine (AZT) to HIV-infected macrophages, a conjugate of AZT with tuftsin was synthesized. The AZT-tuftsin chimera possesses the characteristic capacities of its two components. Thus, like AZT, it inhibits reverse transcriptase activity and HIV-antigen expression, and similarly to tuftsin, it stimulates IL-1 release from mouse macrophages and augments the immunogenic function of the cells. Importantly, the conjugate is not cytotoxic to T-cells. The results suggest that the AZT-tuftsin conjugate might have potential use in AIDS therapy.

Monocyte/macrophage-derived CC chemokines and their modulation by HIV-1 and cytokines: a complex network of interactions influencing viral replication and AIDS pathogenesis.

Monocytes/macrophages are cells of the innate arm of the immune system and exert important regulatory effects on adaptive immune response. These cells also represent major targets of HIV infection and one of the main reservoirs. Notably, macrophage-tropic viruses are responsible for the initial infection, predominate in the asymptomatic phase, and persist throughout infection, even after the emergence of dual-tropic and T-tropic variants. Functional impairment of HIV-infected macrophages plays an important role in the immune dysregulation typical of AIDS. Recent studies have underlined the pivotal role of chemokines, cytokines, and their receptors in HIV pathogenesis. It is becoming increasingly apparent that the expression level of chemokine receptors, serving as HIV coreceptors, influences the susceptibility of a CD4+ cell to viral infection and to certain HIV envelope-induced alterations in cellular functions. Numerous pathogens, including HIV, can stimulate the production of chemokines and cytokines, which in turn can modulate coreceptor availability, resulting in differential replication potential for R5 and X4 strains, depending on the microenvironment milieu. Thus, a complex network of interactions involving immune mediators produced by monocytes/macrophages and other cell types as a direct/indirect consequence of HIV infection is operative at all stages of the disease and may profoundly influence the extent of viral replication, dissemination, and pathogenesis.

Drug-loaded red blood cell-mediated clearance of HIV-1 macrophage reservoir by selective inhibition of STAT1 expression.

Current highly active antiretroviral therapy (HAART) cannot eliminate HIV-1 from infected persons, mainly because of the existence of refractory viral reservoir(s). Beyond latently-infected CD4+-T lymphocytes, macrophages (M/M) are important persistent reservoirs for HIV in vivo, that represent a major obstacle to HIV-1 eradication. Therefore, a rational therapeutic approach directed to the selective elimination of long-living HIV-infected M/M may be relevant in the therapy of HIV infection. Here we report that HIV-1 chronic infection of human macrophages results in the marked increase of expression and phosphorylation of STAT1, a protein involved in the regulation of many functions such as cell growth, differentiation, and maintenance of cellular homeostasis, thereby providing a new molecular target for drug development. A single and brief exposure to 9-(beta-D-arabinofuranosyl)-2-fluoroadenine 5'-monophosphate (FaraAMP, Fludarabine), a potent antileukemic nucleoside analog active against STAT1 expressing cells, selectively kills macrophage cultures infected by HIV-1 without affecting uninfected macrophages. Furthermore, encapsulation of Fludarabine into autologous erythrocytes (RBC) and targeting to macrophages through a single-18 h treatment with drug-loaded RBC, not only abolishes the Fludarabine-mediated toxic effect on non-phagocytic cells, but also enhances the selective killing of HIV-infected macrophages. As a final result, a potent (>98%) and long-lasting (at least 4 weeks without rebound) inhibition of virus release from drug-loaded RBC-treated chronically-infected macrophages was achieved. Taken together, the evidence of HIV-1-induced increase of STAT1, and the availability of a selective drug targeting system, may prove useful in the design of new pharmacological treatments to clear the HIV-1 macrophage reservoir.

Characterization of HIV-related periodontitis in AIDS patients: HIV-infected macrophage exudate in gingival crevicular fluid as a hallmark of distinctive etiology.

In an attempt to clarify the immunobiological events featuring periodontitis lesions of AIDS patients in the late stage of the disease, peripheral blood (PB) and gingival crevicular fluid (GCF) leucocytes from periodontitis lesions of 23 late-stage AIDS patients were analysed by three-colour flow cytometry for detection and identification of intracytoplasmic p24+ cell fractions. The cells were reacted with CD14 and CD68 for mononuclear phagocytes or with CD4 and CD14 for Th cells, then with anti-p24 MoAb. To detect HIV proviral sequences and intracellular p24 RNA sequences, genomic DNA and cellular RNA from leucocytes were extracted for semi-nested polymerase chain reaction (PCR) amplification. CD68+/p24+ and CD14+/CD68+/p24+ fractions were larger in GCF than in PB (P<0.0001; P < 0.003). CD14+/p24+ fraction was lower in GCF than in PB (P < 0.05). The fluorescence intensities (FI) for intracellular p24 in CD68+ and CD14+/CD68+ cells were higher in GCF than in PB (P < 0.003; P < 0.02), whereas those of CD14+ macrophages did not differ. The p24 FI of CD68+ macrophages in GCF correlated with CD4+ lymphocyte counts in PB (P < 0.005). p24 FI levels of CD14+ monocytes in GCF and PB significantly correlated (P < 0.02), whereas that of CD68+ macrophages did not. PCR and reverse transcriptase (RT)-PCR of cellular DNA and RNA yielded positive signals, demonstrating viral integration and production in GCF leucocytes. These results show that periodontitis lesions in AIDS patients can be characterized by a rapid macrophage turnover, and these HIV-infected macrophage exudates in GCF may be considered as a within-mouth source of virus.

HIV-induced metalloproteinase processing of the chemokine stromal cell derived factor-1 causes neurodegeneration.

The mechanisms of neurodegeneration that result in human immunodeficiency virus (HIV) type 1 dementia have not yet been identified. Here, we report that HIV-infected macrophages secrete the zymogen matrix metalloproteinase-2 (MMP-2), which is activated by exposure to MT1-MMP on neurons. Stromal cell-derived factor 1 alpha (SDF-1), a chemokine overexpressed by astrocytes during HIV infection, was converted to a highly neurotoxic protein after precise proteolytic processing by active MMP-2, which removed the N-terminal tetrapeptide. Implantation of cleaved SDF-1(5-67) into the basal ganglia of mice resulted in neuronal death and inflammation with ensuing neurobehavioral deficits that were abrogated by neutralizing antibodies to SDF-1 and an MMP inhibitor drug. Hence, this study identifies a new in vivo neurotoxic pathway in which cleavage of a chemokine by an induced metalloproteinase results in neuronal apoptosis that leads to neurodegeneration.

Anti-nerve growth factor Ab abrogates macrophage-mediated HIV-1 infection and depletion of CD4+ T lymphocytes in hu-SCID mice.

Infection by HIV-1 causes persistent, long-term high virus production in macrophages. Major evidence, both in humans and in primate models, shows the crucial role of macrophages in sustaining virus production and in mediating a cytopathic effect on bystander CD4+ T lymphocytes and neuronal cells. In the present study, we used severe combined immunodeficient (SCID) mice engrafted with human peripheral blood lymphocytes (hu-PBL-SCID mice) to investigate the in vivo effect of HIV-1-infected macrophages on virus spread and CD4+ T lymphocyte depletion, and the ability of a mAb against nerve growth factor (NGF, a neurokine essential for the survival of HIV-1-infected macrophages) to suppress the pathogenetic events mediated by infected macrophages. Injection of mice with as few as 500 HIV-exposed macrophages causes (i) complete depletion of several millions of autologous CD4+ T lymphocytes, (ii) sustained HIV viremia, and (iii) spreading of HIV-1 DNA in mouse lymphoid organs. In contrast, in vivo treatment with an anti-NGF Ab completely abrogates all effects mediated by HIV-infected macrophages. Taken together, the results demonstrate the remarkable power of macrophages in sustaining in vivo HIV-1 infection, and that such a phenomenon can be specifically abrogated by an anti-NGF Ab. This may open new perspectives of experimental approaches aimed at selectively eliminating persistently infected macrophages from the bodies of HIV-infected patients.

Phosphatidylserine on HIV envelope is a cofactor for infection of monocytic cells.

HIV-1 is an enveloped retrovirus that acquires its outer membrane as the virion exits the cell. Because of the association of apoptosis with the progression of AIDS, HIV-1-infected T cells or macrophages might be expected to express elevated levels of surface phosphatidylserine (PS), a hallmark of programmed cell death. Virions produced by these cells would also be predicted to have PS on the surface of their envelopes. In this study, data are presented that support this hypothesis and suggest that PS is required for macrophage infection. The PS-specific protein annexin V was used to enrich for virus particles and to inhibit HIV-1 replication in primary macrophages, but not T cells. HIV-1 replication was also significantly inhibited with vesicles consisting of PS, but not phosphatidylcholine. PS is specifically required for HIV-1 infection because viruses pseudotyped with vesicular stomatitis virus G and amphotropic murine leukemia virus envelopes were not inhibited by PS vesicles or annexin V. These data indicate that PS is an important cofactor for HIV-1 infection of macrophages.

Regulated expression of sodium-dependent glutamate transporters and synthetase: a neuroprotective role for activated microglia and macrophages in HIV infection?

It is now widely accepted that neuronal damage in HIV infection results mainly from microglial activation and involves apoptosis, oxidative stress and glutamate-mediated neurotoxicity. Glutamate toxicity acts via 2 distinct pathways: an excitotoxic one in which glutamate receptors are hyperactivated, and an oxidative one in which cystine uptake is inhibited, resulting in glutathione depletion and oxidative stress. A number of studies show that astrocytes normally take up glutamate, keeping extracellular glutamate concentration low in the brain and preventing excitotoxicity. This action is inhibited in HIV infection, probably due to the effects of inflammatory mediators and viral proteins. Other in vitro studies as well as in vivo experiments in rodents following mechanical stimulation, show that activated microglia and brain macrophages express high affinity glutamate transporters. These data have been confirmed in chronic inflammation of the brain, particularly in SIV infection, where activated microglia and brain macrophages also express glutamine synthetase. Recent studies in humans with HIV infection show that activated microglia and brain macrophages express the glutamate transporter EAAT-1 and that expression varies according to the disease stage. This suggests that, besides their recognized neurotoxic properties in HIV infection, these cells also have a neuroprotective function, and may partly make up for the inhibited astrocytic function, at least temporarily. This hypothesis might explain the discrepancy between microglial activation which occurs early in the disease, and neuronal apoptosis and neuronal loss which is a late event. In this review article, we discuss the possible neuroprotective and neurotrophic roles of activated microglia and macrophages that may be generated by the expression of high affinity glutamate transporters and glutamine synthetase, 2 major effectors of glial glutamate metabolism, and the implications for HIV-induced neuronal dysfunction, the underlying cause of HIV dementia.

Activation of NR1a/NR2B receptors by monocyte-derived macrophage secretory products: implications for human immunodeficiency virus type one-associated dementia

The final pathways for neuronal injury in human immunodeficiency virus type one (HIV-1)-associated dementia (HAD) were investigated in Xenopus oocytes expressing recombinant NR1a/NR2B N-methyl- d-aspartate (NMDA) receptors exposed to secretory products from HIV-infected macrophages. Pressure ejection of HIV-1-infected and CD40 ligand-stimulated human monocyte-derived macrophage (MDM) fluids produced inward currents in oocytes expressing NR1a/NR2B (30.2±5.1 nA, n=42, mean±SE), but not in uninjected cells. In contrast, control (uninfected MDM) fluids induced currents of 4.5±0.5 nA ( n=17). Infected or stimulated MDM without virus showed intermediate responses. The induced currents were MDM fluid dose-dependant and blocked by the NMDA receptor antagonist 2-amino-5-phosphnovalerate (50 μM), but not by 6-cyano-7-nitroquinoxaline-2,3-dione (20 μM). Although low levels of glutamate were detected in the culture fluids, the addition of l-glutamate decarboxylase to the MDM did not significantly change the level of induced inward currents. Our experiments demonstrate that secretory factors from HIV-1-infected MDM activate NMDA receptors NR1a/NR2B and may contribute to neuronal demise during HAD.

HIV-1 Tat-mediated apoptosis in human brain microvascular endothelial cells.

The integrity of the blood-brain barrier (BBB) is critical for normal brain function. Neuropathological abnormalities in AIDS patients have been associated with perivascular HIV-infected macrophages, gliosis, and abnormalities in the permeability of the BBB. The processes by which HIV causes these pathological conditions are not well understood. To characterize the mechanism by which HIV-1 Tat protein modulates human brain microvascular endothelial cell (HBMEC) functions, we studied the effects of HIV-1 Tat in modulating HBMEC apoptosis and permeability. Treatment of HBMEC with HIV-1 Tat led to Flk-1/KDR and Flt-4 receptor activation and the release of NO. The protein levels of endothelial NO synthase (NOS) and inducible NOS were increased by HIV-1 Tat stimulation. Importantly, HIV-1 Tat caused apoptosis of HBMEC, as evidenced by changes in the cleavage of poly(A)DP-ribose polymerase, DNA laddering, and incorporation of fluorescein into the nicked chromosomal DNA (TUNEL assay). HIV-1 Tat-mediated apoptosis in HBMEC was significantly inhibited in the presence of N-nitro-L-arginine methyl ester (an inhibitor of NOS) and wortmannin (a phosphoinositol 3-kinase inhibitor). Furthermore, HIV-1 Tat treatment significantly increased HBMEC permeability, and pretreatment with both N-nitro-L-arginine methyl ester and wortmannin inhibited the Tat-induced permeability. Taken together, these results indicate that dysregulated production of NO by HIV-1 Tat plays a pivotal role in brain endothelial injury, resulting in the irreversible loss of BBB integrity, which may lead to enhanced infiltration of virus-carrying cells across the BBB.

Double-edged effect of Vgamma9/Vdelta2 T lymphocytes on viral expression in an in vitro model of HIV-1/mycobacteria co-infection.

A reciprocal influence exists between mycobacteria and HIV: HIV-infected individuals are more susceptible to mycobacterial infections and, on the other hand, mycobacterial infection results inacceleration of HIV disease progression. Vgamma9/Vdelta2 T lymphocytes are known to participate in the defense against intracellular pathogens, including Mycobacterium tuberculosis. Indeed, they kill mycobacteria-infected macrophages and, upon recognition of mycobacterial Ag, release TNF-alpha and IFN-gamma, which are also up-regulators of HIV expression. To assess whether mycobacteria-activated gamma delta T lymphocytes contribute to the enhancement of HIV replication, we established an in vitro model mimicking HIV and mycobacteria co-infection with the latently HIV-infected promonocytic U1 cell line and Vgamma9/Vdelta2 peripheral lymphocytes stimulated with mycobacterial Ag. gamma delta T cell activation determined two distinct, but connected effects, namely U1cell death and HIV expression. Both effects were mainly mediated by release of TNF-alpha and IFN-gamma from activated gamma delta lymphocytes, although Fas-FasL interaction also contributed to U1 apoptosis. The final outcome on U1 survival, and thus, on HIV expression, highly depended on mycobacterial Ag concentration coupled to the differential secretory potency of gamma delta cells. In particular, the induction of viral expression prevailed at low Ag concentration and with lower cytokine production by mycobacteria-activated gamma delta cells. Notably, during the course of HIV infection, Vgamma9/Vdelta2 lymphocytes are reported to be functionally impaired and may thus indirectly influence the progression of HIV disease. In addition, a predominant inhibition of viral replication was encountered when mycobacteria-activated gamma delta T cells were co-cultured with primary HIV-infected macrophages. Thus, we suggest that specific recognition of mycobacterial Ag by gamma delta T lymphocytes in co-infected individuals may modulate viral replication through the complex array of soluble factors released.

Neuroprotective effects of IGF-I against TNFalpha-induced neuronal damage in HIV-associated dementia.

Human immunodeficiency virus type 1 (HIV-1) infection often results in disorders of the central nervous system, including HIV-associated dementia (HAD). It is suspected that tumor necrosis factor-alpha (TNFalpha) released by activated and/or infected macrophages/microglia plays a role in the process of neuronal damage seen in AIDS patients. In light of earlier studies showing that the activation of the insulin-like growth factor I receptor (IGF-IR) exerts a strong neuroprotective effect, we investigated the ability of IGF-I to protect neuronal cells from HIV-infected macrophages. Our results demonstrate that the conditioned medium from HIV-1-infected macrophages, HIV/CM, causes loss of neuronal processes in differentiated PC12 and P19 neurons and that these neurodegenerative effects are associated with the presence of TNFalpha. Furthermore, we demonstrate that IGF-I rescues differentiated neurons from both HIV/CM and TNFalpha-induced damage and that IGF-I-mediated neuroprotection is strongly enhanced by overexpression of the wt IGF-IR cDNA and attenuated by the antisense IGF-IR cDNA. Finally, IGF-I-mediated antiapoptotic pathways are continuously functional in differentiated neurons exposed to HIV/CM and are likely supported by TNFalpha-mediated phosphorylation of I(kappa)B. All together these results suggest that the balance between TNFalpha and IGF-IR signaling pathways may control the extent of neuronal injury in this HIV-related experimental setting.

Macrophage activation and HIV infection: can the Trojan horse turn into a fortress?

Macrophages are infected early during HIV infection and are thought to play the role of a Trojan horse by spreading infection in tissues. Most recent studies point out to a more complex role for macrophages in HIV infection: macrophages could contribute to both host defense and viral persistence and pathogenesis. Infected macrophages are a reservoir for HIV and modulate apoptosis of T cells present in their vicinity. Also, a functional impairment of HIV-infected macrophages may play a role in AIDS pathogenesis. Nevertheless, both activation and differentiation of monocyte/macrophages can interfere with susceptibility of these cells to infection. Therefore, a wide variety of stimuli result in HIV suppression through macrophage activation. At present times, a dynamic view on the role of macrophages in HIV infection arises which indicates that macrophages are a target for the virus and at the same time regulate its replication. Therefore, macrophages are at the cross-road between protection and pathogenesis in HIV infection due to their involvement both as a viral target and a key modulator of non-specific and specific immune responses. Future studies will help unravel the cellular and molecular mechanisms that underlie HIV-macrophage interactions and might result in new vaccine and/or therapeutic strategies.

Long-term survival and virus production in human primary macrophages infected by human immunodeficiency virus.

The role of macrophages in the pathogenesis and progression of human immunodeficiency virus (HIV)-related infection is substantiated by in vitro and in vivo evidence. The unique ability to survive HIV infection and produce viral particles for long periods is postulated. Detailed studies of this phenomenon are lacking. The dynamics of HIV-1 replication and cumulative virus production was studied in long-term cultures of macrophages in the presence or in the absence of antiviral drugs. Multiply spliced and unspliced HIV-RNA production was assessed by quantitative PCR, and the number of infected cells was monitored by FACS analysis. Cumulative HIV-1 production was determined by a trapezoidal equation, including such parameters as times of collection and experimental values of genomic-RNA and p24 gag antigen. Unspliced and multiply spliced HIV-RNA increased linearly after macrophage infection; reached levels of 1.5 x 10(8) and 2.8 x 10(5) copies/10(5) cells, respectively, at day 10; and then remained stable throughout the course of the experiment. Cumulative production of genomic-RNA and p24 gag antigen was 10(10) copies/10(6) cells and 10(7) pg/10(6) cells, respectively, with an average of >200 virus particles produced daily by each macrophage. AZT decreased the cumulative production of both genomic-RNA and p24 gag antigen down to 2.5 x 10(9) copies and 1.1 x 10(6) pg/10(6) cells (73.8% and 88.9% inhibition, respectively) up to day 50 without virus breakthrough. Ritonavir had a limited, but consistent, efficacy on the release of mature virus proteins (about 40% inhibition), but not on HIV-RNA production. In conclusion, the long-term dynamics and the high cumulative virus production that characterize HIV-1 infection of macrophages underscore the peculiar role of these cells as a persistently infected reservoir of HIV.

Development of a human neuronal cell model for human immunodeficiency virus (HIV)-infected macrophage-induced neurotoxicity: apoptosis induced by HIV type 1 primary isolates and evidence for involvement of the Bcl-2/Bcl-xL-sensitive intrinsic apoptosis pathway.

Neuronal apoptosis within the central nervous system (CNS) is a characteristic feature of AIDS dementia, and it represents a common mechanism of neuronal death induced by neurotoxins (e.g., glutamate) released from human immunodeficiency virus (HIV)-infected macrophages (HIV/macrophage-induced neurotoxicity). Neuronal apoptosis may result from activation of the intrinsic (mitochondrial/bcl-2 regulated) or extrinsic (death receptor) pathways, although which pathway predominates in CNS HIV infection is unknown. Apoptosis initiated by the intrinsic pathway is typically blocked by antiapoptosis Bcl-2 family proteins, such as Bcl-2 and Bcl-xL, but whether these can block HIV/macrophage-induced neuronal apoptosis is unknown. To determine the potential role of the Bcl-2 family in HIV/macrophage-induced neuronal apoptosis, we developed a unique in vitro model, utilizing the NT2 neuronal cell line, primary astrocytes and macrophages, and primary CNS HIV type 1 (HIV-1) isolates. We validated our model by demonstrating that NT2.N neurons are protected against HIV-infected macrophages by N-methyl-D-aspartate (NMDA) glutamate receptor antagonists, similar to effects seen in primary neurons. We then established stable NT2.N neuronal lines that overexpress Bcl-2 or Bcl-xL (NT2.N/bcl-2 and NT2.N/bcl-xL, respectively) and determined their sensitivity to macrophages infected with primary R5, X4, and R5/X4 HIV-1 isolates. We found that NT2.N/bcl-2 and NT2.N/bcl-xL neurons were resistant to apoptosis induced by either R5, X4, or R5/X4 isolates and that resistance was abrogated by a Bcl-2 antagonist. Thus, the NMDA receptor/bcl-2-regulated apoptotic pathway contributes significantly to HIV/macrophage-induced neuronal apoptosis, and Bcl-2 family proteins protect neurons against the spectrum of primary HIV-1 isolates. Modulation of bcl-2 gene expression may therefore offer adjunctive neuroprotection against development of AIDS dementia.