HIV-infected Monocytes Research Articles

Anti-HIV effect of beta subunit of human chorionic gonadotropin (βhCG) in vitro

Human chorionic gonadotropin (hCG)—a pregnancy-associated immunomodulating hormone—has been recently shown in vitro to suppress reverse transcriptase activity in chronically HIV-infected lymphocytes and monocytes and to block viral transmission resulting from cell-cell contact between virus-carrying lymphocytes and placental trophoblasts. In further pursuit of the query into the mechanism of action, purified α and β subunits of hCG were tested for the inhibition of p24 gag protein synthesis in virus-producing ACH-2 lymphocytes and U1 monocytes. Unlike the α subunit, β-hCG displayed a distinct U-shaped dose response, characteristic of the effect of dimer hCG. Maximum inhibition of viral expression has been achieved at 10–100 ng/ml, the concentration corresponding to blood levels of β-hCG in pregnant women. The doses that were several logs higher of normal levels seemed to increase viral production in monocytes. The data presented supports our original observations regarding the effect of intact hCG on HIV replication. While the mechanism of action remains to be established, the results suggest that the virus-interfering activity of hCG is determined by hormone-specific β chain but not by the α subunit—shared with the family of glycoprotein hormones from the pituitary—follicle-stimulating hormone, luteinizing hormone and thyrotropin.

Interleukin 4 and human immunodeficiency virus stimulate LFA-1-ICAM-1-mediated aggregation of monocytes and subsequent giant cell formation

The effects of recombinant interleukin 4 (IL-4) on cell cluster and multinucleated giant cell (MGC) formation from human immunodeficiency virus (HIV)-infected and uninfected monocytes were examined. Human blood monocytes were isolated by centrifugal elutriation and monoclonal antibody-complement-dependent lysis of residual T cells, and infected with low passage HIV strains. Monocytes were exposed to recombinant IL-4 (1 to 20 ng/ml), continuously after inoculation with HIV. Monocyte expression of ICAM-1 but not LFA-1 was significantly enhanced by IL-4 although substrate adherence was a more potent stimulus. Monocyte cluster and MGC formation was quantified after fixation and staining with Giemsa. Clusters of HIV-infected and uninfected monocytes were consistently and significantly increased at 4 to 7 days after IL-4 stimulation. The combination of HIV and IL-4 was more stimulatory than either treatment alone. In two out of five uninfected and three out of seven HIV-infected monocyte cultures, MGC formation was also markedly increased at 10 to 14 days after stimulation. Incubation with anti-LFA-1 (anti-CD11a, anti-CD18) and anti-ICAM-1 (anti-CD54) monoclonal antibodies reduced IL-4-stimulated aggregation in HIV-infected and uninfected monocytes and subsequently reduced MGC formation. Anti-ICAM-1 was not as effective as anti-CD11a or anti-CD18 in inhibiting aggregation of HIV-infected monocytes and in these cultures anti-ICAM-2 was also inhibitory. Extracellular HIV antigen concentrations were not consistently reduced by anti-CD11a or anti-ICAM-1. Hence IL-4 markedly enhanced monocyte aggregation in both HIV-infected and uninfected monocytes, probably through enhanced LFA-1-ICAM-1 interactions in all cultures and LFA-1-ICAM-2 interactions in infected monocytes, leading subsequently to MGC formation in some cultures.

Macrophage-colony stimulating factor (M-CSF) stimulation induces cell death in HIV-infected human monocytes.

We show here that HIV-infected monocyte-macrophages stimulated by macrophage-colony stimulating factor (M-CSF) undergo massive syncytia formation and die. The M-CSF-stimulated HIV-infected monocyte-macrophages (M/M) destroy themselves by blebbing out particles (resembling apoptotic bodies) which may contain condensed and marginated chromatin. The death of monocyte-macrophages is also characterized by the expression of "Tissue" Transglutaminase (tTG) which is one of the genes specifically expressed and activated in apoptising cells. Noteworthy, when the syncytia formation and consequently death is prevented, infected monocyte-macrophages remain viable and produce large amounts of virus for an extended period. The concentrations of M-CSF (1000 U/ml) used in this work are similar to those that stimulate macrophages in vivo. This suggests that HIV killing of M/M in the presence of M-CSF could lead, in vivo, to a greater than expected loss of immune cells and may contribute to explain the complex derangement of the immune function observed in HIV-infected patients.

Interferon-gamma protects primary monocytes against infection with human immunodeficiency virus type 1.

Monocytes treated with 500 IU/ml human recombinant interferon-gamma (rIFN-gamma) 1 day before and continuously after human immunodeficiency virus (HIV) infection showed no evidence of virus replication 7 days after addition of the viral inoculum. There was no HIV-associated cytopathic effect, no reverse transcriptase (RT) activity or p24 detected in culture fluids, and no HIV RNA or DNA in cell lysates. Furthermore, no evidence of HIV infection was evident in replicate cultures in which all IFN-gamma was removed at 7 days and the cells were cultured for an additional 3 weeks without IFN-gamma. The 50% inhibitory dose for reduction of maximum RT activity in HIV-infected monocyte cultures was about 1 IU/ml IFN-gamma. No increase in HIV replication was evident in monocytes treated with IFN-gamma at any concentration (0 to 5000 IU/ml) or at any time (7 days before to 10 days after HIV infection). In side-by-side experiments with identical monocytes and HIV-1 stock, rIFN-gamma was 10 to 20 times more effective than rIFN-alpha 2b for induction of antiviral activity. With both interferons, significant antiviral activity was evident with monocytes treated 1 day before, at the time of, or up to 3 days after infection. At 7 to 10 days after infection (a time at which less than 20% of total cells were infected with HIV) addition of even high concentrations of IFN-alpha or IFN-gamma had no effect on virus replication. These data suggest that the principal action of IFN-alpha and IFN-gamma was directed against the fluid-phase virus. Cell-cell spread of infection within the HIV-infected monocyte culture and extent of virus replication in HIV-infected cells were not affected by interferon treatment.

IL-4 and IL-13 have overlapping but distinct effects on HIV production in monocytes.

In HIV-1-infected monocytes and monocytoid cell lines, viral expression can be observed as high-level production, restricted (chronic low-level) expression, and latency (no viral expression). Interleukin-13 (IL-13) and IL-4, which have remarkedly similar deactivating effects on inflammatory monocyte functions, were studied for their regulation of HIV expression in monocytes. Pretreatment of peripheral monocytes for 48-72 h with IL-13 markedly decreased acute HIV infection, whereas IL-4 increased it. Similar effects were seen when the U1 and R-THP-1 monocytoid cell lines with restricted HIV expression were treated with these cytokines. However, when these continuously producing cell lines were chronically treated with cytokines, IL-13 increased HIV production. Neither IL-4 nor IL-13 stimulated HIV expression in latently infected cells. In chronically infected cells, several cytokines reduced viral mRNA. Both IL-4 and IL-13 increased monocyte aggregate formation, but only IL-4 ultimately stimulated cytolysis of HIV-infected monocytes as well as increased apoptosis of U1. In the presence of tumor necrosis factor alpha or IL-6, which upregulate HIV expression, IL-13 could no longer suppress HIV expression. These results indicate that IL-4 and IL-13, although closely related in modulating monocyte function, can have divergent effects on HIV expression in monocytes. Collectively, these data suggest that there exists a complex cytokine tissue environment with positive regulators of HIV expression able to override negative regulators.

Platelet-activating factor: a candidate human immunodeficiency virus type 1-induced neurotoxin.

The pathogenesis of central nervous system disease during human immunodeficiency virus type 1 (HIV-1) infection revolves around productive viral infection of brain macrophages and microglia. Neuronal losses in the cortex and subcortical gray matter accompany macrophage infection. The question of how viral infection of brain macrophages ultimately leads to central nervous system (CNS) pathology remains unanswered. Our previous work demonstrated high-level production of tumor necrosis factor alpha, interleukin 1 beta, arachidonic acid metabolites, and platelet-activating factor (PAF) from HIV-infected monocytes and astroglia (H. E. Gendelman, P. Genis, M. Jett, and H. S. L. M. Nottet, in E. Major, ed., Technical Advances in AIDS Research in the Nervous System, in press; P. Genis, M. Jett, E. W. Bernton, H. A. Gelbard, K. Dzenko, R. Keane, L. Resnick, D. J. Volsky, L. G. Epstein, and H. E. Gendelman, J. Exp. Med. 176:1703-1718, 1992). These factors, together, were neurotoxic. The relative role(s) of each of these candidate neurotoxins in HIV-1-related CNS dysfunction was not unraveled by these initial experiments. We now report that PAF is produced during HIV-1-infected monocyte-astroglia interactions. PAF was detected at high levels in CSF of HIV-1-infected patients with immunosuppression and signs of CNS dysfunction. The biologic significance of the results for neurological disease was determined by addition of PAF to cultures of primary human fetal cortical or rat postnatal retinal ganglion neurons. Here, PAF at concentrations of > or = 300 pg/ml produced neuronal death. The N-methyl-D-aspartate receptor antagonist MK-801 or memantine partially blocked the neurotoxic effects of PAF. The identification of PAF as an HIV-1-induced neurotoxin provides new insights into how HIV-1 causes neurological impairment and how it may ultimately be ameliorated.

HIV-1 infection of in vitro cultured human monocytes: early events and influence of anti HIV-1 antibodies.

To characterize the role of the humoral immune response on HIV-1 infection of monocytes and macrophages (M phi s) we examined the susceptibility of in vitro cultured monocyte/M phi s to various HIV-1 isolates and the influence of heterologous and particularly autologous anti HIV-1 sera on this infection. Depending on the period of in vitro cultivation and the virus isolate used different patterns of susceptibility were detected. One week old monocyte/M phi s were highly susceptible to HIV-1 infection, in contrast to monocyte/M phi s cultured 4 weeks. The infection by virus isolated immediately after seroconversion lead to persistent infection with high level of antigen production in contrast to infection by homologous virus isolated later. MAb against the V3-IIIB loop and sCD4 inhibited the infection of monocyte/M phi s in a dose dependent manner, indicating that infection requires binding to CD4 and that post binding events may be common to the infection of lymphocytes. Anti HIV-1 sera showed neutralizing activity against heterologous and even autologous escape virus. This finding, together with the observation that monocytes and M phi s are infected in vivo, suggests that protection against HIV-1 infection of monocytes and M phi s in vivo may not be obtainable by the humoral immune response alone.

Regulation of interferon-alpha-inducible cellular genes in human immunodeficiency virus-infected monocytes.

Cellular mechanisms that control susceptibility to opportunistic infection in human immunodeficiency virus (HIV)-infected individuals remain poorly understood. HIV may induce certain cellular genes that restrict HIV replication and protect cells against other superinfecting viral pathogens. Indeed, HIV-infected monocytes resist infection by vesicular stomatitis virus (VSV). HIV-induced VSV interference in monocytes increases with time after HIV infection. Such interference was evident 6 h after HIV infection and reached maximal levels at 14 days. Monocytotropic but not T cell-tropic HIV strains elicited these effects, signaling a requirement for viral entry and/or replication. Viral interference was independent of interferon (IFN) and was unaffected by addition of neutralizing IFN-alpha and -beta antibodies. The well-described IFN-alpha-inducible antiviral pathways were examined to determine their relationship to the cellular mechanism(s) underlying VSV interference. HIV and IFN-alpha both induced the expression of 2-5A synthetase and Mx gene. In contrast, the guanylate-binding protein (GBP), 6-16, and 9-27 cellular genes were up-regulated by IFN-alpha but not HIV. MxA was detected in HIV-infected monocytes but not in uninfected monocytes. The association between Mx expression and resistance to VSV, coupled with previously described anti-VSV activities by human MxA, suggested that Mx may be an effector molecule for the HIV-induced anti-VSV activities. These results, taken together, suggest that HIV can induce antiviral cellular gene expression, independent of IFN.

The role of the blood-brain barrier in HIV infection of the central nervous system

The blood-brain barrier (BBB) functions to regulate the entry of macromolecules, microbial pathogens, and circulating leukocytes into the central nervous system (CNS). It consists, in part, of the microvascular endothelium and associated astrocyte foot processes, found in close apposition to the abluminal side of the vascular endothelial cells (EC). During the pathogenesis of certain nervous system diseases with inflammatory components, the BBB may function to facilitate the entry of leukocytes into the CNS parenchyma. A common histologic observation in human immunodeficiency virus type-1 (HIV) encephalitis is the localization of HIV proteins to multinucleated giant cells that co-immunolabel with antibodies specific for cells of the monocyte/macrophage lineage, suggesting that HIV can enter the CNS as cell-associated virus. We previously characterized a tissue culture model of the BBB that consists of the co-culture of autologous EC and astrocytes. In this presentation, we used this model to examine the expression of adhesion molecules by both the EC and astrocyte components of this BBB model, and to characterize the interactions between HIV-infected monocytes and EC. The data presented in this review of our work demonstrates that astrocytes upregulate the expression of intercellular adhesion molecule (ICAM-1) by EC. In a parallel study, western blot analysis demonstrated that ICAM-1 is also expressed in the developing human CNS. When exposed to the proinflammatory cytokine tumor necrosis factor alpha (TNF), both EC cocultured with astrocytes and astrocytes cultured alone expressed the adhesion proteins IG9, ICAM-1, vascular cell adhesion molecule 1 (VCAM) and E-selection.(ABSTRACT TRUNCATED AT 250 WORDS)

Suppression by HIV of IL-1 and IL-6 Secretion in Accessory Cells: AC Function Defect Partially Corrected with Exogenous IL-1 and IL-6

To determine the effect of HIV infection on the accessory cell function of monocytes we measured the ability of HIV-infected monocytes to restore PHA-induced and soluble anti-CD3-induced T cell blastogenesis. These T cells were highly purified and depleted of monocytes (<0.5%) and activated T cells. Monocytes were isolated using gelatin-fibronectin-coated flasks (<1% T cells) and after 4 days in culture with granulocyte/macrophage-colony stimulating factor, they were infected with HIV. Accessory cell (AC) function was tested 2 and 7 days later, employing autologous cryopreserved T lymphocytes. Monocytes infected with HIV for 2 days lacked the ability to permit phytohemagglutinin (PHA) and anti-CD3-induced T cell blastogenesis. Noninfected monocytes restored the proliferative response of purified T cells. Interleukin 1 (IL-1) and interleukin 6 (IL-6) levels in culture supernatants were low when compared to cultures with noninfected AC. Preincubation of monocytes with human anti-HIV neutralizing antibodies did not restore either of the responses. AC treated with heat-inactivated HIV had normal accessory cell function. The addition of IL-1 and/or IL-6 partially restored the AC function for PHA stimulation, but not for anti-CD3 stimulation. We conclude that HIV infection of monocytes suppressed their accessory cell function in the T cell blastogenesis assay. The response was partially restored with IL-1 and/or IL-6, suggesting that HIV infection down-regulated the monocyte production of both cytokines.

Neurotoxic effects of tumor necrosis factor alpha in primary human neuronal cultures are mediated by activation of the glutamate AMPA receptor subtype: implications for AIDS neuropathogenesis.

Human immunodeficiency virus type 1 (HIV) infection of the central nervous system is characterized by neuronal loss in discrete areas of the central nervous system. We have previously demonstrated that HIV-infected monocytes in culture with astroglial cells produce high levels (> or = 200 pg/ml) of the cytokine tumor necrosis factor-alpha (TNF alpha). We now demonstrate that TNF alpha (> or = 200 pg/ml) is neurotoxic to cultured primary human fetal cortical neurons at both light and electron microscopic levels. Subtoxic doses of TNF alpha (50 pg/ml) are neurotoxic in combination with the glutamate (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) subtype receptor agonist AMPA (100 microM). The neurotoxic effects of TNF alpha (200 pg/ml) are blocked in part by the AMPA receptor antagonist, 6-cyano-7-nitroquinoxaline-2, 3-dione (10 microM). This suggests that TNF alpha may exert neurotoxic effects on human neurons by indirect activation of AMPA receptors, which may be important in the pathogenesis and treatment of HIV-mediated encephalopathy.

Adhesion proteins on human microglial cells and modulation of their expression by IL1α and TNFα

Expression of adhesion proteins on human microglial cells was studied by immunocytochemistry. Both microglial cells and peripheral blood monocytes expressed beta 2 integrins and molecules of the immunoglobulin superfamily at similar levels whereas the expression of the beta 1 integrins (alpha 2-VLA (very late antigen), alpha 4-VLA, alpha 5-VLA, alpha 6-VLA) was higher on microglial cells than on monocytes. Stimulation of microglial cells with interleukin-1 alpha and tumour necrosis factor-alpha, the main cytokines detected in HIV1-infected central nervous system (CNS), increased the microglial expression of alpha 1-VLA, intercellular adhesion molecule-1, vascular cell adhesion molecule-1 and beta 2-LFA-1 (leukocyte-function-associated molecule-1) but not of alpha L-LFA-1. Such an induction of adhesion molecules could facilitate penetration of HIV1-infected monocytes into brain parenchyma and their adhesion to CNS cells, and could maintain a chronic inflammation during human immunodeficiency virus-1 (HIV1) encephalopathy.

Cytokines and arachidonic metabolites produced during human immunodeficiency virus (HIV)-infected macrophage-astroglia interactions: implications for the neuropathogenesis of HIV disease.

Human immunodeficiency virus (HIV) infection of brain macrophages and astroglial proliferation are central features of HIV-induced central nervous system (CNS) disorders. These observations suggest that glial cellular interactions participate in disease. In an experimental system to examine this process, we found that cocultures of HIV-infected monocytes and astroglia release high levels of cytokines and arachidonate metabolites leading to neuronotoxicity. HIV-1ADA-infected monocytes cocultured with human glia (astrocytoma, neuroglia, and primary human astrocytes) synthesized tumor necrosis factor (TNF-alpha) and interleukin 1 beta (IL-1 beta) as assayed by coupled reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, and biological activity. The cytokine induction was selective, cell specific, and associated with induction of arachidonic acid metabolites. TNF-beta, IL-1 alpha, IL-6, interferon alpha (IFN-alpha), and IFN-gamma were not produced. Leukotriene B4, leukotriene D4, lipoxin A4, and platelet-activating factor were detected in large amounts after high-performance liquid chromatography separation and correlated with cytokine activity. Specific inhibitors of the arachidonic cascade markedly diminished the cytokine response suggesting regulatory relationships between these factors. Cocultures of HIV-infected monocytes and neuroblastoma or endothelial cells, or HIV-infected monocyte fluids, sucrose gradient-concentrated viral particles, and paraformaldehyde-fixed or freeze-thawed HIV-infected monocytes placed onto astroglia failed to induce cytokines and neuronotoxins. This demonstrated that viable monocyte-astroglia interactions were required for the cell reactions. The addition of actinomycin D or cycloheximide to the HIV-infected monocytes before coculture reduced, > 2.5-fold, the levels of TNF-alpha. These results, taken together, suggest that the neuronotoxicity associated with HIV central nervous system disorders is mediated, in part, through cytokines and arachidonic acid metabolites, produced during cell-to-cell interactions between HIV-infected brain macrophages and astrocytes.

A novel coculture system for evaluating anti-HIV drugs.

To develop a useful system for evaluating novel anti-HIV drugs. The activity of most antiviral compounds in cell-free HIV infection systems has been evaluated. However, the inhibitory effects on both the process of HIV induction and viral dissemination to uninfected cells have not been fully investigated. We have therefore developed a new cocultivation system using chronically HIV-infected monocytes and CD4+ T-lymphocytes in the presence of tumor necrosis factor (TNF). We designed a cocultivation system using flow cytometry with U1 cells and Molt-4 cells in the presence of TNF. The antiviral activities of several compounds in the cocultivation system and other assay systems were compared. Only pradimicin A and glycyrrhizin showed strong inhibitory activity in the cocultivation system in the presence of TNF, whereas dextran sulfate, curdlan sulfate and N-acetylcysteine exhibited moderate or weak inhibitory activity in the system. 3'-azido-2',3'-dideoxythymidine and 2',3'-dideoxyadenosine were completely ineffective in the system. These results support the suggestion that our cocultivation system includes HIV induction in chronically infected monocytes, and the resulting cell-to-cell infection between HIV-infected monocytes and Molt-4 cells or Molt-4 cells and their HIV-converted counterparts. Our new cocultivation system may constitute a useful tool in the identification of novel anti-HIV compounds.

Effect of human chorionic gonadotropin (hCG) on reverse transcriptase activity in HIV-1 infected lymphocytes and monocytes

The majority of infants born to HIV-positive mothers are not infected in utero, suggesting that the pregnancy factors produced by fetal trophoblasts may provide protection against HIV-1 infection. Except for steroid female hormones, little is know of other pregnancy factors that may regulate either resistance or susceptibility to HIV-1. Human chorionic gonadotropin (hCG) — the major glycoprotein produced by the placental trophoblast throughout the pregnancy — was tested on reverse transcriptase activity in HIV-infected ACH-2 lymphocytes and U1 monocytes. The results suggest that low non-cytotoxic doses of hCG (0.01–1.0 IU range) may inhibit viral replication in maternal blood cells.

Interactions between HIV-infected monocytes and the extracellular matrix: HIV-infected monocytes secrete neutral metalloproteases that degrade basement membrane protein matrices.

The frequency of human immunodeficiency virus (HIV)-infected monocytes that spread on a model basement membrane was about twofold greater than that of an equal number of uninfected control cells through the initial 12 to 18 h of culture. By 24 h, virtually all HIV-infected and uninfected control cells spread on the basement membrane gel. The frequency of spread cells in the uninfected control population was less than 10% of total cells by 12 days. In contrast, 30 to 40% of HIV-infected monocytes remained spread through this time interval and formed a dense interdigitated network of cell processes on and into the gel matrix. Invasion of the basement membrane matrix by HIV-infected monocytes suggested increased secretion of proteases able to digest the gel. Indeed, levels of neutral protease activity in culture fluids from HIV-infected monocytes were significantly higher than those from equal numbers of uninfected control cells. High levels of protease activity in culture fluids of HIV-infected monocytes required productive virus infection and were not observed with cells exposed to T cell-tropic HIV isolates. The predominant protease activity in these cultures was a 92-kd neutral metallogelatinase. HIV-induced changes in monocyte metalloprotease activity may be important for extravasation of infected cells to tissue or for the development of AIDS-associated neuropathology, carcinogenesis, and opportunistic infection.

Interactions between HIV-infected monocytes and the extracellular matrix: increased capacity of HIV-infected monocytes to adhere to and spread on extracellular matrix associated with changes in extent of virus replication and cytopathic effects in infected cells.

Monocytes express cell surface receptors for extracellular matrix (ECM) proteins of basement membranes. These receptors are engaged during extravasation of cells through capillary endothelium into tissue. The number of human immunodeficiency virus (HIV)-infected monocytes that adhered to ECM over 2 h was threefold higher than that of uninfected control cells. This difference was ECM specific and was not observed with a bovine serum albumin substrate. Enhanced adhesion to ECM was evident in monocytes by 4 days after HIV infection and increased through 10 days. Monocytes exposed to a T cell-tropic HIV strain that binds to but does not replicate in monocytes showed no changes in adherence to ECM. Thus, productive infection of monocytes by HIV induces a significant increase in the capacity of these cells to interact with ECM. Enhanced adhesion of HIV-infected monocytes to ECM was associated with increased spreading: at 12 h, sixfold more HIV-infected monocytes were spread on ECM than were uninfected control cells. Cell processes of HIV-infected monocytes formed a complex network on ECM: many of these cells expressed HIV proteins as detected by indirect immunofluorescence. HIV-associated cytopathic effects and levels of virion-associated reverse transcriptase activity depended on the substrate to which monocytes were attached. Virus replication and cytopathic effects in monocytes adhered to ECM, fibronectin, or plastic alone were comparable. In contrast, HIV-infected monocytes attached to laminin showed a significant increase in virus replication and in extent of cytopathic effects through 2 weeks after infection. The lowest levels of HIV replication and cytopathic effects were in monocytes attached to collagen IV. Interactions between monocytes and ECM profoundly affect the manner in which these cells control HIV infection: HIV infection changes the capacity of infected monocytes to attach and spread on ECM; attachment to ECM alters the extent of virus replication in infected cells.

No direct neuronotoxicity by HIV-1 virions or culture fluids from HIV-1-infected T cells or monocytes.

Macrophages and microglia are the principal target cells for human immunodeficiency virus (HIV) in brain, and as such, are likely participants in the neuropathology of HIV infection. In a model system for this process, we found that fluids from human monocyte cultures enhanced survival and differentiation of the neurons in fetal rat brain explants. In contrast, fluids from HIV-infected monocyte cultures were strongly toxic to neurons and paradoxically enhanced the proliferation of glial cells. Further, neuronotoxic activity in these fluids was mediated through activation of NMDA binding receptors on the neurons and was inhibited by any of several different NMDA antagonists. Neuronotoxic activity was directly related to contamination of the HIV virus stock with Mycoplasma arginini and M. hominis. Pure cultures of mycoplasma, bacterial lipopolysaccharide (LPS), or murine recombinant tumor necrosis factor alpha (rTNF alpha) each induced neuronotoxicity which exactly mirrored that induced by the contaminated HIV stock. It is likely that mycoplasma or components of the mycoplasma plasma membrane stimulate TNF alpha production by the glial cells in the brain explants. Indeed, careful depletion of glial cells in these explants prevented mycoplasma or LPS-mediated neuronotoxicity. No neuronotoxicity was evident with HIV-1 virus stock, HIV-1 gp120, or culture fluids from HIV-infected T cells or monocytes when these preparations were free of contamination by mycoplasma and LPS. These findings suggest caution in interpretation of those experiments in which similar contamination has not been rigorously excluded.

Interferons in the persistence, pathogenesis, and treatment of HIV infection.

Interferon (IFN) plays an important role in the treatment and pathogenesis of HIV disease. Recent studies show beneficial effects of IFN alpha in the treatment of HIV-associated Kaposi's Sarcoma and early HIV-infection. Moreover, cell culture studies support these beneficial effects. HIV infection of monocytes is blocked by IFN alpha administered at the time of viral challenge. The IFN alpha-treated cells show no evidence of HIV infection. Viral gene products produced in monocytes infected with HIV then treated with IFN alpha gradually decrease to baseline. Large quantities of proviral DNA are seen in the HIV-infected IFN alpha-treated cells with little evidence for viral transcription suggesting true microbiological latency. While most viral infections of cells result in IFN production, HIV is a notable exception. Indeed, HIV does not induce monocytes to produce IFN alpha and blocks its production following poly(I).poly(c) stimulation. This allows HIV yet another mechanism to evade an important host antiviral response. Paradoxically, the appearance of IFN activity in sera of HIV-infected patients is associated with disease progression, not resolution. Recent observations showing that the interaction between HIV-infected monocytes and PBMC results in the production of IFN alpha s with reduced anti-HIV activity may help explain this paradox. Thus, IFN alpha plays an important but complex role in HIV disease. The elucidation of cellular factors that regulate the antiretroviral effects of IFN alpha may lead to the development of novel therapeutic strategies for HIV infection.

Induction of IFN-alpha in peripheral blood mononuclear cells by HIV-infected monocytes. Restricted antiviral activity of the HIV-induced IFN.

PBMC cocultured with HIV-infected monocytes for 12 to 48 h released high levels of IFN activity. IFN titers were directly dependent upon time after virus infection and level of HIV replication in infected cells. IFN induction in PBMC was evident with HIV-infected monocytes and PBMC and with myeloid and lymphoblastoid cell lines with at least three different HIV strains. In HIV-infected cell line pairs in which virus infection occurs in both productive and restricted forms, IFN induction in PBMC occurred only with productive infection. IFN activity was acid stable and completely neutralized by antibodies against IFN-alpha. Induction of IFN required cell-cell contact between HIV-infected cells and PBMC, but was independent of MHC compatibility. With PBMC co-cultured with autologous HIV-infected monocytes, IFN induction was highly selective: IL-1 beta, IL-6, or TNF-alpha activity and mRNA were not detected. Cell surface determinants on HIV-infected monocytes that induced IFN in PBMC remained active after fixation in 4% paraformaldehyde. Both adherent and nonadherent PBMC produced IFN after coculture with HIV-infected monocytes. Ability to produce IFN by PBMC was not affected by depletion of T cell, NK cell, B cell, or monocyte subpopulations. The IFN activity produced by PBMC cocultured with HIV-infected cells was about 20-fold less active than equal quantities of rIFN-alpha 2b for inhibition of HIV replication in monocytes and at low concentrations enhanced virus growth. Clinical studies with HIV-infected patients and parallel findings in animal lentivirus disease suggest an adverse role for IFN in disease progression. Conditions for induction of IFN in the culture system described in this report may mimic those in the HIV-infected patient. Defining the molecular basis for IFN induction, the cells that produce IFN, and the altered biologic activity of this important cytokine may provide insight into the pathogenesis of HIV disease.