Jim A. Turpin

Macrophages and the human immunodeficiency virus

Abstract Mononuclear phagocytes are major participants in human immunodeficiency virus (HIV) disease. These cells function as susceptible targets, persistent reservoirs for virus in tissue and key immunoregulatory elements that control the level of virus replication and the extent of disease. In this review, the second of the series, Monte Meltzer and colleagues review the distinct interactions between HIV and monocytes and between HIV and T cells. Understanding this dualism may more clearly define both the pathogenesis of HIV disease and strategies for therapeutic intervention.

Potent anti-influenza activity of cyanovirin-N and interactions with viral hemagglutinin.

ABSTRACT The novel antiviral protein cyanovirin-N (CV-N) was initially discovered based on its potent activity against the human immunodeficiency virus (HIV). Subsequent studies identified the HIV envelope glycoproteins gp120 and gp41 as molecular targets of CV-N. More recently, mechanistic studies have shown that certain high-mannose oligosaccharides (oligomannose-8 and oligomannose-9) found on the HIV envelope glycoproteins comprise the specific sites to which CV-N binds. Such selective, carbohydrate-dependent interactions may account, at least in part, for the unusual and unexpected spectrum of antiviral activity of CV-N described herein. We screened CV-N against a broad range of respiratory and enteric viruses, as well as flaviviruses and herpesviruses. CV-N was inactive against rhinoviruses, human parainfluenza virus, respiratory syncytial virus, and enteric viruses but was moderately active against some herpesvirus and hepatitis virus (bovine viral diarrhea virus) strains (50% effective concentration [EC50] = ∼1 μg/ml) while inactive against others. Remarkably, however, CV-N and related homologs showed highly potent antiviral activity against almost all strains of influenza A and B virus, including clinical isolates and a neuraminidase inhibitor-resistant strain (EC50 = 0.004 to 0.04 μg/ml). When influenza virus particles were pretreated with CV-N, viral titers were lowered significantly (>1,000-fold). Further studies identified influenza virus hemagglutinin as a target for CV-N, showed that antiviral activity and hemagglutinin binding were correlated, and indicated that CV-N′s interactions with hemagglutinin involved oligosaccharides. These results further reveal new potential avenues for antiviral therapeutics and prophylaxis targeting specific oligosaccharide-comprised sites on certain enveloped viruses, including HIV, influenza virus, and possibly others.

Macrophage - HIV interaction: Viral isolation and target cell tropism

Viral isolates were recovered by cocultivation on macrophage colony-stimulatingfactor (MCSF)-treated monocyte target cells from peripheral blood mononuclear cells (PBMCs) in 25 out of 27 patients seropositive or at risk for HIV infection. Frequency of virus recovery was independent of the patients age, sex, numbers of CD4+ T cells, clinical stage or zidovudine (azidothymidine) therapy. Sixteen out of 19 HIV isolates were serially passaged in MCSF- treated monocytes. Five out of five virus isolates were also passaged in phytohemagglutinin/interleukin-2 (PHA/IL-2)-treated lymphoblasts. In lymphoblasts, no qualitative or quantitative differences were observed between these isolates and human T-cell leukemia virus IIIB (HTLV-IIIB) for (1) release of p24 antigen reverse transcriptase, and infectious virus, (2) induction of typical cytopathic effects (cell syncytia in 3–10% of cells) and cell lysis, (3) frequency of infected cells (5–20% of PBMC) as detected by in situ hybridization for HIV RNA, (4) down-modulation of T cell plasma membrane CD4, and (5) site of progeny virion assembly and budding (plasma membrane only with no intracytoplasmic accumulation of virus). Progeny virus recovered from infected lymphoblasts was fully infectious for other lymphoblasts, but failed to infect MCSF-treated monocytes. Detailed analysis of target cell tropism among HIV isolates showed that HIV isolated in monocytes infected both monocytes and lymphoblasts; progeny virus isolated in lymphoblasts infected only T cells. HIV interacts differently with monocytes and T cells. Understanding this interaction may more clearly define both the pathogenesis of HIV disease and strategies for therapeutic intervention.

In vivo antiviral activity of novel human immunodeficiency virus type 1 nucleocapsid p7 zinc finger inhibitors in a transgenic murine model.

Control of human immunodeficiency virus through the use of inexpensive chemotherapeutics, with minimal side effects and decreased potential for engendering resistant virus, is a long-term therapeutic goal. In principle, this goal can be accomplished if viral replication in reservoirs of chronically and latently infected cells is addressed. As a first step, we have developed novel antiviral compounds based on a 2-mercaptobenzamide thioester chemotype, including the pyridinioalkanoyl thioesters, which specifically target the zinc fingers of the human immunodeficiency virus nucleocapsid protein (NCp7). Using these compounds in a murine transgenic model, in which infectious human immunodeficiency virus is induced from an integrated provirus, we show inhibition of transgenic spleen cell p24 expression with potencies comparable to acute infection assays using human peripheral blood lymphocytes. More importantly, transgenic mice treated in vivo with two 2-mercaptobenzamide thioesters expressed significantly lower plasma p24, and splenocytes from these animals produced fewer infectious virions. Thus, these thioesters may provide an effective means for inhibiting the expression of human immunodeficiency virus from integrated viral reservoirs.

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

Monocytes treated with 500 IU/ml human recombinant interferon‐γ (rIFN‐γ) 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‐γ was removed at 7 days and the cells were cultured for an additional 3 weeks without IFN‐γ. The 50% inhibitory dose for reduction of maximum RT activity in HIV‐infected monocyte cultures was about 1 IU/ml IFN‐γ. No increase in HIV replication was evident in monocytes treated with IFN‐γ 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‐γ was 10 to 20 times more effective than rIFN‐α2b 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‐α or IFN‐γ had no effect on virus replication. These data suggest that the principal action of IFN‐α and IFN‐γ 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. J. Leukoc. Biol. 56: 362–368; 1994.

Preclinical Evaluation of a Zinc Finger Inhibitor Targeting Lentivirus Nucleocapsid Protein in SIV-Infected Monkeys

There is a continued need to develop inexpensive and effective drugs specific for novel targets of human immunodeficiency virus type 1 (HIV-1). The HIV-1 nucleocapsid p7 (NCp7) protein plays a critical role in early and late stages of the virus life cycle and possesses two highly conserved retroviral zinc fingers that are essential for its function. We have previously shown that zinc finger inhibitors (ZFI) based on the S-acyl 2-mercaptobenzamide thioester (SAMT) chemotype specifically target HIV NCp7 and are effective at reducing levels of infectious virus in an HIV-1-transgenic mouse model. Here, we did an initial proof-of-concept study to test the potential of a lead SAMT compound to reduce virus infectivity in the simian immunodeficiency virus (SIV) nonhuman primate model. SAMT-19 had potent antiviral and virucidal effects against the primary pathogenic isolate SIV/DeltaB670 and was non-cytotoxic in vitro. Cynomolgus macaques were infected intrarectally with SIV/DeltaB670 and treated with a low dose of SAMT-19 by continuous infusion from day 8 to day 28 post infection. Monkeys in the treatment group had significantly lower levels of infectious virus in peripheral blood mononuclear cells during the course of therapy as compared to monkeys in the control group, although therapy had no demonstrable effect on virus load. SAMT-19 therapy did not alter liver, kidney or immunologic function and was well tolerated by all treated monkeys. These data demonstrate that SAMT-19 is safe and virucidal in the nonhuman primate model. Further studies directed at optimizing SAMT bioavailability and pharmacokinetics likely will result in enhanced therapeutic efficacy of this promising HIV therapeutic.

Interleukin-2 suppresses activated macrophage intracellular killing activity by inducing macrophages to secrete TGF-β

Phorbol myristate acetate (PMA) treatment of an EL‐4 thymoma cell line (EL‐4farrar) induced secretion of a factor that inhibited intracellular killing of Leishmania major amastigotes by activated macrophages. Analysis of the cytokines produced by EL‐4 cells after PMA stimulation identified interleukin‐2 (IL‐2, 2500 U/ml), IL‐4 (1280 U/ml), interferon‐γ (IFN‐γ; 100 U/ml), and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF; 50 U/ml). Neither tumor necrosis factor nor transforming growth factor β (TGF‐β) was detected. Each of the cytokines present in EL‐4 fluids was assessed for capacity to activate macrophages for destruction of parasites or to suppress intracellular killing. IFN‐γ and GM‐CSF both activated macrophages to kill Leishmania; I L‐2 and IL‐4 had no activity for induction of this anti‐microbial effector function. IL‐2 and IL‐4 were tested for their capacity to inhibit lymphokine‐ or IFN‐γ‐induced destruction of L. major by macrophages: IL‐4 was ineffective, but IL‐2 markedly suppressed the activation of macrophages for intracellular killing. Addition of ≥ 10 U/ml of IL‐2 at the time of infection, or up to 4 h before, blocked up to 100% of the capacity of activated macrophages to kill intracellular amastigotes. Immunoaffinity treatment of EL‐4 fluids with anti‐IL‐2 antibody resulted in >80% reduction in suppression of intracellular killing. The suppressive effects of IL‐2 were not direct, but mediated by TGF‐β. IL‐2 induced resident peritoneal macrophages to secrete >5000 pg/ml TGF‐β1, a quantity that is > 500‐fold higher than constitutive background levels (20–40 pg/ml) and is sufficient to block intracellular killing activities. This increase in secretion of TGF‐β was not dependent increases in TGF‐β1 mRNA. Treatment of cultures with EL‐4 fluids or recombinant IL‐2 in the presence of antibody to TGF‐β1 blocked the suppressive activity of both. Thus, IL‐2 was the major suppressor factor in EL‐4 fluids, and it acted indirectly through the induction and autocrine action of TGF‐β. J. Leukoc. Biol. 55: 81–90; 1994.

Phorbol ester reduces constitutive nuclear NF kappa B and inhibits HIV-1 production in mature human monocytic cells.

NFχB is a potent mediator of specific gene expression in human monocytes and has been shown to play a role in transcription of the HIV‐1 genome in promonocyte leukemias. There is little information available on the response of NFχB to cytokines in normal human monocytes. We have used a 32P‐labeled oligonucleotide derived from human immunodeficiency virus (HIV‐1) long terminal repeat, which contains a tandem repeat of the NFχB binding sequence, as a probe in a gel retardation assay to study this transcription factor. Using this assay, we have detected NFXB in extracts of nuclei from normal human monocytes. Treatment of normal monocytes with 12‐0‐tetradecanoyl phorbol‐13‐acetate (TPA) for 4‐24 h caused the complete disappearance of NFχB from nuclear extracts of monocytes. A similar result was obtained with the mature monocytic leukemia cell line THP‐1. The constitutive transcription factor SPI was unaffected by addition of TPA. The disappearance of NFXB from the nucleus was concentration dependent between 10 and 50 ng/ml of phorbol ester. In THP‐1 cells, TPA also induced a new, faster‐migrating NFXB species not induced in monocytes. Protein kinase C inhibitor staurosporine, but not cyclic nucleotide‐dependent protein kinase inhibitor HA‐1004, also dramatically reduced constitutive levels of nuclear NFχB. Finally, TPA addition to monocytes infected with HIV‐1 inhibited HIV‐1 replication, as determined by reverse transcriptase assays, in a concentration‐dependent manner. These results are in striking contrast to the increase in nuclear NFχB and HIV‐1 replication induced by phorbol esters in promonocyte leukemia cells U937 and HL‐60, and emphasize the importance of studying cytokine regulation of HIV‐1 in normal monocytes.

Regulation of Cytokine and Viral Gene Expression in Monocytes Infected with the Human Immunodeficiency Virus

Monocytes treated with interferon-alpha (IFN-alpha) at virus challenge show no evidence of human immunodeficiency virus (HIV) infection: no p24 antigen or reverse transcriptase (RT) activity, no viral mRNA and no proviral DNA. Levels of p24 antigen and RT activity in monocytes infected with HIV 1-3 weeks before IFN-alpha treatment gradually decrease to baseline. HIV-induced cytopathic changes are markedly reduced, as are levels of HIV mRNA: the frequency of productively infected cells is less than or equal to 1%. But, levels of proviral DNA in the IFN-alpha-treated and control HIV-infected cells are indistinguishable, and remain so through 3 weeks. Large quantities of proviral DNA in IFN-alpha-treated cells with little active transcription suggest true microbiological latency. The major potential source for IFN-alpha in HIV-infected patients is the macrophage. With any of 15 virus isolates, tumor necrosis factor-alpha, interleukin-1 beta, interleukin-6, IFN-omega or IFN-beta are not detected nor the mRNA expressed in HIV-infected or uninfected monocytes. Both uninfected and HIV-infected monocytes produce high levels of these cytokines after treatment with synthetic double-stranded RNA (poly-I:C). Uninfected monocytes also produce high levels of IFN-alpha after treatment with Poly-I:C, Newcastle disease virus or herpes simplex virus. In marked contrast, HIV-infected monocytes express no IFN-alpha activity or mRNA before or after treatment with any of these agents. The markedly diminished capacity of HIV-infected monocyte to produce IFN-alpha reflects a specific transcriptional block and may be an adaptive mechanism of virus to alter basic microbicidal functions of this cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-2 and the Regulation of Activated Macrophage Cytotoxic Activities

In 1976, Morgan and colleagues reported that conditioned media from mitogen-stimulated mononuclear cells contained a factor which maintained the exponential proliferative growth of human leukemic blood or bone marrow cells (1); the proliferative cells were identified as normal T lymphocytes (2). Isolation, characterization, and subsequent purification of this factor in the conditioned medium lead to the identification of a T cell growth factor (TCGF) now known as IL-2 (3), a glycoprotein of 15.5 kD with a slightly basic isoelectric point (4). Shortly thereafter, Taniguchi et al. (5) isolated a cDNA clone for IL-2. Although IL-2 was initially described as the ultimate mitogenic signal for both antigenically and polyclonally activated T cells, aiding in their cell cycle transition from G1 to S phase (6), subsequent studies showed that IL-2 stimulates NK and LAK cell activity (7, 8), induces B cell differentiation and proliferation (9), and activates macrophage cytotoxicity (10). IL-2 also participates in induction of T cell synthesis of cytokines, such as IFN-γ (11) and B cell growth factor-1, or IL-4 (12).