Interleukin-8 fails to induce human immunodeficiency virus-1 expression in chronically infected promonocytic U1 cells but differentially modulates induction by proinflammatory cytokines

This study addresses the role of interleukin (IL)-8, a CXC-chemokine, the level of which is reported to be raised in the peripheral circulation of human immunodeficiency virus-1 (HIV-1)-infected individuals, during the induction of HIV-1 expression from latency and during cytokine-mediated HIV-1 up-regulation. IL-8 at the higher concentrations tested (> or = 100 ng/ml) was unable to induce HIV-1 expression in the chronically infected promonocytic U1 cell line, as measured by p24 antigen enzyme-linked immunosorbent assay (ELISA), whereas at lower concentrations of 1 and 10 ng/ml, constitutive HIV-1 expression was only marginally reduced. HIV-1 replication in acutely infected U937 cells was also significantly reduced by IL-8. The potent up-regulation of HIV-1 expression in U1 cells by tumour necrosis factor-alpha (TNF-alpha) remained unaffected by the addition of IL-8. HIV-1 induction by IL-1beta, IL-6 and TNF-beta, cytokines grouped here as intermediate HIV-1 inducers, was suppressed by IL-8 at concentrations of 1 and 10 ng/ml. However, IL-8 at 100 ng/ml did not significantly alter the effect of IL-1beta, synergized with IL-6 in enhancing, and marginally suppressed TNF-beta-induced HIV-1 expression. IL-8 suppressed granulocyte-macrophage colony-stimulating factor (GM-CSF) and enhanced interferon-gamma (IFN-gamma)-induced HIV-1 expression in a dose-dependent manner. Pretreatment of U1 cells with IL-8 did not alter the IL-8-mediated effects on cytokine-induced HIV-1 expression, suggesting that this chemokine exerts its effect at the time of HIV-1 induction or at a postinduction stage. Furthermore, IL-8 was itself induced by cytokines that up-regulate HIV-1 expression in U1 cells and the levels produced correlated directly with the levels of p24 antigen produced, suggesting common pathways for cytokine induction of both HIV-1 and IL-8. These results show that IL-8, typically a non-inducer, can differentially modulate HIV-1 expression in U1 cells and that this is dependent on the inducing cytokine and on the concentration of IL-8.     

Human immunodeficiency virus infection of monocytic and T-lymphocytic cells: Receptor modulation and differentiation induced by phorbol ester

The monocytic leukemic cell line U937 can be infected with human immunodeficiency virus type 1 (HIV-1) to become permanently infected virus producers. Uninfected U937 cells express T4 (CD4) antigen and form syncytia when mixed with HIV-1 producing cells. Anti-T4 monoclonal antibodies block syncytium formation indicating that the HIV-1 receptors on U937 cells include T4 antigen. The promyelocytic leukemic cell line HL60, while expressing only low amounts of surface T4 and not forming syncytia on exposure to HIV-1, can be infected by HIV-1 at lower efficiency than U937 and T-cell lines. 12-O-Tetradecanoylphorbol-13-acetate (TPA) treatment induces the differentiation of U937 cells into macrophages. HIV-infected U937 cells retain the ability to differentiate, though less efficiently, as shown by the appearance of monocyte/macrophage surface markers. T4 antigen on both U937 and T-cell lines is down regulated by TPA treatment. Functional receptors for HIV-1, assayed by syncytium induction and pseudotype plating, are lost concomitantly with T4 antigen following TPA treatment of U937 cells and T cells.     

Incomplete HIV-1 activation in latently infected U1 cells demonstrated by double in situ hybridization

Triple combination antiretroviral therapy can reduce HIV-1 infection to a relatively small pool of latently infected cells. To eliminate this residual source of virus, new therapies designed to activate latently infected cells are currently being tested. We therefore investigated the kinetics of in vitro HIV-1 RNA induction using chronically infected U1 cells. A new two-probe fluorescence in situ hybridization (double ISH) method was devised to simultaneously assess total HIV-1 RNA (T-RNA) and unspliced HIV-1 RNA (U-RNA) expression in individual cells. Activation of the U1 cells resulted in increasing expression of T-RNA between 0 and 24 h with lagging expression of U-RNA between 6 and 30 h. Both the positive area per cell and the number of positive cells increased with time. Although activation induced 98.5% of the cells to express HIV-1 T-RNA by 24 h, 52% remained negative for U-RNA. In contrast, 100% of 8E5 cells, which constitutively express HIV-1, scored positive for U-RNA as well as T-RNA with the double ISH. This study provides, for the first time, a semiquantitative cell-by-cell analysis of HIV-1 mRNA subsets in latently infected cells. Our results establish the advantages of using double fluorescence ISH to study gene expression and demonstrate that chronically infected U1 cells remain in a partially induced state despite potent activation.     

Inhibition of HIV-1 transmission by interferon and 3'-azido-3'-deoxythymidine during de novo infection of promonocytic cells.

HIV-1 infection of promonocytic U937 cells was used to examined induction of IFN-alpha/beta gene expression and to determine the inhibitory effects of IFN-alpha 2 and/or AZT on de novo HIV-1 infection, initiated either by coculture of virus-shedding U9-IIIB cells with uninfected cells or by incubation of U937 cells with virus-containing supernatants. Usually 21-28 days were required to transmit virus to greater than 90% of the cell culture. HIV-1 infection did not stimulate constitutive production of endogenous IFN-alpha 1/alpha 2 or IFN-beta genes, although induction of IFN RNA was observed following coinfection with the paramyxovirus Sendai. Exogenous rIFN-alpha 2 treatment decreased the intracellular accumulation of viral RNA 5- to 20-fold as determined by Northern blot analysis and the extracellular levels of HIV-1 as measured by p24 ELISA antigen capture. The effect was most dramatic at a time coincident with the rapid increase in virus spread through the cell culture (Days 10-18). During the fourth week of infection, HIV-1 multiplication was able to overcome the IFN-induced block in virus spread. AZT was not effective in limiting virus spread in the cocultivation experiments. When U937 cells were infected with virus-containing supernatants from U9-IIIB cells, IFN and AZT acted in combination to limit the number of infected cells and to inhibit intracellular accumulation of viral RNA. These experiments suggest that subtle differences in the mode of virus transmission in monocytic cells may alter the efficacy of combination antiretroviral therapy.     

Role of mycoplasma infection in the cytopathic effect induced by human immunodeficiency virus type 1 in infected cell lines.

In addition to previously reported tetracycline analogs, other antibiotics known for antimycoplasmal activities inhibited the cytopathic effect in CEM cl13 cells infected with human immunodeficiency virus type 1 (HIV-1) or HIV-2 but were unable to block virus replication. A contaminating mycoplasma was isolated from our CEM cl13 cells and identified as a strain of Mycoplasma fermentans. Following infection of lymphoblastoid (CEM) or promonocytic (U937 and THP1) cell lines with HIV-1, cytopathic effect was observed only in association with mycoplasmal contamination. Moreover, HIV-1 infection of U937 cells after experimental inoculation with a human isolate of M. fermentans led to pronounced cell killing. We have verified that this effect is not merely an artifact caused by arginine and/or glucose depletion in the cell culture medium. These results confirm that mollicutes, in particular M. fermentans, are able to act synergistically with HIV-1 to kill infected cells in some in vitro systems.     

Cell surface antigens and function of monocytes and a monocyte-like cell line before and after infection with HIV

The human immunodeficiency virus (HIV-1) preferentially infects cells that express the CD4 molecule, including monocytes and cells of the monocyte lineage. The monocyte-like cell line U937 and monocytes isolated from peripheral blood lymphocytes (PBL) were infected with HIV-1. Cell surface antigen expression was determined in infected and noninfected cells as was the ability to stimulate in mixed lymphocyte reaction. The CD4 antigen decreased in infected cells U937 and PBL monocytes. MHC class II antigens HLA-DR, HLA-DQ, and HLA-DP increased in HIV-1 infected U937 cells. In infected PBL-derived monocytes, HLA-DR increased, HLA-DQ decreased, and HLA-DP was unchanged. Infected U937 and PBL monocytes were capable of stimulating allogeneic lymphocytes, thus demonstrating retention of the alloantigen presentation function of HIV-1-infected monocytes.     

Interference patterns of human immunodeficiency viruses HIV-1 and HIV-2

The ability of cells infected with a retrovirus to interfere with superinfection by another retrovirus usually involves blockage, by the primary virus, of the receptors for the superinfecting virus. Retroviruses using different receptors do not interfere with each other, and this property has been used to classify various types of retroviruses. Different isolates of human immunodeficiency virus (HIV) were subjected to this type of analysis, and it was found that all HIV-1s cross-interfere with each other in T cells as well as in U937 promonocytic cells, substantiating further that all isolates use the same receptor on these cells. An HIV-2 isolate was found to interfere with HIV-1s, but HIV-1s only partially interfered with HIV-2 superinfection, indicating that inherent differences in receptor interactions exist between HIV-1s and HIV-2. For comparison, interference patterns of D-type primate retroviruses (SRVs) and murine amphotropic and xenotropic retroviruses revealed that each virus fell within distinct interference groups demonstrating that human T cells possess at least four distinct receptors for retroviruses. The mechanism of HIV interference was found to be due to receptor blockage in productively infected cells and to receptor elimination in latently infected T cells. Our findings that all HIV-1s completely interfere with each other and that interference occurs rapidly following acute infection suggests that a cell infected with HIV-1 will not permit reinfection by progeny or by other exogenous HIVs. This, in turn, suggests that progeny reinfection may not be the source of the large amount of unintegrated viral DNA observed following HIV cytopathic infection.     

Restriction of HIV-1 replication in promonocytic cells: a role for IFN-alpha

A comparative study of the replication kinetics of human immunodeficiency virus type 1 (HIV-1) was performed in the promonocytic U937 cells and in the T lymphoblastoid H9 cells. If a productive HIV-1 infection of both cell types could be established, the time which elapses before most of the cells could express viral proteins is always proportionally longer for U937 cells than for H9 cells. Indeed, when U937 cells are infected with HIV-1, this nonproductive phase is followed by a lag phase during which the percentage of virus-producing cells is slowly increasing when compared to H9 cells. The restriction of HIV-1 replication in U937 cells might be consecutive to the lower adsorption of viral particles to these cells, even though the same percentage of U937 and H9 cells was expressing the CD4 molecule. Furthermore, we demonstrate that HIV-1 replication in U937 cells is mainly restricted by endogenous IFN-alpha. Indeed, addition of anti-IFN-alpha antibodies at the time of infection, during the nonproductive phase of the viral replication cycle, or during the lag phase leads to an earlier expression of viral proteins and/or to a rapid increase in the percentage of virus-producing cells. Likewise, the treatment of cultures of HIV-1 chronically infected U937 cells with the same antibodies induces an increased production of viral particles. Thus, IFN-alpha appears to be involved in the persistence of HIV-1 in the monocytes/macrophages of infected individuals.     

Chronically HIV-1 infected human monocyte culture U937 as a model for assessing the efficacy of anti-HIV agents]

Cell culture U937 chronically infected with HIV-1 is suggested as a model for adequate evaluation of antiviral activity of HIV inhibitors. Azidothimidine (AZT) notable decreased HIV-1 reproduction in chronically infected U937 cells to passages 15-18. Glycirrhizic acid (GA) effectively inhibited the virus production during the first four passages, while in subsequent passages (up to passage 20) decreased the virus production by only 60% in comparison with the control. If a combination of AZT and GA (1:1000) was used, p24 was not detected in the culture fluid by passage 20. Culturing of U937 cells with AZT led to a 10-fold decrease in the amount of DNA 2-LTR in comparison with the total content of proviral DNA, the content of HIV-1 DNA 1-LTR remaining virtually unchanged. Culturing of U937 with a combination of AZT and GA resulted in a notable decrease in the content of proviral DNA 2-LTR after passage 3, while after passage 9 this form of HIV-1 DNA was not detected at all.     

Recombinant vaccinia viruses expressing an immunodominant epitope of HIV-1 envelope protein within an influenza hemagglutinin cassette predominantly prime epitope-specific CD8+ CTL

Summary.  We constructed recombinant vaccinia viruses (RVV) expressing a 15-residue peptide (P18IIIB; RIQRGPGRAFVTIGK) of gp160 envelope protein from a human immunodeficiency virus type-1 (HIV-1) IIIB isolate using an H1 influenza virus hemagglutinin (HA) gene cassette. Immunofluorescent tests with antisera against both H1N1 influenza virus and P18IIIB localized chimeric HA molecules comprising influenza virus HA and P18IIIB peptide intracellularly, but the P18IIIB could not be seen on the outer surfaces of infected cells though weak fluorescence was detected regarding HA molecule. Consistent with these findings, Western blotting confirmed the expression of a polypeptide of about 74-kDa protein representing chimeric HA molecule in the infected cells. These recombinants markedly primed CD8⁺ cytotoxic T lymphocytes (CTL) specific for P18IIIB as well as HA protein of the influenza virus, but failed to elicit P18IIIB-specific antibody despite stimulating production of HA-specific antibody. In addition, the P18IIIB-specific CTL could strongly lyse target cells expressing the whole HIV-1 envelope gene of IIIB strain. Thus, the influenza virus chimeric HA cassette vector system used in the present study appeared to be a useful tool for constructing vaccine candidates which will predominantly prime CD8⁺ CTL specific for immunodominant determinants of various infectious agents. Received February 19, 1999 Accepted April 20, 1999     

Efficient induction of HIV-1 replication in latently infected cells through contact with CD4+ T cells: involvement of NF-kappaB activation

Reservoir cells latently infected with HIV-1 pose one of the major obstacles that hamper ultimate eradication of HIV-1 from infected patients. In this report, we showed that direct contact with MOLT-4 T cells induced HIV-1 replication in J(22)-HL-60 latently infected cells without any additional stimulus. Neutralization experiments revealed that pro-inflammatory cytokines, whose production was increased following cell-cell contact, were unlikely to be primarily involved in the induced HIV-1 replication. Cell-cell contact, but not soluble components in the culture supernatant, caused a rapid phosphorylation and degradation of IkappaBalpha, which led to elevated NF-kappaB DNA binding activity in J(22)-HL-60 cells. Furthermore, forced expression of a super-repressor form of IkappaBalpha or pretreatment with ritonavir efficiently blocked the activation of NF-kappaB and HIV-1 replication in J(22)-HL-60 cells co-cultured with MOLT-4 T cells. Moreover, either resting or PHA stimulated primary CD4(+) T cells induced HIV-1 replication in J(22)-HL-60 cells in a similar way with that of MOLT-4 cells. These results indicated that direct contact with CD4(+) T cells induced HIV-1 replication in latently infected cells and provide insight into the molecular mechanism of virus release from myeloid progenitor cells latently infected with HIV-1.     

Dual role of TNF-alpha in NK/LAK cell-mediated lysis of chronically HIV-infected U1 cells. Concomitant enhancement of HIV expression and sensitization of cell-mediated lysis

The U937-derived chronically HIV-infected U1 cell line and uninfected U937 cell clones were efficiently lysed by both unstimulated (NK) and IL-2-stimulated (lymphokine-activated killer; LAK) peripheral blood mononuclear cells (PBMC) of healthy HIV-seronegative donors. Pretreatment of target cells with IFN-gamma down-modulated killing of both U1 cells and two U937 cell clones, and up-regulated MHC class I expression. In contrast, TNF-alpha enhanced the sensitivity of infected U1 cells, but not of U937 cell clones to NK / LAK cell lysis. Co-cultivation of IL-2-stimulated PBMC with U1 cells triggered expression and replication of HIV by cell-cell contact, and this effect was inhibited by anti-TNF-alpha antibodies (Ab); virus production was partially inhibited by zidovudine. Of interest, anti-TNF-alpha Ab protected U1 cells from LAK cell activity. Thus, TNF-alpha can induce HIV expression from chronically infected U1 cells, but also plays an important role in sensitizing these cells to lysis.     

Antibody to native human immunodeficiency virus type 1 envelope glycoproteins induced by IIIB and MN recombinant gp120 vaccines. The NIAID AIDS Vaccine Evaluation Group.

The ability of antibody induced by MN and IIIB recombinant gp120 (rgp120) human immunodeficiency virus type 1 (HIV-1) vaccines the bind to oligomeric native and monomeric recombinant HIV-1 envelope glycoproteins (rgp 120) was measured in 25 uninfected, healthy adult volunteers. A major focus was to evaluate the effect of simultaneous and sequential immunization with vaccines representing different strains of HIV-1 on the ability to broaden cross-reactivity of antibodies against these and other HIV-1 strains. A flow cytometric indirect immunofluorescence assay (FIFA) to detect vaccine-induced antibody to envelope glycoprotein expressed by infected and rgp120-coated target cells was used, MN rgp120 HIV-1 vaccine given alone and coadministered with IIIB rgp120 HIV-1 vaccine elicited antibody which bound to cells infected with HIV-1MN, HIV-IIIB, HIV-1RF, and HIV-1-SF2. The presence of envelope glycoprotein-binding antibody detected by FIFA correlated to a moderate degree with functional antibody against HIV-1MN and HIV-IIIB. Priming immunization with IIIB rgp120 HIV-1 vaccine followed by booster injections of MN rgp120 HIV-1 vaccine resulted in increased cross-reactive antibody binding to these and heterologous clade B HIV-1 strains infecting cells. MN rgp120 HIV-1 vaccine given alone was better able to induce cross-reactive antibody to cells infected with heterologous HIV-1 laboratory strains than was IIIB rgp120 HIV-1 vaccine given alone. The vaccines induced binding antibody to rgp120 possessing the amino acid sequence of a clade E HIV-1 strain as measured by enzyme-linked immunosorbent assay. Levels of antibody binding to cells infected with clade B HIV-1 and cells coated with monomeric rgp120 were greater than that induced by HIV-1IIIB-based gp160 vaccines in previous studies.     

Human herpesvirus 6 variant a infects human term syncytiotrophoblasts in vitro and induces replication of human immunodeficiency virus type 1 in dually infected cells

Human herpesvirus 6 (HHV-6) and human immunodeficiency virus type 1 (HIV-1) may interact during transplacental transmission of HIV-1. The placental syncytiotrophoblast layer serves as the first line of defense of the fetus against viruses. Patterns of replication of HHV-6 variant A (HHV-6A) and HIV-1 were analyzed in singly and dually infected human term syncytiotrophoblast cells cultured in vitro. For this purpose, the GS strain of HHV-6A and the Ba-L and IIIB strains of HIV-1 were used. HHV-6A replication was restricted at the level of early gene products in singly infected syncytiotrophoblasts, whereas no viral protein expression was found in cells infected with HIV-1 alone. Coinfection of syncytiotrophoblast cells with HHV-6A and HIV-1 resulted in production of infectious HIV-1. In contrast, no enhancement of HHV-6A expression was observed in cell cultures infected with both viruses. Uninfected syncytiotrophoblast cells were found to express CXCR4 and CCR3 but not CD4 or CCR5 receptors. Infection of syncytiotrophoblasts with HHV-6A did not induce CD4 expression and had no influence on chemokine receptor expression. Activation of HIV-1 from latency in coinfected cells was mediated by the immediate-early (IE)-A and IE-B gene products of HHV-6A. Open reading frames U86 and U89 of the IE-A region were able to activate HIV-1 replication in a synergistic manner. The data suggest that in vivo double infection of syncytiotrophoblast cells with HHV-6A and HIV-1 could contribute to the transplacental transmission of HIV-1 but not HHV-6A.